<?xml version="1.0" encoding="UTF-8"?>
<!DOCTYPE article PUBLIC "-//NLM//DTD Journal Publishing DTD v2.3 20070202//EN" "journalpublishing.dtd">
<article article-type="research-article" dtd-version="2.3" xml:lang="EN" xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">
<front>
<journal-meta>
<journal-id journal-id-type="publisher-id">Front. Pharmacol.</journal-id>
<journal-title>Frontiers in Pharmacology</journal-title>
<abbrev-journal-title abbrev-type="pubmed">Front. Pharmacol.</abbrev-journal-title>
<issn pub-type="epub">1663-9812</issn>
<publisher>
<publisher-name>Frontiers Media S.A.</publisher-name>
</publisher>
</journal-meta>
<article-meta>
<article-id pub-id-type="publisher-id">1397116</article-id>
<article-id pub-id-type="doi">10.3389/fphar.2024.1397116</article-id>
<article-categories>
<subj-group subj-group-type="heading">
<subject>Pharmacology</subject>
<subj-group>
<subject>Original Research</subject>
</subj-group>
</subj-group>
</article-categories>
<title-group>
<article-title>Natural compound Alternol actives multiple endoplasmic reticulum stress-responding pathways contributing to cell death</article-title>
<alt-title alt-title-type="left-running-head">Liu et al.</alt-title>
<alt-title alt-title-type="right-running-head">
<ext-link ext-link-type="uri" xlink:href="https://doi.org/10.3389/fphar.2024.1397116">10.3389/fphar.2024.1397116</ext-link>
</alt-title>
</title-group>
<contrib-group>
<contrib contrib-type="author" equal-contrib="yes">
<name>
<surname>Liu</surname>
<given-names>Wang</given-names>
</name>
<xref ref-type="aff" rid="aff1">
<sup>1</sup>
</xref>
<xref ref-type="author-notes" rid="fn001">
<sup>&#x2020;</sup>
</xref>
<uri xlink:href="https://loop.frontiersin.org/people/2018660/overview"/>
<role content-type="https://credit.niso.org/contributor-roles/investigation/"/>
<role content-type="https://credit.niso.org/contributor-roles/methodology/"/>
<role content-type="https://credit.niso.org/contributor-roles/writing-original-draft/"/>
<role content-type="https://credit.niso.org/contributor-roles/data-curation/"/>
<role content-type="https://credit.niso.org/contributor-roles/formal-analysis/"/>
</contrib>
<contrib contrib-type="author" equal-contrib="yes">
<name>
<surname>He</surname>
<given-names>Chenchen</given-names>
</name>
<xref ref-type="aff" rid="aff2">
<sup>2</sup>
</xref>
<xref ref-type="author-notes" rid="fn001">
<sup>&#x2020;</sup>
</xref>
<uri xlink:href="https://loop.frontiersin.org/people/2167434/overview"/>
<role content-type="https://credit.niso.org/contributor-roles/data-curation/"/>
<role content-type="https://credit.niso.org/contributor-roles/methodology/"/>
<role content-type="https://credit.niso.org/contributor-roles/writing-original-draft/"/>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Li</surname>
<given-names>Changlin</given-names>
</name>
<xref ref-type="aff" rid="aff3">
<sup>3</sup>
</xref>
<uri xlink:href="https://loop.frontiersin.org/people/1077519/overview"/>
<role content-type="https://credit.niso.org/contributor-roles/data-curation/"/>
<role content-type="https://credit.niso.org/contributor-roles/writing-original-draft/"/>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Ye</surname>
<given-names>Shazhou</given-names>
</name>
<xref ref-type="aff" rid="aff4">
<sup>4</sup>
</xref>
<role content-type="https://credit.niso.org/contributor-roles/data-curation/"/>
<role content-type="https://credit.niso.org/contributor-roles/writing-original-draft/"/>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Zhao</surname>
<given-names>Jiang</given-names>
</name>
<xref ref-type="aff" rid="aff5">
<sup>5</sup>
</xref>
<uri xlink:href="https://loop.frontiersin.org/people/1987456/overview"/>
<role content-type="https://credit.niso.org/contributor-roles/data-curation/"/>
<role content-type="https://credit.niso.org/contributor-roles/writing-original-draft/"/>
<role content-type="https://credit.niso.org/contributor-roles/methodology/"/>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Zhu</surname>
<given-names>Cunle</given-names>
</name>
<xref ref-type="aff" rid="aff5">
<sup>5</sup>
</xref>
<uri xlink:href="https://loop.frontiersin.org/people/2050756/overview"/>
<role content-type="https://credit.niso.org/contributor-roles/data-curation/"/>
<role content-type="https://credit.niso.org/contributor-roles/writing-original-draft/"/>
</contrib>
<contrib contrib-type="author" corresp="yes">
<name>
<surname>Wang</surname>
<given-names>Xiangwei</given-names>
</name>
<xref ref-type="aff" rid="aff5">
<sup>5</sup>
</xref>
<xref ref-type="corresp" rid="c001">&#x2a;</xref>
<uri xlink:href="https://loop.frontiersin.org/people/2212340/overview"/>
<role content-type="https://credit.niso.org/contributor-roles/funding-acquisition/"/>
<role content-type="https://credit.niso.org/contributor-roles/investigation/"/>
<role content-type="https://credit.niso.org/contributor-roles/validation/"/>
<role content-type="https://credit.niso.org/contributor-roles/Writing - review &#x26; editing/"/>
</contrib>
<contrib contrib-type="author" corresp="yes">
<name>
<surname>Ma</surname>
<given-names>Qi</given-names>
</name>
<xref ref-type="aff" rid="aff4">
<sup>4</sup>
</xref>
<xref ref-type="corresp" rid="c001">&#x2a;</xref>
<role content-type="https://credit.niso.org/contributor-roles/funding-acquisition/"/>
<role content-type="https://credit.niso.org/contributor-roles/investigation/"/>
<role content-type="https://credit.niso.org/contributor-roles/validation/"/>
<role content-type="https://credit.niso.org/contributor-roles/Writing - review &#x26; editing/"/>
</contrib>
<contrib contrib-type="author" corresp="yes">
<name>
<surname>Li</surname>
<given-names>Benyi</given-names>
</name>
<xref ref-type="aff" rid="aff1">
<sup>1</sup>
</xref>
<xref ref-type="corresp" rid="c001">&#x2a;</xref>
<uri xlink:href="https://loop.frontiersin.org/people/172873/overview"/>
<role content-type="https://credit.niso.org/contributor-roles/conceptualization/"/>
<role content-type="https://credit.niso.org/contributor-roles/funding-acquisition/"/>
<role content-type="https://credit.niso.org/contributor-roles/investigation/"/>
<role content-type="https://credit.niso.org/contributor-roles/methodology/"/>
<role content-type="https://credit.niso.org/contributor-roles/project-administration/"/>
<role content-type="https://credit.niso.org/contributor-roles/supervision/"/>
<role content-type="https://credit.niso.org/contributor-roles/validation/"/>
<role content-type="https://credit.niso.org/contributor-roles/visualization/"/>
<role content-type="https://credit.niso.org/contributor-roles/writing-original-draft/"/>
<role content-type="https://credit.niso.org/contributor-roles/Writing - review &#x26; editing/"/>
</contrib>
</contrib-group>
<aff id="aff1">
<sup>1</sup>
<institution>Department of Urology</institution>, <institution>The University of Kansas Medical Center</institution>, <addr-line>Kansas City</addr-line>, <addr-line>KS</addr-line>, <country>United States</country>
</aff>
<aff id="aff2">
<sup>2</sup>
<institution>Department of Radiation Oncology</institution>, <institution>The First Affiliated Hospital of Xi&#x2019;an Jiaotong University School of Medicine</institution>, <addr-line>Xi&#x2019;an</addr-line>, <country>China</country>
</aff>
<aff id="aff3">
<sup>3</sup>
<institution>Tianjin Institute of Urology</institution>, <institution>The Second Hospital of Tianjin Medical University</institution>, <addr-line>Tianjin</addr-line>, <country>China</country>
</aff>
<aff id="aff4">
<sup>4</sup>
<institution>Translational Research Laboratory for Urology</institution>, <institution>The First Affiliated Hospital of Ningbo University</institution>, <addr-line>Ningbo</addr-line>, <addr-line>Zhejiang</addr-line>, <country>China</country>
</aff>
<aff id="aff5">
<sup>5</sup>
<institution>Department of Urology</institution>, <institution>The Affiliated Hospital of Guangdong Medical University</institution>, <addr-line>Zhanjiang</addr-line>, <country>China</country>
</aff>
<author-notes>
<fn fn-type="edited-by">
<p>
<bold>Edited by:</bold> <ext-link ext-link-type="uri" xlink:href="https://loop.frontiersin.org/people/819454/overview">Wagdy Mohamed Eldehna</ext-link>, Kafrelsheikh University, Egypt</p>
</fn>
<fn fn-type="edited-by">
<p>
<bold>Reviewed by:</bold> <ext-link ext-link-type="uri" xlink:href="https://loop.frontiersin.org/people/2687292/overview">Shimaa Abass</ext-link>, Kafrelsheikh University, Egypt</p>
<p>
<ext-link ext-link-type="uri" xlink:href="https://loop.frontiersin.org/people/502962/overview">Xiaolin Zi</ext-link>, University of California, Irvine, United States</p>
<p>
<ext-link ext-link-type="uri" xlink:href="https://loop.frontiersin.org/people/602502/overview">Hari K. Koul</ext-link>, Louisiana State University, United States</p>
</fn>
<corresp id="c001">&#x2a;Correspondence: Benyi Li, <email>bli@kumc.edu</email>; Qi Ma, <email>fyymaqi@nbu.edu.cn</email>; Xiangwei Wang, <email>winn0324@gmail.com</email>
</corresp>
<fn fn-type="equal" id="fn001">
<label>
<sup>&#x2020;</sup>
</label>
<p>These authors have contributed equally to this work</p>
</fn>
</author-notes>
<pub-date pub-type="epub">
<day>20</day>
<month>05</month>
<year>2024</year>
</pub-date>
<pub-date pub-type="collection">
<year>2024</year>
</pub-date>
<volume>15</volume>
<elocation-id>1397116</elocation-id>
<history>
<date date-type="received">
<day>06</day>
<month>03</month>
<year>2024</year>
</date>
<date date-type="accepted">
<day>02</day>
<month>05</month>
<year>2024</year>
</date>
</history>
<permissions>
<copyright-statement>Copyright &#xa9; 2024 Liu, He, Li, Ye, Zhao, Zhu, Wang, Ma and Li.</copyright-statement>
<copyright-year>2024</copyright-year>
<copyright-holder>Liu, He, Li, Ye, Zhao, Zhu, Wang, Ma and Li</copyright-holder>
<license xlink:href="http://creativecommons.org/licenses/by/4.0/">
<p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</p>
</license>
</permissions>
<abstract>
<p>
<bold>Background:</bold> Alternol is a small molecular compound isolated from the fermentation of a mutant fungus obtained from Taxus brevifolia bark. Our previous studies showed that Alternol treatment induced reactive oxygen species (ROS)-dependent immunogenic cell death. This study conducted a comprehensive investigation to explore the mechanisms involved in Alternol-induced immunogenic cell death.</p>
<p>
<bold>Methods:</bold> Prostate cancer PC-3, C4-2, and 22RV1 were used in this study. Alternol interaction with heat shock proteins (HSP) was determined using CETSA assay. Alternol-regulated ER stress proteins were assessed with Western blot assay. Extracellular adenosine triphosphate (ATP) was measured using ATPlite Luminescence Assay System.</p>
<p>
<bold>Results:</bold> Our results showed that Alternol interacted with multiple cellular chaperone proteins and increased their expression levels, including endoplasmic reticulum (ER) chaperone hypoxia up-regulated 1 (HYOU1) and heat shock protein 90 alpha family class B member 1 (HSP90AB1), as well as cytosolic chaperone heat shock protein family A member 8 (HSPA8). These data represented a potential cause of unfolded protein response (UPR) after Alternol treatment. Further investigation revealed that Alternol treatment triggered ROS-dependent (ER) stress responses via R-like ER kinase (PERK), inositol-requiring enzyme 1&#x03B1; (IRE1&#x03B1;). The double-stranded RNA-dependent protein kinase (PKR) but not activating transcription factor 6 (ATF6) cascades, leading to ATF-3/ATF-4 activation, C/EBP-homologous protein (CHOP) overexpression, and X-box binding protein XBP1 splicing induction. In addition, inhibition of these ER stress responses cascades blunted Alternol-induced extracellular adenosine triphosphate (ATP) release, one of the classical hallmarks of immunogenic cell death.</p>
<p>
<bold>Conclusion: </bold>Taken together, our data demonstrate that Alternol treatment triggered multiple ER stress cascades, leading to immunogenic cell death.</p>
</abstract>
<kwd-group>
<kwd>Er stress</kwd>
<kwd>PERK</kwd>
<kwd>IRE1&#x3b1;</kwd>
<kwd>prostate cancer</kwd>
<kwd>heat-shock proteins</kwd>
<kwd>ATP release</kwd>
</kwd-group>
<custom-meta-wrap>
<custom-meta>
<meta-name>section-at-acceptance</meta-name>
<meta-value>Pharmacology of Anti-Cancer Drugs</meta-value>
</custom-meta>
</custom-meta-wrap>
</article-meta>
</front>
<body>
<sec id="s1">
<title>Introduction</title>
<p>ER is a crucial organelle with a lot of functions, including storage and buffering of calcium ions (Ca<sup>2&#x2b;</sup>), lipid biosynthesis, and folding and assembly of secretory and transmembrane proteins (<xref ref-type="bibr" rid="B3">Celik et al., 2023</xref>). However, due to physical and chemical factors, cell homeostasis is easy to be destroyed and cellular proteins cannot be properly folded, causing a series of physiological responses, including lack of Ca<sup>2&#x2b;</sup> deficiency, molecular chaperone or cellular energy, and increased reactive oxygen species (ROS), protein variation and disulfide bond reduction (<xref ref-type="bibr" rid="B27">Oakes and Papa, 2015</xref>; <xref ref-type="bibr" rid="B32">Rufo et al., 2017</xref>). For maintaining ER homeostasis, cells have developed an adaptation mechanism through a series of adaptive pathways called the unfolded protein response (UPR) (<xref ref-type="bibr" rid="B13">Hetz et al., 2020</xref>).</p>
<p>The UPR aims to recover the ER-related protein folding ability by increasing the expression of ER-related chaperones and attenuating global protein translation (<xref ref-type="bibr" rid="B32">Rufo et al., 2017</xref>). In mammalian cells, the UPR is controlled by three ER stress sensors, namely, IRE1&#x3b1;, PERK, and activating transcription factor 6 (ATF6). In homeostasis conditions, these proteins are kept in an inactive state by the master regulator of the UPR, the glucose-regulated protein-78 (GRP78, also known as BiP) (<xref ref-type="bibr" rid="B7">Ernst et al., 2024</xref>). PERK protein responds to ER stress by inducing eIF2&#x3b1; phosphorylation, resulting in increased expression of ATF4 protein and CHOP (<xref ref-type="bibr" rid="B33">Saaoud et al., 2024</xref>). IRE1&#x3b1; protein cleaves the transcription factor X-box binding protein (XBP1) mRNA to generate a spliced XBP1 variant (XBP1s), which triggers ER stress by up-regulating a large number of genes involved in the UPR (<xref ref-type="bibr" rid="B29">Park et al., 2021</xref>). During ER stress, the ATF6 protein is translocated from the ER to the Golgi apparatus for cleavage by S1P and S2P. The cleaved N-terminal region of ATF6 protein is an active transcription factor for ER chaperones and XBP1 (<xref ref-type="bibr" rid="B5">de la Calle et al., 2022</xref>).</p>
<p>Immunogenic cell death (ICD) is a subtype of cell death that triggers an adaptive immune response against remaining tumor cells (<xref ref-type="bibr" rid="B1">Aria and Rezaei, 2023</xref>; <xref ref-type="bibr" rid="B36">Sprooten et al., 2023</xref>). Certain chemo-drugs, radiation therapy, and photodynamic therapy were reported to induce ICD on treated tumor cells by eliciting ER stress and subsequent secretion of damage-associated molecular patterns (DAMPs), including calreticulin membrane translocation, ATP and HMGB1 release, type-I interferon production, etc. (<xref ref-type="bibr" rid="B8">Galluzzi et al., 2020</xref>). These DAMPs then recruit innate immune cells such as dendritic cells to stimulate tumor-specific cytotoxic T lymphocytes to eliminate remaining tumors. We recently demonstrated that the chemo-drug Mitoxantrone was a <italic>bona-fade</italic> ICD inducer for prostate cancer by activating eIF2&#x3b1; <italic>via</italic> PERK/GCN2-dependent ER stress cascade (<xref ref-type="bibr" rid="B17">Li et al., 2020</xref>). We also discovered that Alternol, a novel small chemical compound, induced a strong ICD response in prostate cancer <italic>via</italic> releasing large amounts of inflammatory cytokines while the molecular mechanism was not determined (<xref ref-type="bibr" rid="B19">Li et al., 2021</xref>).</p>
<p>Alternol was isolated from the fermentation of a mutant fungus obtained from <italic>Taxus brevifolia</italic> bark (<xref ref-type="bibr" rid="B24">Liu et al., 2020</xref>). Previous studies from our group and others demonstrated that Alternol treatment in prostate cancer cells caused a dramatic increase in reactive oxygen species and subsequent cell death (<xref ref-type="bibr" rid="B37">Tang et al., 2014</xref>; <xref ref-type="bibr" rid="B48">Zuo et al., 2017</xref>; <xref ref-type="bibr" rid="B41">Xu et al., 2019</xref>; <xref ref-type="bibr" rid="B42">Xu et al., 2020</xref>). We also discovered that Alternol interacted with 14 cellular proteins including five mitochondrial and ER-residing chaperone proteins, indicating a potential link between Alternol-induced ICD (inflammatory response) (<xref ref-type="bibr" rid="B19">Li et al., 2021</xref>) and ER stress. To determine if Alternol-induced ICD responses were due to chaperone protein disruption and ER stress, we conducted a series of experiments to investigate ER stress-related cascades in Alternol-treatment prostate cancer cells. Our data confirmed that Alternol treatment elicited multiple ER stress cascades and subsequent immunogenic ATP release.</p>
</sec>
<sec sec-type="materials|methods" id="s2">
<title>Materials and methods</title>
<sec id="s2-1">
<title>Cell lines, reagents, antibodies, and siRNA</title>
<p>Human prostate cancer C4-2B, 22RV1, PC-3 cells, and Benign Prostatic Hyperplasia-1 (BPH1) cells were recently obtained from ATCC (Manassas, VA) and authenticated by ATCC before shipment. Cells were cultured in RPMI1640 medium (Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco) plus 100&#xa0;U/mL penicillin/streptomycin and 2&#xa0;mM&#xa0;L-glutamine.</p>
<p>Antibodies of ATF-3 (&#x23;33593), ATF-6 (&#x23;65880), IRE1&#x3b1; (&#x23;3284), ATF4 (&#x23;11815), eIF2&#x3b1; (&#x23;9722), eIF2&#x3b1;/pS51 (&#x23;9721), CHOP (&#x23;2895), BiP (&#x23;3177), XBP1s (&#x23;27091), PERK (&#x23;5683), phospho-PERK (T980, &#x23;3179), PARP (&#x23;9542), HSP60 (D6F1, &#x23;12165), HSPA8 (&#x23;8444), HYOU1 (&#x23;13452), NF-&#x3ba;B/p65 (&#x23;3034), phospho-NF-&#x3ba;B (S563, &#x23;3031), I-&#x3ba;Ba (&#x23;4024), and IKK (&#x23;2684) were obtained from Cell Signaling (Danvers, MA, United States of America). Antibodies of HSP90B1 (&#x23;H9010) and phospho-IRE1&#x3b1; (Ser724) (&#x23; PA1-16927) were purchased from Thermo Fisher (Waltham, MA, United States of America). Antibodies of HSP90A1 (sc-13119) and &#x3b2;-Actin (sc-47778) were obtained from Santa Cruz (Dallas, TX, United States of America).</p>
<p>Alternol was obtained as a gift from Sungen Biosciences (Shantou, China). PERK inhibitor AMG44 (&#x23;SML3049), and ATF-6 inhibitor CEAPIN-A7 (SML2330) were obtained from Millipore Sigma (Burlington, MA, United States of America). PKR inhibitor Imoxin (S9668), NF-&#x3ba;B inhibitor SN50 (S6672), and IRE1&#x3b1; inhibitor MKC8866 (S8875) were obtained from Sellectchem (Huston, TX, United States of America). ROS scavenger n-acetylcysteine (N-Ac) was obtained from Cayman Chemicals (Ann Arbor, MI). The small interfering RNAs (siRNAs) for IRE1a, PERK, and PKR were obtained from Horizon Discovery Ltd (Cambridge, UK). ATPlite&#x2122; luminescence assay system (catalog &#x23;6016941) was purchased from PerkinElmer (Waltham, MA).</p>
</sec>
<sec id="s2-2">
<title>Western blot assays</title>
<p>Western blot assay of protein expression was conducted as described (<xref ref-type="bibr" rid="B16">Li et al., 2019</xref>). Briefly, total cellular protein lysates were extracted using the radioimmunoprecipitation assay (RIPA) buffer supplemented with a protease inhibitor cocktail. An equal amount of proteins was subjected to SDS-PAGE separation, followed by transferring onto the PVDF membrane. After blocking in 5% nonfat milk for 1h, the membranes were incubated with primary antibodies overnight at 4&#xb0;C. Protein bands were visualized using the horseradish peroxidase-linked (HRP-linked) secondary antibody for 2&#xa0;h and the ECL solution (Santa Cruz Biotech).</p>
</sec>
<sec id="s2-3">
<title>ATP level assay</title>
<p>ATP level was measured using ATPlite&#x2122; Luminescence Assay System following the manufacturer&#x2019;s instructions, as described (<xref ref-type="bibr" rid="B19">Li et al., 2021</xref>). Briefly, C4-2B or PC-3 cells were seeded in a 6-well plate overnight, and then treated as described. The cell pellets were collected and lysed in RIPA buffer. The cellular lysate was then diluted with the assay buffer, and mixed with the substrate solution (at a 4:1 ratio). The luminescence signal was measured using the Lumat LB9501 reader (Berthold, Oak Ridge, TN).</p>
</sec>
<sec id="s2-4">
<title>Cellular thermal shift assay (CETSA)</title>
<p>Alternol binding with the chaperone proteins was examined using the CETSA assay, as described previously (<xref ref-type="bibr" rid="B12">He et al., 2017</xref>). Briefly, C4-2B cells were incubated with Alternol (10&#xa0;&#x3bc;M) for 2&#xa0;h. Cell pellets were washed with PBS followed by two repeated freeze-thaw cycles with liquid nitrogen. The lysates were then aliquoted into 9 vials for heating at 37, 41, 45, 49, 53, 57, 61, 65, and 69&#xb0;C for 3&#xa0;min, and then cooled down on ice for 2&#xa0;min. The cell lysates were briefly vortexed and then centrifuged at 18,000&#xa0;g for 20&#xa0;min at 4&#xb0;C. The supernatant was loaded onto SDS-PAGE gel followed by Western blot analysis.</p>
</sec>
<sec id="s2-5">
<title>Statistical analysis</title>
<p>Data were present as the mean &#xb1; SEM from at least three experiments. Representative images of non-quantitative data were shown from multiple experiments. Statistical analysis was conducted using ANOVA analysis and student <italic>t</italic>-test to compare two groups with SPSS software (Chicago, IL). A <italic>p</italic>-value of 0.05 or less was considered as a significant difference.</p>
</sec>
</sec>
<sec sec-type="results" id="s3">
<title>Results</title>
<sec id="s3-1">
<title>Alternol interacts with multiple heat-shock proteins</title>
<p>We and others have shown that Alternol induces oxidative stress-dependent cell death preferentially in malignant cells (<xref ref-type="bibr" rid="B37">Tang et al., 2014</xref>; <xref ref-type="bibr" rid="B48">Zuo et al., 2017</xref>; <xref ref-type="bibr" rid="B41">Xu et al., 2019</xref>; <xref ref-type="bibr" rid="B24">Liu et al., 2020</xref>; <xref ref-type="bibr" rid="B42">Xu et al., 2020</xref>). In our previous report (<xref ref-type="bibr" rid="B16">Li et al., 2019</xref>), to identify Alternol-interacting proteins we utilized biotin-labeled Alternol and pulled down 14 cellular proteins. Among these proteins, there were five chaperone proteins, including two ER-residing HYOU1 (also known as HSP120&#x3b1; or GRP170) and HSP90B1 (or GRP94), mitochondrial HSPD1 (or HSP60), cytosolic HSP90AB1 (or HSP84) and heat-shock cognate HSPA8 (HSC70). To further verify their interaction with Alternol, we conducted a CETSA assay (<xref ref-type="bibr" rid="B25">Martinez Molina et al., 2013</xref>) in prostate cancer PC-3 cells. As shown in <xref ref-type="fig" rid="F1">Figures 1A&#x2013;E</xref>, these proteins displayed a clear curve-shifting pattern after Alternol treatment with a &#x394;T<sub>m</sub>50 value between 3.48C and 12.01C. These data indicated that Alternol interacted with these proteins in cells. In addition, Alternol treatment increased the expression levels of HYOU1, HSP90B1, HSP90AB1, and HSPA8 proteins (<xref ref-type="fig" rid="F3">Figures 3F, G</xref>). Since these chaperone proteins are involved in protein folding and ER stress protection (<xref ref-type="bibr" rid="B22">Lindenmeyer et al., 2008</xref>; <xref ref-type="bibr" rid="B30">Rachidi et al., 2015</xref>; <xref ref-type="bibr" rid="B20">Lin et al., 2023</xref>; <xref ref-type="bibr" rid="B38">Tao et al., 2024</xref>), these data indicate that Alternol treatment potentially caused an unfolded protein response (UPR) and subsequent ER stress.</p>
<fig id="F1" position="float">
<label>FIGURE 1</label>
<caption>
<p>Alternol interacts with multiple chaperone proteins. <bold>(A&#x2013;E)</bold> C4-2B cells were treated with Alternol (10&#xa0;&#x3bc;M) for 2&#xa0;h. Cell pellets were washed with cold PBS, and then lysed through two freeze-thaw cycles in liquid nitrogen. Cell lysates were aliquoted to 9 tubes and then heated at 37, 41, 45, 49, 53, 57, 61, 65, and 69&#xb0;C for 3&#xa0;min. After colling down on the ice for 2&#xa0;min, the lysates were centrifuged for 20&#xa0;min. The supernatants were collected for Western blot analysis. Average band density from three independent experiments was used for the curve fitting analysis as described previously in our publication (<xref ref-type="bibr" rid="B16">Li et al., 2019</xref>). <bold>(F,G)</bold> C4-2B cells were treated with Alternol in different concentrations (0, 2.5, 5, 10&#xa0;&#x3bc;M) or periods (0, 2, 4, 8&#xa0;h) at 10&#xa0;&#x3bc;M. Cells were collected for Western blot analysis. &#x3b2;-Actin blot was used as the protein loading control.</p>
</caption>
<graphic xlink:href="fphar-15-1397116-g001.tif"/>
</fig>
</sec>
<sec id="s3-2">
<title>Alternol induces ER stress responses via a ROS-dependent mechanism</title>
<p>To elucidate the detail of Alternol-induced UPR and ER stress response, we re-analyzed the RNA-seq data generated from Alternol-treated PC-3 cells as described in our recent publication (<xref ref-type="bibr" rid="B19">Li et al., 2021</xref>). As shown in <xref ref-type="table" rid="T1">Table 1</xref>, the most upregulated genes after Alternol treatment were inflammatory cytokines and ER stress responding factors, including CXCL8, IL1A, IL6, CXCL3, CCL20, CXCL2, DDIT3 (CHOP), IL1B, and ATF3. Gene set enrichment analysis (GSEA) revealed that unfolded protein response (UPR) and PERK/ATF4-related ER stress response pathways were highly activated after Alternol treatment (<xref ref-type="fig" rid="F2">Figure 2</xref>; <xref ref-type="table" rid="T2">Table 2</xref>). These data strongly suggest that Alternol treatment caused UPR and ER stress.</p>
<table-wrap id="T1" position="float">
<label>TABLE 1</label>
<caption>
<p>The most significantly upregulated genes after Alternol treatment.</p>
</caption>
<table>
<thead valign="top">
<tr>
<th align="right">Symbol</th>
<th align="right">log2FC</th>
<th align="right">P adj</th>
<th align="left">functional significance</th>
</tr>
</thead>
<tbody valign="top">
<tr>
<td align="right">
<bold>CXCL8</bold>
</td>
<td align="right">3.069614276</td>
<td align="right">8.25E-148</td>
<td align="left">chemotactic factor for the neutrophils to infection site and a potent angiogenic factor</td>
</tr>
<tr>
<td align="right">
<bold>CYP1A1</bold>
</td>
<td align="right">2.989899427</td>
<td align="right">4.64E-29</td>
<td align="left">ER protein for PAHs metabolism to carcinogens</td>
</tr>
<tr>
<td align="right">
<bold>IL1A</bold>
</td>
<td align="right">2.903908725</td>
<td align="right">3.17E-26</td>
<td align="left">A pleiotropic cytokine produced by monocytes and macrophages in response to cell injury and involved in immune responses, inflammatory processes and hematopoiesis</td>
</tr>
<tr>
<td align="right">
<bold>FGF21</bold>
</td>
<td align="right">2.818916616</td>
<td align="right">1.30E-27</td>
<td align="left">A secreted metabolic regulator stimulating the uptake of glucose in adipose tissue</td>
</tr>
<tr>
<td align="right">
<bold>IL6</bold>
</td>
<td align="right">2.524239099</td>
<td align="right">7.52E-137</td>
<td align="left">A cytokine in inflammation and the maturation of B cells</td>
</tr>
<tr>
<td align="right">
<bold>CLDN1</bold>
</td>
<td align="right">2.07557688</td>
<td align="right">1.09E-15</td>
<td align="left">An integral membrane protein and a component of tight junction strands</td>
</tr>
<tr>
<td align="right">
<bold>CXCL3</bold>
</td>
<td align="right">2.060143538</td>
<td align="right">4.46E-15</td>
<td align="left">A secreted CXCR2 ligand for inflammation and a chemoattractant for neutrophils</td>
</tr>
<tr>
<td align="right">
<bold>SPOCD1</bold>
</td>
<td align="right">2.033185799</td>
<td align="right">1.45E-18</td>
<td align="left">A TFIIS family protein of transcription elongation factor</td>
</tr>
<tr>
<td align="right">
<bold>EREG</bold>
</td>
<td align="right">2.030978558</td>
<td align="right">8.37E-40</td>
<td align="left">A secreted EGF family protein structurally related to ERBB4 and involved in inflammation, wound healing, oocyte maturation, and cell proliferation</td>
</tr>
<tr>
<td align="right">
<bold>CCL20</bold>
</td>
<td align="right">1.988531829</td>
<td align="right">2.44E-06</td>
<td align="left">small chemotactic antimicrobial cytokine CC gene for lymphocytes but repressing proliferation of myeloid progenitors</td>
</tr>
<tr>
<td align="right">
<bold>CXCL2</bold>
</td>
<td align="right">1.984503994</td>
<td align="right">4.12E-37</td>
<td align="left">a CXC subfamily antimicrobial secreted protein expressed at sites of inflammation suppressing hematopoietic progenitor cell proliferation</td>
</tr>
<tr>
<td align="right">
<bold>MMP3</bold>
</td>
<td align="right">1.856126057</td>
<td align="right">6.01E-05</td>
<td align="left">an enzyme for fibronectin, laminin, collagens III, IV, IX, and X, and cartilage proteoglycans degradation invovled in wound repair, progression of atherosclerosis and tumor initiation</td>
</tr>
<tr>
<td align="right">
<bold>INHBE</bold>
</td>
<td align="right">1.837234608</td>
<td align="right">6.66E-25</td>
<td align="left">TGF-beta superfamily regulating cell proliferation, apoptosis, immune response and hormone secretion under ER stress</td>
</tr>
<tr>
<td align="right">
<bold>DDIT3</bold>
</td>
<td align="right">1.823943929</td>
<td align="right">4.86E-83</td>
<td align="left">C/EBPzeta or CHOP activated after ER stress for apoptosis</td>
</tr>
<tr>
<td align="right">
<bold>TMEM191C</bold>
</td>
<td align="right">1.785536372</td>
<td align="right">1.17E-04</td>
<td align="left">transmembrane protein</td>
</tr>
<tr>
<td align="right">
<bold>IL1B</bold>
</td>
<td align="right">1.760248754</td>
<td align="right">1.57E-04</td>
<td align="left">produced by macrophages and processed CASP1 involved in cell proliferation, differentiation, and apoptosis</td>
</tr>
<tr>
<td align="right">
<bold>DHRS9</bold>
</td>
<td align="right">1.753826235</td>
<td align="right">1.75E-04</td>
<td align="left">a moonlighting protein in short-chain dehydrogenases-reductases (SDR) family</td>
</tr>
<tr>
<td align="right">
<bold>ATF3</bold>
</td>
<td align="right">1.751932311</td>
<td align="right">9.22E-74</td>
<td align="left">a CREB protein family involved in the complex process of cellular stress response</td>
</tr>
<tr>
<td align="right">
<bold>IER3</bold>
</td>
<td align="right">1.734525965</td>
<td align="right">4.10E-239</td>
<td align="left">protection of cells from Fas- or TNF alpha-induced apoptosis</td>
</tr>
<tr>
<td align="right">
<bold>PNPLA1</bold>
</td>
<td align="right">1.698159315</td>
<td align="right">4.79E-10</td>
<td align="left">patatin-like phospholipase family protein in lipid and glycerophospholipid metabolism</td>
</tr>
<tr>
<td align="right">
<bold>PHLDA1</bold>
</td>
<td align="right">1.692770358</td>
<td align="right">3.85E-131</td>
<td align="left">a proline-histidine rich nuclear protein in the anti-apoptotic effects of IGF-1</td>
</tr>
<tr>
<td align="right">
<bold>DUSP1</bold>
</td>
<td align="right">1.647561862</td>
<td align="right">1.99E-102</td>
<td align="left">a phosphatase with dual specificity for tyrosine and threonine of ERK2 in cellular sress and negative regulation of cellular proliferation</td>
</tr>
<tr>
<td align="right">
<bold>CPA4</bold>
</td>
<td align="right">1.643863159</td>
<td align="right">1.37E-17</td>
<td align="left">a carboxypeptidase A/B subfamily protein involved in histone hyperacetylation pathway</td>
</tr>
<tr>
<td align="right">
<bold>SCN4A</bold>
</td>
<td align="right">1.600392345</td>
<td align="right">2.42E-14</td>
<td align="left">sodium voltage-gated channel alpha subunit 4 Nav1.4</td>
</tr>
<tr>
<td align="right">
<bold>C11orf96</bold>
</td>
<td align="right">1.579647153</td>
<td align="right">1.64E-05</td>
<td align="left">1242&#xa0;nt</td>
</tr>
<tr>
<td align="right">
<bold>ARHGAP9</bold>
</td>
<td align="right">1.566966431</td>
<td align="right">9.75E-04</td>
<td align="left">Rho GAP9 towards Rho-family GTPases <italic>in vitro</italic> regulating adhesion of hematopoietic cells to the extracellular matrix</td>
</tr>
<tr>
<td align="right">
<bold>DPP4</bold>
</td>
<td align="right">1.566467628</td>
<td align="right">1.57E-10</td>
<td align="left">CD26, an intrinsic membrane glycoprotein and a serine exopeptidase that cleaves X-proline dipeptides from the N-terminus of polypeptides</td>
</tr>
<tr>
<td align="right">
<bold>GLIPR1</bold>
</td>
<td align="right">1.566363867</td>
<td align="right">4.99E-39</td>
<td align="left">decreased expression of this gene through gene methylation is associated with prostate cancer hodling proapoptotic activities in prostate and bladder cancer cells</td>
</tr>
<tr>
<td align="right">
<bold>PSG1</bold>
</td>
<td align="right">1.564068691</td>
<td align="right">1.00E-03</td>
<td align="left">a major product of the syncytiotrophoblast in placenta</td>
</tr>
<tr>
<td align="right">
<bold>PPP1R15A</bold>
</td>
<td align="right">1.562368114</td>
<td align="right">5.17E-72</td>
<td align="left">proapoptotic protein induced by stressful growth arrest conditions and treatment with DNA-damaging agents regardless of p53 status</td>
</tr>
<tr>
<td align="right">
<bold>HIST1H1E</bold>
</td>
<td align="right">1.558191745</td>
<td align="right">9.08E-05</td>
<td align="left">a linker histone H1 interacts with linker DNA between nucleosomes H1.4</td>
</tr>
<tr>
<td align="right">
<bold>GDF15</bold>
</td>
<td align="right">1.544564896</td>
<td align="right">6.35E-72</td>
<td align="left">a TGF-beta family ligand in activation of SMAD transcription factors involved in the stress response of hypoxia, inflammation, acute injury and oxidative stress</td>
</tr>
<tr>
<td align="right">
<bold>MPZ</bold>
</td>
<td align="right">1.538122369</td>
<td align="right">6.18E-04</td>
<td align="left">a type I transmembrane glycoprotein specifically expressed in Schwann cells of the peripheral nervous system that is a major structural protein of the peripheral myelin sheath</td>
</tr>
<tr>
<td align="right">
<bold>PSG4</bold>
</td>
<td align="right">1.524354694</td>
<td align="right">2.61E-31</td>
<td align="left">a member of the carcinoembryonic antigen (CEA) gene family and may play a role in regulation of the innate immune system</td>
</tr>
<tr>
<td align="right">
<bold>HIST1H1D</bold>
</td>
<td align="right">1.521422522</td>
<td align="right">1.62E-04</td>
<td align="left">H1D-H1.3</td>
</tr>
<tr>
<td align="right">
<bold>MAGEC2</bold>
</td>
<td align="right">1.507672547</td>
<td align="right">1.47E-03</td>
<td align="left">expressed only in tumors of various histological types</td>
</tr>
<tr>
<td align="right">
<bold>AREG</bold>
</td>
<td align="right">1.503432175</td>
<td align="right">3.00E-18</td>
<td align="left">an EGF member autocrine and mitogen interacts with EGFR/TGF-a receptor to promote the growth of epithelial cells but inhibits aggressive carcinoma cell lines</td>
</tr>
</tbody>
</table>
</table-wrap>
<fig id="F2" position="float">
<label>FIGURE 2</label>
<caption>
<p>GSEA analysis of RNA-seq data for gene expression alterations after Alternol treatment. Activation of the unfolded protein response <bold>(A)</bold>, ATF4 <bold>(B)</bold>, and PERK <bold>(C)</bold> pathways was noticed in PC-3 cells after Alternol treatment.</p>
</caption>
<graphic xlink:href="fphar-15-1397116-g002.tif"/>
</fig>
<table-wrap id="T2" position="float">
<label>TABLE 2</label>
<caption>
<p>GSEA enrichment of ER stress and inflammatory pathways after Alternol treatment.</p>
</caption>
<table>
<thead valign="top">
<tr>
<th align="center">Pathway ID and description</th>
<th align="center">setSize</th>
<th align="center">enrichmentScore</th>
<th align="right">NES</th>
<th align="right">P-value</th>
<th align="right">p.adjust</th>
<th align="right">qvalue</th>
<th align="left">core_enrichment</th>
</tr>
</thead>
<tbody valign="top">
<tr>
<td align="left">REACTOME_UNFOLDED_PROTEIN_RESPONSE_UPR</td>
<td align="center">90</td>
<td align="center">0.403810332095922</td>
<td align="right">1.61739253010353</td>
<td align="right">0.00371964709976059</td>
<td align="right">0.0347455407380737</td>
<td align="right">0.0278176790447212</td>
<td align="left">CXCL8/DDIT3/ATF3/CREB3L3/CREB3L1/ERN1/CEBPB/ASNS/EXOSC8/EXOSC7/EXOSC1/EXOSC3/CREB3/DNAJB9/LMNA/ATP6V0D1/SRPRB/ZBTB17/GFPT1/EXOSC2/EIF2S1/XBP1/EXOSC9</td>
</tr>
<tr>
<td align="left">REACTOME_ATF4_ACTIVATES_GENES_IN_RESPONSE_TO_ENDOPLASMIC_RETICULUM_STRESS</td>
<td align="center">26</td>
<td align="center">0.669544754867975</td>
<td align="right">2.08679020953492</td>
<td align="right">0.000136088569553141</td>
<td align="right">0.00324726190715911</td>
<td align="right">0.00259979518489743</td>
<td align="left">CXCL8/DDIT3/ATF3/CEBPB/ASNS/EXOSC8/EXOSC7/EXOSC1/EXOSC3</td>
</tr>
<tr>
<td align="left">REACTOME_PERK_REGULATES_GENE_EXPRESSION</td>
<td align="center">31</td>
<td align="center">0.624849485874999</td>
<td align="right">2.03523436113533</td>
<td align="right">0.000185971399602142</td>
<td align="right">0.00409290845155759</td>
<td align="right">0.00327682952247437</td>
<td align="left">CXCL8/DDIT3/ATF3/CEBPB/ASNS/EXOSC8/EXOSC7/EXOSC1/EXOSC3</td>
</tr>
<tr>
<td align="left">REACTOME_RESPONSE_OF_EIF2AK1_HRI_TO_HEME_DEFICIENCY</td>
<td align="center">15</td>
<td align="center">0.831017551110207</td>
<td align="right">2.25397975547525</td>
<td align="right">1.73707362397832E-06</td>
<td align="right">0.00016745389735151</td>
<td align="right">0.000134065513800095</td>
<td align="left">DDIT3/ATF3/PPP1R15A/CHAC1/TRIB3/ATF5/CEBPB/ASNS</td>
</tr>
<tr>
<td align="left">WP_OVERVIEW_OF_PROINFLAMMATORY_AND_PROFIBROTIC_MEDIATORS</td>
<td align="center">69</td>
<td align="center">0.666293872828877</td>
<td align="right">2.54058809095234</td>
<td align="right">1E-10</td>
<td align="right">0.0000000482</td>
<td align="right">3.85894736842105E-08</td>
<td align="left">CXCL8/IL1A/IL6/CXCL3/CCL20/CXCL2/MMP3/IL1B/AREG/CCL26/MMP1/LIF/CCL24/IL11</td>
</tr>
<tr>
<td align="left">WP_PHOTODYNAMIC_THERAPYINDUCED_NFKB_SURVIVAL_SIGNALING</td>
<td align="center">31</td>
<td align="center">0.769362889109306</td>
<td align="right">2.50593754735177</td>
<td align="right">5.27116095919563E-09</td>
<td align="right">2.11724965194358E-06</td>
<td align="right">1.69509439266765E-06</td>
<td align="left">CXCL8/IL1A/IL6/CXCL2/MMP3/IL1B/MMP1/BIRC3/CCND1/NFKB2/ICAM1</td>
</tr>
<tr>
<td align="left">REACTOME_INTERLEUKIN_10_SIGNALING</td>
<td align="center">36</td>
<td align="center">0.728492156095692</td>
<td align="right">2.41319894329377</td>
<td align="right">3.77827702097557E-08</td>
<td align="right">7.49878624272726E-06</td>
<td align="right">6.00361440616688E-06</td>
<td align="left">CXCL8/IL1A/IL6/CCL20/CXCL2/IL1B/LIF</td>
</tr>
<tr>
<td align="left">WP_LTF_DANGER_SIGNAL_RESPONSE_PATHWAY</td>
<td align="center">16</td>
<td align="center">0.828588190440065</td>
<td align="right">2.29128292141293</td>
<td align="right">3.93571600941655E-06</td>
<td align="right">0.000271002159505539</td>
<td align="right">0.000216967441962723</td>
<td align="left">CXCL8/IL1A/IL6/IL1B</td>
</tr>
<tr>
<td align="left">REACTOME_DECTIN_1_MEDIATED_NONCANONICAL_NF_KB_SIGNALING</td>
<td align="center">59</td>
<td align="center">0.591165749821468</td>
<td align="right">2.20455108416999</td>
<td align="right">2.24146074500624E-06</td>
<td align="right">0.000200071125757964</td>
<td align="right">0.000160179241543721</td>
<td align="left">MAP3K14/PSMD6/PSMC1/PSMD13/PSMD14/PSMB6/PSMC4/PSMD8/PSMC2/NFKB2/PSMB7/PSMB3/PSMB1/PSMB4/PSMB5/PSMA7/PSME3/PSMD12/PSMD2/PSMA1/PSMC5/PSMD4/PSMD3/PSMA5/UBE2M/PSMA3/PSMD11/PSMB2/PSMF1/PSMA2/PSMC3/PSMC6/PSMA4/PSMD7</td>
</tr>
<tr>
<td align="left">REACTOME_TNFR2_NON_CANONICAL_NF_KB_PATHWAY</td>
<td align="center">94</td>
<td align="center">0.469873707004391</td>
<td align="right">1.88843053915875</td>
<td align="right">0.000112012261310524</td>
<td align="right">0.00293423423650394</td>
<td align="right">0.00234918163595183</td>
<td align="left">TNFRSF12A/TNFSF18/TNFRSF9/BIRC3/MAP3K14/PSMD6/PSMC1/PSMD13/PSMD14/PSMB6/PSMC4/PSMD8/PSMC2/NFKB2/PSMB7/LTBR/PSMB3/PSMB1/PSMB4/PSMB5/PSMA7/PSME3/PSMD12/PSMD2/PSMA1/PSMC5/PSMD4/PSMD3/PSMA5/UBE2M/PSMA3/PSMD11/PSMB2/PSMF1/PSMA2/PSMC3/PSMC6/TNFRSF1A/PSMA4</td>
</tr>
<tr>
<td align="left">REACTOME_SIGNALING_BY_INTERLEUKINS</td>
<td align="center">404</td>
<td align="center">0.372894448305077</td>
<td align="right">1.82136922961858</td>
<td align="right">4.35614138581666E-08</td>
<td align="right">7.49878624272726E-06</td>
<td align="right">6.00361440616688E-06</td>
<td align="left">CXCL8/IL1A/IL6/CCL20/CXCL2/MMP3/IL1B/IL7R/MEF2C/SERPINB2/STAT4/IL2RB/MMP1/HMOX1/LIF/CDKN1A/MAOA/IL12RB1/CD36/IRAK2/CSF1R/ANXA1/IL11/PSMD6/DUSP4/SNRPA1/PSMC1/JUN/GAB2/HNRNPDL/PSMD13/FOXO1/DUSP6/PSMD14/PSMB6/PSMC4/IL21R/SOCS1/PSMD8/PSMC2/BOLA2B/BOLA2/S1PR1/CCND1/HAVCR2/NFKB2/NFKBIB/PSMB7/ICAM1/ANXA2/PSMB3/PSMB1/PSMB4/S100A12/TGFB1/PSMB5/PSMA7/LCN2/PSME3/PSMD12/PSMD2/JAK1/PSMA1/ITGAX/VEGFA/ARF1/SOD1/PSMC5/ITGAM/PSMD4/PTPN2/SQSTM1/PSMD3/IL16/PSMA5/CSF3/MAP2K3/EBI3/PSMA3/STX1A/PSMD11/IL13RA2/PSMB2/PSMF1/PSMA2/IL15/CNN2/PSMC3/PSMC6/MAPK10/PPP2R1A/CD80</td>
</tr>
<tr>
<td align="left">WP_INTERLEUKIN1_INDUCED_ACTIVATION_OF_NFKB</td>
<td align="center">10</td>
<td align="center">0.670824274298685</td>
<td align="right">1.60513022644812</td>
<td align="right">0.0212616867687299</td>
<td align="right">0.118612650723702</td>
<td align="right">0.0949626506995174</td>
<td align="left">IL1A/AJUBA/SQSTM1</td>
</tr>
<tr>
<td align="left">REACTOME_INTERLEUKIN_1_FAMILY_SIGNALING</td>
<td align="center">133</td>
<td align="center">0.432313543114489</td>
<td align="right">1.83653655910291</td>
<td align="right">3.613182646093E-05</td>
<td align="right">0.00126979828293217</td>
<td align="right">0.00101661509177317</td>
<td align="left">IL1A/IL1B/IRAK2/PSMD6/PSMC1/PSMD13/PSMD14/PSMB6/PSMC4/PSMD8/PSMC2/NFKB2/NFKBIB/PSMB7/PSMB3/PSMB1/PSMB4/S100A12/PSMB5/PSMA7/PSME3/PSMD12/PSMD2/PSMA1/PSMC5/PSMD4/PTPN2/SQSTM1/PSMD3/PSMA5/PSMA3/PSMD11/PSMB2/PSMF1/PSMA2/PSMC3/PSMC6</td>
</tr>
<tr>
<td align="left">REACTOME_INTERLEUKIN_4_AND_INTERLEUKIN_13_SIGNALING</td>
<td align="center">96</td>
<td align="center">0.465496512696374</td>
<td align="right">1.87979168950261</td>
<td align="right">8.53180753047583E-05</td>
<td align="right">0.00232828257097992</td>
<td align="right">0.00186404977183061</td>
<td align="left">CXCL8/IL1A/IL6/MMP3/IL1B/MMP1/HMOX1/LIF/CDKN1A/MAOA/CD36/ANXA1/FOXO1/SOCS1/S1PR1/CCND1/ICAM1/TGFB1/LCN2/JAK1/ITGAX/VEGFA/ITGAM</td>
</tr>
<tr>
<td align="left">BIOCARTA_IL10_PATHWAY</td>
<td align="center">12</td>
<td align="center">0.8339679706654</td>
<td align="right">2.13295111435416</td>
<td align="right">2.7558613693034E-05</td>
<td align="right">0.00107122998387439</td>
<td align="right">0.00085763903054892</td>
<td align="left">IL1A/IL6/HMOX1/BLVRB</td>
</tr>
<tr>
<td align="left">WP_IL10_ANTIINFLAMMATORY_SIGNALING_PATHWAY</td>
<td align="center">11</td>
<td align="center">0.844548136912348</td>
<td align="right">2.07961312966775</td>
<td align="right">6.50967140375665E-05</td>
<td align="right">0.00189015760036789</td>
<td align="right">0.00151328188751883</td>
<td align="left">IL1A/IL6/HMOX1/BLVRB</td>
</tr>
<tr>
<td align="left">REACTOME_INTERLEUKIN_12_FAMILY_SIGNALING</td>
<td align="center">54</td>
<td align="center">0.465672585039885</td>
<td align="right">1.7178504278712</td>
<td align="right">0.00347140036911967</td>
<td align="right">0.0331987098792794</td>
<td align="right">0.0265792684903774</td>
<td align="left">SERPINB2/STAT4/IL12RB1/SNRPA1/HNRNPDL/BOLA2B/BOLA2/ANXA2/JAK1/ARF1/SOD1/EBI3/CNN2/TCP1</td>
</tr>
<tr>
<td align="left">BIOCARTA_IL17_PATHWAY</td>
<td align="center">10</td>
<td align="center">0.757360892312669</td>
<td align="right">1.81219271149915</td>
<td align="right">0.00265695294069546</td>
<td align="right">0.0273643443892139</td>
<td align="right">0.021908208458366</td>
<td align="left">CXCL8/IL6</td>
</tr>
<tr>
<td align="left">WP_IL18_SIGNALING_PATHWAY</td>
<td align="center">242</td>
<td align="center">0.426822735994919</td>
<td align="right">1.96172264663375</td>
<td align="right">1.99205143391354E-08</td>
<td align="right">4.80084395573163E-06</td>
<td align="right">3.84361081933002E-06</td>
<td align="left">CXCL8/IL6/CLDN1/CXCL3/CCL20/CXCL2/MMP3/IL1B/ATF3/IER3/PTX3/MMP1/HMOX1/BMP2/TGM2/ARL4D/CD36/FUT1/BIRC3/RUNX2/IRF1/EPS8/NR1H3/JUN/TOMM40/SDC4/TNFAIP3/ZC3H12A/RND2/BID/FAM186B/RAE1/NFKB2/CEBPB/ICAM1</td>
</tr>
<tr>
<td align="left">PID_IL23_PATHWAY</td>
<td align="center">29</td>
<td align="center">0.589289764820712</td>
<td align="right">1.87852782402297</td>
<td align="right">0.00156416869931681</td>
<td align="right">0.0192328906395588</td>
<td align="right">0.0153980731785592</td>
<td align="left">IL6/IL1B/STAT4/IL12RB1</td>
</tr>
<tr>
<td align="left">PID_IL27_PATHWAY</td>
<td align="center">23</td>
<td align="center">0.612243789203322</td>
<td align="right">1.84681905495019</td>
<td align="right">0.00260985355445942</td>
<td align="right">0.0269946226019193</td>
<td align="right">0.0216122049483809</td>
<td align="left">IL6/IL1B/STAT4/IL12RB1</td>
</tr>
<tr>
<td align="left">REACTOME_PYROPTOSIS</td>
<td align="center">25</td>
<td align="center">0.63039573734784</td>
<td align="right">1.93675836401405</td>
<td align="right">0.00107381975398576</td>
<td align="right">0.0142192615775037</td>
<td align="right">0.0113841041587964</td>
<td align="left">IL1A/IL1B/IRF1/BAK1/CASP4/CYCS/TP63/CHMP4B/IL18</td>
</tr>
<tr>
<td align="left">REACTOME_PROGRAMMED_CELL_DEATH</td>
<td align="center">191</td>
<td align="center">0.356288705819984</td>
<td align="right">1.59704620046667</td>
<td align="right">0.000457089959077232</td>
<td align="right">0.00739320000923577</td>
<td align="right">0.00591908085474085</td>
<td align="left">IL1A/IL1B/AVEN/BIRC3/IRF1/PSMD6/PSMC1/BCAP31/PSMD13/PSMD14/PSMB6/PSMC4/PSMD8/BID/PSMC2/DBNL/PSMB7/PSMB3/PSMB1/PSMB4/PSMB5/PSMA7/PSME3/PSMD12/PSMD2/BAK1/PSMA1/CASP4/PSMC5/PSMD4/DAPK3/CDH1/PMAIP1/PSMD3/PSMA5/YWHAQ/PSMA3/PSMD11/PSMB2/PSMF1/PSMA2/OMA1/DSG3/LMNA/PSMC3/PSMC6/OCLN/C1QBP/PSMA4/PSMD7/CYCS/HSP90AA1/UNC5B/STUB1/TP63/FADD/CHMP4B/IL18/PSMB10/STK24/PSMA6/DAPK2/CDC37</td>
</tr>
<tr>
<td align="left">WP_OXIDATIVE_STRESS_RESPONSE</td>
<td align="center">30</td>
<td align="center">0.601541821039662</td>
<td align="right">1.94574696830302</td>
<td align="right">0.00101767559125042</td>
<td align="right">0.0136255454161861</td>
<td align="right">0.0109087681798948</td>
<td align="left">CYP1A1/HMOX1/MAOA/NOX4/GPX3/UGT1A6/MGST1/TXNRD1/NQO1/TXN2/SOD1/NOX5/NFE2L2/MAPK10/GPX1/JUNB/XDH</td>
</tr>
<tr>
<td align="left">WP_PHOTODYNAMIC_THERAPYINDUCED_NFKB_SURVIVAL_SIGNALING</td>
<td align="center">31</td>
<td align="center">0.769362889109306</td>
<td align="right">2.50593754735177</td>
<td align="right">5.27116095919563E-09</td>
<td align="right">2.11724965194358E-06</td>
<td align="right">1.69509439266765E-06</td>
<td align="left">CXCL8/IL1A/IL6/CXCL2/MMP3/IL1B/MMP1/BIRC3/CCND1/NFKB2/ICAM1</td>
</tr>
<tr>
<td align="left">BIOCARTA_NFKB_PATHWAY</td>
<td align="center">21</td>
<td align="center">0.519034166151175</td>
<td align="right">1.52617701855322</td>
<td align="right">0.0409492634325475</td>
<td align="right">0.178458815320867</td>
<td align="right">0.142876177542323</td>
<td align="left">IL1A/MAP3K14/TNFAIP3</td>
</tr>
</tbody>
</table>
</table-wrap>
<p>It is well known that UPR and ER stress responses are monitored by three major cascades modulated by PERK, IRE1&#x3b1;, and ATF6 (<xref ref-type="bibr" rid="B13">Hetz et al., 2020</xref>). Activation of these pathways induces modifications on downstream effector proteins, leading to translational alteration and ER stress responses (<xref ref-type="bibr" rid="B39">Wiseman et al., 2022</xref>). We analyzed Alternol-induced changes of these ER stress-responding proteins (<xref ref-type="bibr" rid="B35">Sicari et al., 2020</xref>). Our results from three different prostate cancer cell lines showed that PERK and IRE1&#x3b1; were phosphorylated at their activating domain (PERK/T980 and IRE1&#x3b1;/S724) as early as 2&#xa0;h after Alternol treatment (<xref ref-type="fig" rid="F3">Figures 3A&#x2013;C</xref>). Meanwhile, PERK downstream effector eIF2&#x3b1; protein was phosphorylated at the S51 site (<xref ref-type="bibr" rid="B11">Harding et al., 1999</xref>) and IRE1&#x3b1;/ATF6 downstream effector XBP1s protein level was also largely increased. In addition, the expression levels of classical ER stress-responding ATF4/ATF3/CHOP proteins were drastically increased after Alternol treatment in dose-dependent and time-dependent fashions. Consistent with our previous reports (<xref ref-type="bibr" rid="B37">Tang et al., 2014</xref>; <xref ref-type="bibr" rid="B41">Xu et al., 2019</xref>; <xref ref-type="bibr" rid="B19">Li et al., 2021</xref>), ROS scavenger N-Ac pretreatment abolished these alterations related to ER stress responses (<xref ref-type="fig" rid="F3">Figure 3C</xref>). These data demonstrated that Alternol treatment induced strong UPR and ER stress responses <italic>via</italic> ROS-dependent mechanism (<xref ref-type="bibr" rid="B45">Yoshida et al., 2001</xref>).</p>
<fig id="F3" position="float">
<label>FIGURE 3</label>
<caption>
<p>Alternol triggers ROS-dependent ER stress responses <italic>via</italic> PERK/IRE1&#x3b1; pathways. <bold>(A,B)</bold> PC-3 and 22RV1 cells were seeded in P100 dishes overnight and treated with Alternol at different concentrations or for the indicated period at 10&#xa0;&#x3bc;M. <bold>(C)</bold> C4-2B cells were treated with Alternol at different concentrations as indicated with or without N-Ac (5&#xa0;mM) for 6&#xa0;h. <bold>(D)</bold> PC-3 cells were pre-treated with IRE1&#x3b1; inhibitor MKC8866 (10&#xa0;&#xb5;M), PERK inhibitor AMG44 (1&#xa0;&#xb5;M), and ATF6 inhibitor CEAPIN-A7 (10&#xa0;&#xb5;M) for 30&#xa0;min followed by Alternol (10&#xa0;&#xb5;M) for 4&#xa0;h. Equal amounts of cellular proteins were subjected to western blots with the antibodies as indicated.</p>
</caption>
<graphic xlink:href="fphar-15-1397116-g003.tif"/>
</fig>
<p>We then asked which one or a combination of the three ER stress-responding pathways (<xref ref-type="bibr" rid="B31">Ron and Walter, 2007</xref>) were involved in Alternol treatment-induced ER stress. We utilized pathway-selective pharmacological inhibitors for these three pathways, IRE1&#x3b1; inhibitor MKC8866 (<xref ref-type="bibr" rid="B34">Sheng et al., 2019</xref>), PERK inhibitor AMG44 (<xref ref-type="bibr" rid="B4">Chintha et al., 2019</xref>), and ATF6 inhibitor CEAPIN-A7 (<xref ref-type="bibr" rid="B43">Xue et al., 2021</xref>) to determine their involvement in Alternol-induced ER stress response (<xref ref-type="fig" rid="F3">Figure 3D</xref>). Our results showed that pretreatment with AMG44 reduced the Alternol-induced ATF4 expression while MKC8866 pretreatment suppressed Alternol-induced XBP1s expression. In addition, Alternol-induced CHOP protein expression was largely reduced by either AMG44 or MKC8866. Meantime, Alternol-induced PARP cleavage was also reduced by AMG44 or MKC8866. However, CEAPIN-A7 pre-treatment showed no significant attenuation on Alternol-induced these responses. These data suggest that both PERK and IRE1&#x3b1; cascades were involved in Alternol-induced UPR and ER stress.</p>
</sec>
<sec id="s3-3">
<title>Alternol-induced ER stress is connected to immunogenic cell death-related ATP release</title>
<p>Extracellular release of ATP molecules has been used as one of the hallmark indicators during immunogenic cell death under ER stress conditions (<xref ref-type="bibr" rid="B8">Galluzzi et al., 2020</xref>; <xref ref-type="bibr" rid="B17">Li et al., 2020</xref>; <xref ref-type="bibr" rid="B23">Liu et al., 2024</xref>). Because we recently demonstrated Alternol-elicited ATP molecule release during immunogenic cell death (<xref ref-type="bibr" rid="B19">Li et al., 2021</xref>), we then determined if Alternol-induced ER stress responses were accompanied by immunogenic ATP release. C4-2B cells were pre-treated with IRE1&#x3b1; inhibitor MKC8866, followed by Alternol treatment. As shown in <xref ref-type="fig" rid="F4">Figure 4A</xref>, the Alternol treatment induced a drastic elevation of extracellular ATP level, which was significantly suppressed by MKC8866 pre-treatment. Similarly, PERK inhibitor AMG44 but not ATF-6&#x3b1; inhibitor CEAPIN-A7 suppressed Alternol-induced ATP release in PC-3 cells (<xref ref-type="fig" rid="F4">Figures 4B, C</xref>). These data suggest that Alternol-induced ER stress response was related to immunogenic cell death elicited by Alternol <italic>via</italic> PERK and IRE1&#x3b1; cascades.</p>
<fig id="F4" position="float">
<label>FIGURE 4</label>
<caption>
<p>Alternol induces ATP release through PERK and IRE1&#x3b1;-dependent pathways. C4-2B panel <bold>(A)</bold> or PC-3 panel <bold>(B,C)</bold> cells were seeded in a 96-well plate overnight, and then pre-treated with the solvent, MKC8866 (10&#xa0;&#x3bc;M), AMG44 (1&#xa0;&#x3bc;M), or CEAPIN-A7 (10&#xa0;&#x3bc;M) for 30&#xa0;min, followed by Alternol (10&#xa0;&#x3bc;M) for 6&#xa0;h. The ATP level in the cell culture media was measured using the ATPlite Luminescence Assay System (catalog number 6016941, PerkinElmer). &#x2a;<italic>p</italic> &#x3c; 0.05; &#x2a;&#x2a;<italic>p</italic> &#x3c; 0.01, Student&#x2019;s t-test.</p>
</caption>
<graphic xlink:href="fphar-15-1397116-g004.tif"/>
</fig>
</sec>
<sec id="s3-4">
<title>PKR but not the NF-&#x3ba;B pathway is involved in alternol-induced ER stress responses</title>
<p>NF-&#x3ba;B pathway is a crucial regulator of inflammatory cytokine production (<xref ref-type="bibr" rid="B2">Capece et al., 2022</xref>), and oxidative stress is a common factor of NF-&#x3ba;B activation (<xref ref-type="bibr" rid="B15">Kim et al., 2001</xref>). Oxidative stress-induced NF-&#x3ba;B activation has been implicated in immunogenic cell death (<xref ref-type="bibr" rid="B47">Zhao et al., 2022</xref>). We then asked if Alternol treatment activated the NF-&#x3ba;B pathway. We first re-analyzed the RNA-seq data with the GSEA approach. As expected, GSEA analysis revealed that the NF-&#x3ba;B pathway was enriched in Alternol-induced activation of gene expression (<xref ref-type="table" rid="T2">Table 2</xref>). We then evaluated the changes in major modulators of NF-&#x3ba;B activation including NF-&#x3ba;B/p65, I&#x3ba;B&#x3b1;, and IKK&#x3b2; proteins. As shown in <xref ref-type="fig" rid="F5">Figure 5A</xref>, Alternol treatment reduced the protein levels of I&#x3ba;B-&#x3b1;, the negative regulator of NF-&#x3ba;B activation, in a time-dependent manner. Next, we examined if NF-&#x3ba;B inhibition suppressed Alternol-induced ER stress responses and ATP release. Unexpectedly, pretreatment with NF-&#x3ba;B inhibitor SN50 (<xref ref-type="bibr" rid="B21">Lin et al., 1995</xref>) had no significant suppression on Alternol-induced elevation of XBP1s and CHOP protein levels (<xref ref-type="fig" rid="F5">Figure 5B</xref>), as well ATP release in PC-3 cells (<xref ref-type="fig" rid="F5">Figure 5C</xref>), although SN50 slightly reduced ATF4 expression. As expected, SN50 largely reduced the phosphorylation level of NF-&#x3ba;B/p65, confirming the SN50 action (<xref ref-type="bibr" rid="B40">Wu et al., 2020</xref>). In addition, pretreatment with IRE1&#x3b1; inhibitor MKC8866, PERK inhibitor AMG44, and ATF6 inhibitor CEAPIN-A7 all had no significant effect on NF-kB phosphorylation (<xref ref-type="fig" rid="F3">Figure 3D</xref>). These data indicate that NF-&#x3ba;B activation is not a major player in Alternol-induced ER stress responses and ATP release.</p>
<fig id="F5" position="float">
<label>FIGURE 5</label>
<caption>
<p>PKR but not the NF-&#x3ba;B pathway was involved in Alternol-induced ER stress. <bold>(A)</bold>. PC-3 cells were treated with Alternol (10&#xa0;&#x3bc;M) for 0, 2, 4, 8&#xa0;h. <bold>(B)</bold> C4-2B cells were pre-treated with Imoxin (10&#xa0;&#x3bc;M) or SN50 (10&#xa0;&#x3bc;M) for 30&#xa0;min followed by Alternol (10&#xa0;&#x3bc;M) for 6&#xa0;h. Equal amounts of cellular proteins were subjected to western blots with the antibodies as indicated. &#x3b2;-Actin blots served as the protein loading control. <bold>(C,D)</bold> PC-3 cells were seeded in a 96-well plate overnight and then pre-treated with the solvent, SN50 (panel <bold>C</bold>), or Imoxin (panel <bold>D</bold>) for 30&#xa0;min, followed by Alternol treatment for 6&#xa0;h. ATP level in the cell culture media was measured using the ATPlite&#x2122; Luminescence Assay System. &#x2a;, <italic>p</italic> &#x3c; 0.05, Student&#x2019;s t-test.</p>
</caption>
<graphic xlink:href="fphar-15-1397116-g005.tif"/>
</fig>
<p>Lastly, we evaluated the involvement of protein kinase R (PKR) in Alternol-induced ER stress response, since PKR was recently reported to modulate ER stress-related induction of ATF3, CHOP, and XBP1s expression (<xref ref-type="bibr" rid="B10">Guerra et al., 2006</xref>; <xref ref-type="bibr" rid="B6">Eo and Valentine, 2022</xref>). A PKR-specific inhibitor Imoxin (<xref ref-type="bibr" rid="B26">Nakamura et al., 2014</xref>) was utilized as a pretreatment during Alternol-induced ER stress. As shown in <xref ref-type="fig" rid="F5">Figure 5B</xref>, Imoxin pretreatment largely reduced the protein levels of XBP1s and ATF3 at the basal and Alternol treatment conditions. Meanwhile, Imoxin also blunted the Alternol-induced increase of ATF4 and CHOP proteins. In addition, Imoxin significantly suppressed Alternol-induced ATP release in a concentration-dependent manner (<xref ref-type="fig" rid="F5">Figure 5D</xref>). These data strongly suggest that PKR activation was involved in Alternol-induced ER stress response, leading to immunogenic cell death.</p>
</sec>
</sec>
<sec sec-type="discussion" id="s4">
<title>Discussion</title>
<p>In this study, we demonstrated that Alternol interacted with multiple mitochondrial and ER chaperone proteins and elicited ROS-dependent ER stress responses in prostate cancer cells. Alternol-induced ER stress responses involved three protein kinases, PKR, PERK, and IRE1&#x3b1;, resulting in eIF2&#x3b1; phosphorylation, XBP1s processing, ATF3/ATF4 overexpression, and CHOP protein accumulation. Inhibition of these cascades suppressed immunogenic ATP release. According to the results, we proposed that Alternol induces immunogenic cell death <italic>via</italic> ER stress-related cascades of PKR, PERK, and IRE1&#x3b1; kinases.</p>
<p>Alternol is a novel small molecular compound and preclinical studies from our group and others have shown its potency in specifically killing multiple types of human cancer cells <italic>via</italic> ROS-dependent mechanism (<xref ref-type="bibr" rid="B24">Liu et al., 2020</xref>). Most interestingly, our recent studies discovered that Alternol-induced cancer cell killing elicited a strong immunogenic response that resulted in xenograft tumor suppression in immune-intact mice (<xref ref-type="bibr" rid="B19">Li et al., 2021</xref>). Consistent with the notion that ER stress response is crucial in DAMP release and immunogenic elicitation (<xref ref-type="bibr" rid="B1">Aria and Rezaei, 2023</xref>), in this study, our data confirmed the ER stress responses after Alternol treatment in prostate cancer cells. Our studies discovered that three ER stress-related protein kinases, PKR, PERK, and IRE1a, were involved in Alternol-induced ER stress responses and immunogenic ATP release. Our results also verified our previous report (<xref ref-type="bibr" rid="B16">Li et al., 2019</xref>) that Alternol interacted with five chaperone proteins resided in mitochondria and ER and Alternol treatment increased their expression levels, a potential response due to the UPR and ER stress.</p>
<p>It is well known that there are three sensor kinases, IRE1&#x3b1;, PERK, and ATF-6, responding to ER stress conditions (<xref ref-type="bibr" rid="B13">Hetz et al., 2020</xref>; <xref ref-type="bibr" rid="B35">Sicari et al., 2020</xref>). IRE1&#x3b1; induces the unconventional splicing of XBP1 mRNA to produce a shorter XBP1s protein, PERK kinase induces eIF2&#x3b1; phosphorylation at serine 51 to inactivate protein translation, and ATF6 N-terminal region exerts a transcriptional activity to upregulate UPR-related gene after undergoing proteolytic cleavage (<xref ref-type="bibr" rid="B13">Hetz et al., 2020</xref>; <xref ref-type="bibr" rid="B33">Saaoud et al., 2024</xref>). In this study, our data revealed that PERK and IRE1&#x3b1; but not ATF6 cascades were involved in Alternol-induced ER stress. ATF6 protein did not show a proteolytic change and its specific inhibitor failed to suppress XBP1 expression and immunogenic ATP release after Alternol treatment. A further mechanistic study is warranted to dissect Alternol-induced activation of PERK and IRE1&#x3b1; cascades, although ROS dependency was confirmed (<xref ref-type="bibr" rid="B19">Li et al., 2021</xref>; <xref ref-type="bibr" rid="B44">Yang et al., 2024</xref>).</p>
<p>PKR is one of the four eIF2&#x3b1; kinases (<xref ref-type="bibr" rid="B14">Jackson et al., 2010</xref>) and it is mainly activated after viral infection in mammalian cells (<xref ref-type="bibr" rid="B28">Park et al., 2006</xref>; <xref ref-type="bibr" rid="B46">Zhang and Karijolich, 2024</xref>). However, recent studies showed that PKR activation was also involved in eIF2&#x3b1;/S51 phosphorylation and immunogenic cell death induced by chemo-drugs in melanoma and breast cancer cells (<xref ref-type="bibr" rid="B9">Giglio et al., 2018</xref>; <xref ref-type="bibr" rid="B18">Li et al., 2022</xref>). In addition, PKR-specific inhibitor Imoxin suppressed saturated fatty acid-induced ER stress responses including XBP1s processing, ATF6, and CHOP expression (<xref ref-type="bibr" rid="B6">Eo and Valentine, 2022</xref>). Interestingly, we also found that Imoxin pretreatment almost blunted Alternol-induced XBP1s, largely reduced ATF4 and CHOP expression, and significantly reduced immunogenic ATP release, indicating a signaling crosstalk among the conventional ER stress sensors and PKR during Alternol-induced ROS-dependent immunogenic response.</p>
<p>In conclusion, we demonstrated that Alternol treatment triggered ROS-dependent ER stress responses, linking to immunogenic ATP release. We also proved that Alternol-induced ER stress involved three protein kinases, IRE1&#x3b1;, PERK, and PKR, but not ATF6 protein (<xref ref-type="fig" rid="F6">Figure 6</xref>). Further mechanistic investigation is needed to dissect the crosstalk among these three kinase cascades under oxidative stress.</p>
<fig id="F6" position="float">
<label>FIGURE 6</label>
<caption>
<p>The Schematic drawing for Alternol-induced ER stress in prostate cancer. Alternol treatment causes ROS accumulation, leading to PKR, PERK, and IRE1&#x3b1; activation and subsequent eIF2&#x3b1; phosphorylation, ATF3/ATF4 transactivation, XBP1 splicing, and CHOP expression.</p>
</caption>
<graphic xlink:href="fphar-15-1397116-g006.tif"/>
</fig>
</sec>
</body>
<back>
<sec sec-type="data-availability" id="s5">
<title>Data availability statement</title>
<p>The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: <ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/bioproject/705723">http://www.ncbi.nlm.nih.gov/bioproject/705723</ext-link>.</p>
</sec>
<sec id="s6">
<title>Author contributions</title>
<p>WL: Investigation, Methodology, Writing&#x2013;original draft, Data curation, Formal Analysis. CH: Data curation, Methodology, Writing&#x2013;original draft. CL: Data curation, Writing&#x2013;original draft. SY: Data curation, Writing&#x2013;original draft. JZ: Data curation, Writing&#x2013;original draft, Methodology. CZ: Data curation, Writing&#x2013;original draft. XW: Funding acquisition, Investigation, Validation, Writing&#x2013;review and editing. QM: Funding acquisition, Investigation, Validation, Writing&#x2013;review and editing. BL: Conceptualization, Funding acquisition, Investigation, Methodology, Project administration, Supervision, Validation, Visualization, Writing&#x2013;original draft, Writing&#x2013;review and editing.</p>
</sec>
<sec sec-type="funding-information" id="s7">
<title>Funding</title>
<p>The author(s) declare that financial support was received for the research, authorship, and/or publication of this article. This work was partially supported by grants from the national key R&#x0026;D program (2020YFA0908800) and the 2024 special funding from Guangdong Medical University (4SG24016G) to XW and by Ningbo Clinical Research Center Fund (&#x0023;2019A21001) to QM.</p>
</sec>
<ack>
<p>We are very grateful for the generous gift of Alternol reagent from Dr Jiepeng Chen at Sungen Biosciences (Shantou, China).</p>
</ack>
<sec sec-type="COI-statement" id="s8">
<title>Conflict of interest</title>
<p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p>
<p>The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision.</p>
</sec>
<sec sec-type="disclaimer" id="s9">
<title>Publisher&#x2019;s note</title>
<p>All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.</p>
</sec>
<ref-list>
<title>References</title>
<ref id="B1">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Aria</surname>
<given-names>H.</given-names>
</name>
<name>
<surname>Rezaei</surname>
<given-names>M.</given-names>
</name>
</person-group> (<year>2023</year>). <article-title>Immunogenic cell death inducer peptides: a new approach for cancer therapy, current status and future perspectives</article-title>. <source>Biomed. Pharmacother.</source> <volume>161</volume>, <fpage>114503</fpage>. <pub-id pub-id-type="doi">10.1016/j.biopha.2023.114503</pub-id>
</citation>
</ref>
<ref id="B2">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Capece</surname>
<given-names>D.</given-names>
</name>
<name>
<surname>Verzella</surname>
<given-names>D.</given-names>
</name>
<name>
<surname>Flati</surname>
<given-names>I.</given-names>
</name>
<name>
<surname>Arboretto</surname>
<given-names>P.</given-names>
</name>
<name>
<surname>Cornice</surname>
<given-names>J.</given-names>
</name>
<name>
<surname>Franzoso</surname>
<given-names>G.</given-names>
</name>
</person-group> (<year>2022</year>). <article-title>NF-&#x3ba;B: blending metabolism, immunity, and inflammation</article-title>. <source>Trends Immunol.</source> <volume>43</volume> (<issue>9</issue>), <fpage>757</fpage>&#x2013;<lpage>775</lpage>. <pub-id pub-id-type="doi">10.1016/j.it.2022.07.004</pub-id>
</citation>
</ref>
<ref id="B3">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Celik</surname>
<given-names>C.</given-names>
</name>
<name>
<surname>Lee</surname>
<given-names>S. Y. T.</given-names>
</name>
<name>
<surname>Yap</surname>
<given-names>W. S.</given-names>
</name>
<name>
<surname>Thibault</surname>
<given-names>G.</given-names>
</name>
</person-group> (<year>2023</year>). <article-title>Endoplasmic reticulum stress and lipids in health and diseases</article-title>. <source>Prog. Lipid Res.</source> <volume>89</volume>, <fpage>101198</fpage>. <pub-id pub-id-type="doi">10.1016/j.plipres.2022.101198</pub-id>
</citation>
</ref>
<ref id="B4">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Chintha</surname>
<given-names>C.</given-names>
</name>
<name>
<surname>Carlesso</surname>
<given-names>A.</given-names>
</name>
<name>
<surname>Gorman</surname>
<given-names>A. M.</given-names>
</name>
<name>
<surname>Samali</surname>
<given-names>A.</given-names>
</name>
<name>
<surname>Eriksson</surname>
<given-names>L. A.</given-names>
</name>
</person-group> (<year>2019</year>). <article-title>Molecular modeling provides a structural basis for PERK inhibitor selectivity towards RIPK1</article-title>. <source>RSC Adv.</source> <volume>10</volume> (<issue>1</issue>), <fpage>367</fpage>&#x2013;<lpage>375</lpage>. <pub-id pub-id-type="doi">10.1039/c9ra08047c</pub-id>
</citation>
</ref>
<ref id="B5">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>de la Calle</surname>
<given-names>C. M.</given-names>
</name>
<name>
<surname>Shee</surname>
<given-names>K.</given-names>
</name>
<name>
<surname>Yang</surname>
<given-names>H.</given-names>
</name>
<name>
<surname>Lonergan</surname>
<given-names>P. E.</given-names>
</name>
<name>
<surname>Nguyen</surname>
<given-names>H. G.</given-names>
</name>
</person-group> (<year>2022</year>). <article-title>The endoplasmic reticulum stress response in prostate cancer</article-title>. <source>Nat. Rev. Urol.</source> <volume>19</volume> (<issue>12</issue>), <fpage>708</fpage>&#x2013;<lpage>726</lpage>. <pub-id pub-id-type="doi">10.1038/s41585-022-00649-3</pub-id>
</citation>
</ref>
<ref id="B6">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Eo</surname>
<given-names>H.</given-names>
</name>
<name>
<surname>Valentine</surname>
<given-names>R. J.</given-names>
</name>
</person-group> (<year>2022</year>). <article-title>Saturated fatty acid-induced endoplasmic reticulum stress and insulin resistance are prevented by Imoxin in C2C12 myotubes</article-title>. <source>Front. Physiol.</source> <volume>13</volume>, <fpage>842819</fpage>. <pub-id pub-id-type="doi">10.3389/fphys.2022.842819</pub-id>
</citation>
</ref>
<ref id="B7">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Ernst</surname>
<given-names>R.</given-names>
</name>
<name>
<surname>Renne</surname>
<given-names>M. F.</given-names>
</name>
<name>
<surname>Jain</surname>
<given-names>A.</given-names>
</name>
<name>
<surname>von der Malsburg</surname>
<given-names>A.</given-names>
</name>
</person-group> (<year>2024</year>). <article-title>Endoplasmic reticulum membrane homeostasis and the unfolded protein response</article-title>. <source>Cold Spring Harb. Perspect. Biol.</source> <volume>22</volume>, <fpage>a041400</fpage>. <pub-id pub-id-type="doi">10.1101/cshperspect.a041400</pub-id>
</citation>
</ref>
<ref id="B8">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Galluzzi</surname>
<given-names>L.</given-names>
</name>
<name>
<surname>Vitale</surname>
<given-names>I.</given-names>
</name>
<name>
<surname>Warren</surname>
<given-names>S.</given-names>
</name>
<name>
<surname>Adjemian</surname>
<given-names>S.</given-names>
</name>
<name>
<surname>Agostinis</surname>
<given-names>P.</given-names>
</name>
<name>
<surname>Martinez</surname>
<given-names>A. B.</given-names>
</name>
<etal/>
</person-group> (<year>2020</year>). <article-title>Consensus guidelines for the definition, detection and interpretation of immunogenic cell death</article-title>. <source>J. Immunother. Cancer</source> <volume>8</volume> (<issue>1</issue>), <fpage>e000337</fpage>. <pub-id pub-id-type="doi">10.1136/jitc-2019-000337</pub-id>
</citation>
</ref>
<ref id="B9">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Giglio</surname>
<given-names>P.</given-names>
</name>
<name>
<surname>Gagliardi</surname>
<given-names>M.</given-names>
</name>
<name>
<surname>Tumino</surname>
<given-names>N.</given-names>
</name>
<name>
<surname>Antunes</surname>
<given-names>F.</given-names>
</name>
<name>
<surname>Smaili</surname>
<given-names>S.</given-names>
</name>
<name>
<surname>Cotella</surname>
<given-names>D.</given-names>
</name>
<etal/>
</person-group> (<year>2018</year>). <article-title>PKR and GCN2 stress kinases promote an ER stress-independent eIF2&#x3b1; phosphorylation responsible for calreticulin exposure in melanoma cells</article-title>. <source>Oncoimmunology</source> <volume>7</volume> (<issue>8</issue>), <fpage>e1466765</fpage>. <pub-id pub-id-type="doi">10.1080/2162402X.2018.1466765</pub-id>
</citation>
</ref>
<ref id="B10">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Guerra</surname>
<given-names>S.</given-names>
</name>
<name>
<surname>Lopez-Fernandez</surname>
<given-names>L. A.</given-names>
</name>
<name>
<surname>Garcia</surname>
<given-names>M. A.</given-names>
</name>
<name>
<surname>Zaballos</surname>
<given-names>A.</given-names>
</name>
<name>
<surname>Esteban</surname>
<given-names>M.</given-names>
</name>
</person-group> (<year>2006</year>). <article-title>Human gene profiling in response to the active protein kinase, interferon-induced serine/threonine protein kinase (PKR), in infected cells. Involvement of the transcription factor ATF-3 IN PKR-induced apoptosis</article-title>. <source>J. Biol. Chem.</source> <volume>281</volume> (<issue>27</issue>), <fpage>18734</fpage>&#x2013;<lpage>18745</lpage>. <pub-id pub-id-type="doi">10.1074/jbc.M511983200</pub-id>
</citation>
</ref>
<ref id="B11">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Harding</surname>
<given-names>H. P.</given-names>
</name>
<name>
<surname>Zhang</surname>
<given-names>Y.</given-names>
</name>
<name>
<surname>Ron</surname>
<given-names>D.</given-names>
</name>
</person-group> (<year>1999</year>). <article-title>Protein translation and folding are coupled by an endoplasmic-reticulum-resident kinase</article-title>. <source>Nature</source> <volume>397</volume> (<issue>6716</issue>), <fpage>271</fpage>&#x2013;<lpage>274</lpage>. <pub-id pub-id-type="doi">10.1038/16729</pub-id>
</citation>
</ref>
<ref id="B12">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>He</surname>
<given-names>C.</given-names>
</name>
<name>
<surname>Duan</surname>
<given-names>S.</given-names>
</name>
<name>
<surname>Dong</surname>
<given-names>L.</given-names>
</name>
<name>
<surname>Wang</surname>
<given-names>Y.</given-names>
</name>
<name>
<surname>Hu</surname>
<given-names>Q.</given-names>
</name>
<name>
<surname>Liu</surname>
<given-names>C.</given-names>
</name>
<etal/>
</person-group> (<year>2017</year>). <article-title>Characterization of a novel p110&#x3b2;-specific inhibitor BL140 that overcomes MDV3100-resistance in castration-resistant prostate cancer cells</article-title>. <source>Prostate</source> <volume>77</volume> (<issue>11</issue>), <fpage>1187</fpage>&#x2013;<lpage>1198</lpage>. <pub-id pub-id-type="doi">10.1002/pros.23377</pub-id>
</citation>
</ref>
<ref id="B13">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Hetz</surname>
<given-names>C.</given-names>
</name>
<name>
<surname>Zhang</surname>
<given-names>K.</given-names>
</name>
<name>
<surname>Kaufman</surname>
<given-names>R. J.</given-names>
</name>
</person-group> (<year>2020</year>). <article-title>Mechanisms, regulation and functions of the unfolded protein response</article-title>. <source>Nat. Rev. Mol. Cell Biol.</source> <volume>21</volume> (<issue>8</issue>), <fpage>421</fpage>&#x2013;<lpage>438</lpage>. <pub-id pub-id-type="doi">10.1038/s41580-020-0250-z</pub-id>
</citation>
</ref>
<ref id="B14">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Jackson</surname>
<given-names>R. J.</given-names>
</name>
<name>
<surname>Hellen</surname>
<given-names>C. U.</given-names>
</name>
<name>
<surname>Pestova</surname>
<given-names>T. V.</given-names>
</name>
</person-group> (<year>2010</year>). <article-title>The mechanism of eukaryotic translation initiation and principles of its regulation</article-title>. <source>Nat. Rev. Mol. Cell Biol.</source> <volume>11</volume> (<issue>2</issue>), <fpage>113</fpage>&#x2013;<lpage>127</lpage>. <pub-id pub-id-type="doi">10.1038/nrm2838</pub-id>
</citation>
</ref>
<ref id="B15">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Kim</surname>
<given-names>D. K.</given-names>
</name>
<name>
<surname>Cho</surname>
<given-names>E. S.</given-names>
</name>
<name>
<surname>Lee</surname>
<given-names>B. R.</given-names>
</name>
<name>
<surname>Um</surname>
<given-names>H. D.</given-names>
</name>
</person-group> (<year>2001</year>). <article-title>NF-kappa B mediates the adaptation of human U937 cells to hydrogen peroxide</article-title>. <source>Free Radic. Biol. Med.</source> <volume>30</volume> (<issue>5</issue>), <fpage>563</fpage>&#x2013;<lpage>571</lpage>. <pub-id pub-id-type="doi">10.1016/s0891-5849(00)00504-9</pub-id>
</citation>
</ref>
<ref id="B16">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Li</surname>
<given-names>C.</given-names>
</name>
<name>
<surname>He</surname>
<given-names>C.</given-names>
</name>
<name>
<surname>Xu</surname>
<given-names>Y.</given-names>
</name>
<name>
<surname>Xu</surname>
<given-names>H.</given-names>
</name>
<name>
<surname>Tang</surname>
<given-names>Y.</given-names>
</name>
<name>
<surname>Chavan</surname>
<given-names>H.</given-names>
</name>
<etal/>
</person-group> (<year>2019</year>). <article-title>Alternol eliminates excessive ATP production by disturbing Krebs cycle in prostate cancer</article-title>. <source>Prostate</source> <volume>79</volume> (<issue>6</issue>), <fpage>628</fpage>&#x2013;<lpage>639</lpage>. <pub-id pub-id-type="doi">10.1002/pros.23767</pub-id>
</citation>
</ref>
<ref id="B17">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Li</surname>
<given-names>C.</given-names>
</name>
<name>
<surname>Sun</surname>
<given-names>H.</given-names>
</name>
<name>
<surname>Wei</surname>
<given-names>W.</given-names>
</name>
<name>
<surname>Liu</surname>
<given-names>Q.</given-names>
</name>
<name>
<surname>Wang</surname>
<given-names>Y.</given-names>
</name>
<name>
<surname>Zhang</surname>
<given-names>Y.</given-names>
</name>
<etal/>
</person-group> (<year>2020</year>). <article-title>Mitoxantrone triggers immunogenic prostate cancer cell death via p53-dependent PERK expression</article-title>. <source>Cell Oncol. (Dordr)</source> <volume>43</volume> (<issue>6</issue>), <fpage>1099</fpage>&#x2013;<lpage>1116</lpage>. <pub-id pub-id-type="doi">10.1007/s13402-020-00544-2</pub-id>
</citation>
</ref>
<ref id="B18">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Li</surname>
<given-names>C.</given-names>
</name>
<name>
<surname>Wang</surname>
<given-names>X.</given-names>
</name>
<name>
<surname>Chen</surname>
<given-names>T.</given-names>
</name>
<name>
<surname>Li</surname>
<given-names>W.</given-names>
</name>
<name>
<surname>Zhou</surname>
<given-names>X.</given-names>
</name>
<name>
<surname>Wang</surname>
<given-names>L.</given-names>
</name>
<etal/>
</person-group> (<year>2022</year>). <article-title>Huaier induces immunogenic cell death <italic>via</italic> CircCLASP1/PKR/eIF2&#x3b1; signaling pathway in triple negative breast cancer</article-title>. <source>Front. Cell Dev. Biol.</source> <volume>10</volume>, <fpage>913824</fpage>. <pub-id pub-id-type="doi">10.3389/fcell.2022.913824</pub-id>
</citation>
</ref>
<ref id="B19">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Li</surname>
<given-names>C.</given-names>
</name>
<name>
<surname>Zhang</surname>
<given-names>Y.</given-names>
</name>
<name>
<surname>Yan</surname>
<given-names>S.</given-names>
</name>
<name>
<surname>Zhang</surname>
<given-names>G.</given-names>
</name>
<name>
<surname>Wei</surname>
<given-names>W.</given-names>
</name>
<name>
<surname>Qi</surname>
<given-names>Z.</given-names>
</name>
<etal/>
</person-group> (<year>2021</year>). <article-title>Alternol triggers immunogenic cell death via reactive oxygen species generation</article-title>. <source>Oncoimmunology</source> <volume>10</volume> (<issue>1</issue>), <fpage>1952539</fpage>. <pub-id pub-id-type="doi">10.1080/2162402X.2021.1952539</pub-id>
</citation>
</ref>
<ref id="B20">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Lin</surname>
<given-names>X.</given-names>
</name>
<name>
<surname>Liu</surname>
<given-names>Y. H.</given-names>
</name>
<name>
<surname>Zhang</surname>
<given-names>H. Q.</given-names>
</name>
<name>
<surname>Wu</surname>
<given-names>L. W.</given-names>
</name>
<name>
<surname>Li</surname>
<given-names>Q.</given-names>
</name>
<name>
<surname>Deng</surname>
<given-names>J.</given-names>
</name>
<etal/>
</person-group> (<year>2023</year>). <article-title>DSCC1 interacts with HSP90AB1 and promotes the progression of lung adenocarcinoma via regulating ER stress</article-title>. <source>Cancer Cell Int.</source> <volume>23</volume> (<issue>1</issue>), <fpage>208</fpage>. <pub-id pub-id-type="doi">10.1186/s12935-023-03047-w</pub-id>
</citation>
</ref>
<ref id="B21">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Lin</surname>
<given-names>Y. Z.</given-names>
</name>
<name>
<surname>Yao</surname>
<given-names>S. Y.</given-names>
</name>
<name>
<surname>Veach</surname>
<given-names>R. A.</given-names>
</name>
<name>
<surname>Torgerson</surname>
<given-names>T. R.</given-names>
</name>
<name>
<surname>Hawiger</surname>
<given-names>J.</given-names>
</name>
</person-group> (<year>1995</year>). <article-title>Inhibition of nuclear translocation of transcription factor NF-kappa B by a synthetic peptide containing a cell membrane-permeable motif and nuclear localization sequence</article-title>. <source>J. Biol. Chem.</source> <volume>270</volume> (<issue>24</issue>), <fpage>14255</fpage>&#x2013;<lpage>14258</lpage>. <pub-id pub-id-type="doi">10.1074/jbc.270.24.14255</pub-id>
</citation>
</ref>
<ref id="B22">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Lindenmeyer</surname>
<given-names>M. T.</given-names>
</name>
<name>
<surname>Rastaldi</surname>
<given-names>M. P.</given-names>
</name>
<name>
<surname>Ikehata</surname>
<given-names>M.</given-names>
</name>
<name>
<surname>Neusser</surname>
<given-names>M. A.</given-names>
</name>
<name>
<surname>Kretzler</surname>
<given-names>M.</given-names>
</name>
<name>
<surname>Cohen</surname>
<given-names>C. D.</given-names>
</name>
<etal/>
</person-group> (<year>2008</year>). <article-title>Proteinuria and hyperglycemia induce endoplasmic reticulum stress</article-title>. <source>J. Am. Soc. Nephrol.</source> <volume>19</volume> (<issue>11</issue>), <fpage>2225</fpage>&#x2013;<lpage>2236</lpage>. <pub-id pub-id-type="doi">10.1681/ASN.2007121313</pub-id>
</citation>
</ref>
<ref id="B23">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Liu</surname>
<given-names>P.</given-names>
</name>
<name>
<surname>Zhao</surname>
<given-names>L.</given-names>
</name>
<name>
<surname>Zitvogel</surname>
<given-names>L.</given-names>
</name>
<name>
<surname>Kepp</surname>
<given-names>O.</given-names>
</name>
<name>
<surname>Kroemer</surname>
<given-names>G.</given-names>
</name>
</person-group> (<year>2024</year>). <article-title>Immunogenic cell death (ICD) enhancers-Drugs that enhance the perception of ICD by dendritic cells</article-title>. <source>Immunol. Rev.</source> <volume>321</volume> (<issue>1</issue>), <fpage>7</fpage>&#x2013;<lpage>19</lpage>. <pub-id pub-id-type="doi">10.1111/imr.13269</pub-id>
</citation>
</ref>
<ref id="B24">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Liu</surname>
<given-names>W.</given-names>
</name>
<name>
<surname>Li</surname>
<given-names>J. C.</given-names>
</name>
<name>
<surname>Huang</surname>
<given-names>J.</given-names>
</name>
<name>
<surname>Chen</surname>
<given-names>J.</given-names>
</name>
<name>
<surname>Holzbeierlein</surname>
<given-names>J.</given-names>
</name>
<name>
<surname>Li</surname>
<given-names>B.</given-names>
</name>
</person-group> (<year>2020</year>). <article-title>Alternol/alteronol: potent anti-cancer compounds with multiple mechanistic actions</article-title>. <source>Front. Oncol.</source> <volume>10</volume>, <fpage>568110</fpage>. <pub-id pub-id-type="doi">10.3389/fonc.2020.568110</pub-id>
</citation>
</ref>
<ref id="B25">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Martinez Molina</surname>
<given-names>D.</given-names>
</name>
<name>
<surname>Jafari</surname>
<given-names>R.</given-names>
</name>
<name>
<surname>Ignatushchenko</surname>
<given-names>M.</given-names>
</name>
<name>
<surname>Seki</surname>
<given-names>T.</given-names>
</name>
<name>
<surname>Larsson</surname>
<given-names>E. A.</given-names>
</name>
<name>
<surname>Dan</surname>
<given-names>C.</given-names>
</name>
<etal/>
</person-group> (<year>2013</year>). <article-title>Monitoring drug target engagement in cells and tissues using the cellular thermal shift assay</article-title>. <source>Science</source> <volume>341</volume> (<issue>6141</issue>), <fpage>84</fpage>&#x2013;<lpage>87</lpage>. <pub-id pub-id-type="doi">10.1126/science.1233606</pub-id>
</citation>
</ref>
<ref id="B26">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Nakamura</surname>
<given-names>T.</given-names>
</name>
<name>
<surname>Arduini</surname>
<given-names>A.</given-names>
</name>
<name>
<surname>Baccaro</surname>
<given-names>B.</given-names>
</name>
<name>
<surname>Furuhashi</surname>
<given-names>M.</given-names>
</name>
<name>
<surname>Hotamisligil</surname>
<given-names>G. S.</given-names>
</name>
</person-group> (<year>2014</year>). <article-title>Small-molecule inhibitors of PKR improve glucose homeostasis in obese diabetic mice</article-title>. <source>Diabetes</source> <volume>63</volume> (<issue>2</issue>), <fpage>526</fpage>&#x2013;<lpage>534</lpage>. <pub-id pub-id-type="doi">10.2337/db13-1019</pub-id>
</citation>
</ref>
<ref id="B27">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Oakes</surname>
<given-names>S. A.</given-names>
</name>
<name>
<surname>Papa</surname>
<given-names>F. R.</given-names>
</name>
</person-group> (<year>2015</year>). <article-title>The role of endoplasmic reticulum stress in human pathology</article-title>. <source>Annu. Rev. Pathol.</source> <volume>10</volume>, <fpage>173</fpage>&#x2013;<lpage>194</lpage>. <pub-id pub-id-type="doi">10.1146/annurev-pathol-012513-104649</pub-id>
</citation>
</ref>
<ref id="B28">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Park</surname>
<given-names>S. H.</given-names>
</name>
<name>
<surname>Choi</surname>
<given-names>J.</given-names>
</name>
<name>
<surname>Kang</surname>
<given-names>J. I.</given-names>
</name>
<name>
<surname>Choi</surname>
<given-names>S. Y.</given-names>
</name>
<name>
<surname>Hwang</surname>
<given-names>S. B.</given-names>
</name>
<name>
<surname>Kim</surname>
<given-names>J. P.</given-names>
</name>
<etal/>
</person-group> (<year>2006</year>). <article-title>Attenuated expression of interferon-induced protein kinase PKR in a simian cell devoid of type I interferons</article-title>. <source>Mol. Cells</source> <volume>21</volume> (<issue>1</issue>), <fpage>21</fpage>&#x2013;<lpage>28</lpage>. <pub-id pub-id-type="doi">10.1016/s1016-8478(23)12898-6</pub-id>
</citation>
</ref>
<ref id="B29">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Park</surname>
<given-names>S. M.</given-names>
</name>
<name>
<surname>Kang</surname>
<given-names>T. I.</given-names>
</name>
<name>
<surname>So</surname>
<given-names>J. S.</given-names>
</name>
</person-group> (<year>2021</year>). <article-title>Roles of XBP1s in transcriptional regulation of target genes</article-title>. <source>Biomedicines</source> <volume>9</volume> (<issue>7</issue>), <fpage>791</fpage>. <pub-id pub-id-type="doi">10.3390/biomedicines9070791</pub-id>
</citation>
</ref>
<ref id="B30">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Rachidi</surname>
<given-names>S.</given-names>
</name>
<name>
<surname>Sun</surname>
<given-names>S.</given-names>
</name>
<name>
<surname>Wu</surname>
<given-names>B. X.</given-names>
</name>
<name>
<surname>Jones</surname>
<given-names>E.</given-names>
</name>
<name>
<surname>Drake</surname>
<given-names>R. R.</given-names>
</name>
<name>
<surname>Ogretmen</surname>
<given-names>B.</given-names>
</name>
<etal/>
</person-group> (<year>2015</year>). <article-title>Endoplasmic reticulum heat shock protein gp96 maintains liver homeostasis and promotes hepatocellular carcinogenesis</article-title>. <source>J. Hepatol.</source> <volume>62</volume> (<issue>4</issue>), <fpage>879</fpage>&#x2013;<lpage>888</lpage>. <pub-id pub-id-type="doi">10.1016/j.jhep.2014.11.010</pub-id>
</citation>
</ref>
<ref id="B31">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Ron</surname>
<given-names>D.</given-names>
</name>
<name>
<surname>Walter</surname>
<given-names>P.</given-names>
</name>
</person-group> (<year>2007</year>). <article-title>Signal integration in the endoplasmic reticulum unfolded protein response</article-title>. <source>Nat. Rev. Mol. Cell Biol.</source> <volume>8</volume> (<issue>7</issue>), <fpage>519</fpage>&#x2013;<lpage>529</lpage>. <pub-id pub-id-type="doi">10.1038/nrm2199</pub-id>
</citation>
</ref>
<ref id="B32">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Rufo</surname>
<given-names>N.</given-names>
</name>
<name>
<surname>Garg</surname>
<given-names>A. D.</given-names>
</name>
<name>
<surname>Agostinis</surname>
<given-names>P.</given-names>
</name>
</person-group> (<year>2017</year>). <article-title>The unfolded protein response in immunogenic cell death and cancer immunotherapy</article-title>. <source>Trends Cancer</source> <volume>3</volume> (<issue>9</issue>), <fpage>643</fpage>&#x2013;<lpage>658</lpage>. <pub-id pub-id-type="doi">10.1016/j.trecan.2017.07.002</pub-id>
</citation>
</ref>
<ref id="B33">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Saaoud</surname>
<given-names>F.</given-names>
</name>
<name>
<surname>Lu</surname>
<given-names>Y.</given-names>
</name>
<name>
<surname>Xu</surname>
<given-names>K.</given-names>
</name>
<name>
<surname>Shao</surname>
<given-names>Y.</given-names>
</name>
<name>
<surname>Pratico</surname>
<given-names>D.</given-names>
</name>
<name>
<surname>Vazquez-Padron</surname>
<given-names>R. I.</given-names>
</name>
<etal/>
</person-group> (<year>2024</year>). <article-title>Protein-rich foods, sea foods, and gut microbiota amplify immune responses in chronic diseases and cancers - targeting PERK as a novel therapeutic strategy for chronic inflammatory diseases, neurodegenerative disorders, and cancer</article-title>. <source>Pharmacol. Ther.</source> <volume>255</volume>, <fpage>108604</fpage>. <pub-id pub-id-type="doi">10.1016/j.pharmthera.2024.108604</pub-id>
</citation>
</ref>
<ref id="B34">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Sheng</surname>
<given-names>X.</given-names>
</name>
<name>
<surname>Nenseth</surname>
<given-names>H. Z.</given-names>
</name>
<name>
<surname>Qu</surname>
<given-names>S.</given-names>
</name>
<name>
<surname>Kuzu</surname>
<given-names>O. F.</given-names>
</name>
<name>
<surname>Frahnow</surname>
<given-names>T.</given-names>
</name>
<name>
<surname>Simon</surname>
<given-names>L.</given-names>
</name>
<etal/>
</person-group> (<year>2019</year>). <article-title>IRE1&#x3b1;-XBP1s pathway promotes prostate cancer by activating c-MYC signaling</article-title>. <source>Nat. Commun.</source> <volume>10</volume> (<issue>1</issue>), <fpage>323</fpage>. <pub-id pub-id-type="doi">10.1038/s41467-018-08152-3</pub-id>
</citation>
</ref>
<ref id="B35">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Sicari</surname>
<given-names>D.</given-names>
</name>
<name>
<surname>Delaunay-Moisan</surname>
<given-names>A.</given-names>
</name>
<name>
<surname>Combettes</surname>
<given-names>L.</given-names>
</name>
<name>
<surname>Chevet</surname>
<given-names>E.</given-names>
</name>
<name>
<surname>Igbaria</surname>
<given-names>A.</given-names>
</name>
</person-group> (<year>2020</year>). <article-title>A guide to assessing endoplasmic reticulum homeostasis and stress in mammalian systems</article-title>. <source>FEBS J.</source> <volume>287</volume> (<issue>1</issue>), <fpage>27</fpage>&#x2013;<lpage>42</lpage>. <pub-id pub-id-type="doi">10.1111/febs.15107</pub-id>
</citation>
</ref>
<ref id="B36">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Sprooten</surname>
<given-names>J.</given-names>
</name>
<name>
<surname>Laureano</surname>
<given-names>R. S.</given-names>
</name>
<name>
<surname>Vanmeerbeek</surname>
<given-names>I.</given-names>
</name>
<name>
<surname>Govaerts</surname>
<given-names>J.</given-names>
</name>
<name>
<surname>Naulaerts</surname>
<given-names>S.</given-names>
</name>
<name>
<surname>Borras</surname>
<given-names>D. M.</given-names>
</name>
<etal/>
</person-group> (<year>2023</year>). <article-title>Trial watch: chemotherapy-induced immunogenic cell death in oncology</article-title>. <source>Oncoimmunology</source> <volume>12</volume> (<issue>1</issue>), <fpage>2219591</fpage>. <pub-id pub-id-type="doi">10.1080/2162402X.2023.2219591</pub-id>
</citation>
</ref>
<ref id="B37">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Tang</surname>
<given-names>Y.</given-names>
</name>
<name>
<surname>Chen</surname>
<given-names>R.</given-names>
</name>
<name>
<surname>Huang</surname>
<given-names>Y.</given-names>
</name>
<name>
<surname>Li</surname>
<given-names>G.</given-names>
</name>
<name>
<surname>Huang</surname>
<given-names>Y.</given-names>
</name>
<name>
<surname>Chen</surname>
<given-names>J.</given-names>
</name>
<etal/>
</person-group> (<year>2014</year>). <article-title>Natural compound Alternol induces oxidative stress-dependent apoptotic cell death preferentially in prostate cancer cells</article-title>. <source>Mol. Cancer Ther.</source> <volume>13</volume> (<issue>6</issue>), <fpage>1526</fpage>&#x2013;<lpage>1536</lpage>. <pub-id pub-id-type="doi">10.1158/1535-7163.MCT-13-0981</pub-id>
</citation>
</ref>
<ref id="B38">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Tao</surname>
<given-names>Y.</given-names>
</name>
<name>
<surname>Lu</surname>
<given-names>J.</given-names>
</name>
<name>
<surname>Li</surname>
<given-names>L.</given-names>
</name>
<name>
<surname>Lu</surname>
<given-names>L.</given-names>
</name>
<name>
<surname>Fu</surname>
<given-names>B.</given-names>
</name>
<name>
<surname>Zhang</surname>
<given-names>J.</given-names>
</name>
<etal/>
</person-group> (<year>2024</year>). <article-title>Raltitrexed induces apoptosis through activating ROS-mediated ER stress by impeding HSPA8 expression in prostate cancer cells</article-title>. <source>Biochim. Biophys. Acta Mol. Cell Res.</source> <volume>1871</volume> (<issue>3</issue>), <fpage>119684</fpage>. <pub-id pub-id-type="doi">10.1016/j.bbamcr.2024.119684</pub-id>
</citation>
</ref>
<ref id="B39">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Wiseman</surname>
<given-names>R. L.</given-names>
</name>
<name>
<surname>Mesgarzadeh</surname>
<given-names>J. S.</given-names>
</name>
<name>
<surname>Hendershot</surname>
<given-names>L. M.</given-names>
</name>
</person-group> (<year>2022</year>). <article-title>Reshaping endoplasmic reticulum quality control through the unfolded protein response</article-title>. <source>Mol. Cell</source> <volume>82</volume> (<issue>8</issue>), <fpage>1477</fpage>&#x2013;<lpage>1491</lpage>. <pub-id pub-id-type="doi">10.1016/j.molcel.2022.03.025</pub-id>
</citation>
</ref>
<ref id="B40">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Wu</surname>
<given-names>Y.</given-names>
</name>
<name>
<surname>Wang</surname>
<given-names>Y.</given-names>
</name>
<name>
<surname>Liu</surname>
<given-names>B.</given-names>
</name>
<name>
<surname>Cheng</surname>
<given-names>Y.</given-names>
</name>
<name>
<surname>Qian</surname>
<given-names>H.</given-names>
</name>
<name>
<surname>Yang</surname>
<given-names>H.</given-names>
</name>
<etal/>
</person-group> (<year>2020</year>). <article-title>SN50 attenuates alveolar hypercoagulation and fibrinolysis inhibition in acute respiratory distress syndrome mice through inhibiting NF-&#x3ba;B p65 translocation</article-title>. <source>Respir. Res.</source> <volume>21</volume> (<issue>1</issue>), <fpage>130</fpage>. <pub-id pub-id-type="doi">10.1186/s12931-020-01372-6</pub-id>
</citation>
</ref>
<ref id="B41">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Xu</surname>
<given-names>H.</given-names>
</name>
<name>
<surname>Li</surname>
<given-names>C.</given-names>
</name>
<name>
<surname>Mozziconacci</surname>
<given-names>O.</given-names>
</name>
<name>
<surname>Zhu</surname>
<given-names>R.</given-names>
</name>
<name>
<surname>Xu</surname>
<given-names>Y.</given-names>
</name>
<name>
<surname>Tang</surname>
<given-names>Y.</given-names>
</name>
<etal/>
</person-group> (<year>2019</year>). <article-title>Xanthine oxidase-mediated oxidative stress promotes cancer cell-specific apoptosis</article-title>. <source>Free Radic. Biol. Med.</source> <volume>139</volume>, <fpage>70</fpage>&#x2013;<lpage>79</lpage>. <pub-id pub-id-type="doi">10.1016/j.freeradbiomed.2019.05.019</pub-id>
</citation>
</ref>
<ref id="B42">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Xu</surname>
<given-names>H.</given-names>
</name>
<name>
<surname>Zhou</surname>
<given-names>Z.</given-names>
</name>
<name>
<surname>Li</surname>
<given-names>B.</given-names>
</name>
</person-group> (<year>2020</year>). <article-title>Natural compound Alternol as a novel therapeutic for prostate cancer treatment</article-title>. <source>Am. J. Clin. Exp. Urol.</source> <volume>8</volume> (<issue>3</issue>), <fpage>76</fpage>&#x2013;<lpage>80</lpage>.</citation>
</ref>
<ref id="B43">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Xue</surname>
<given-names>F.</given-names>
</name>
<name>
<surname>Lu</surname>
<given-names>J.</given-names>
</name>
<name>
<surname>Buchl</surname>
<given-names>S. C.</given-names>
</name>
<name>
<surname>Sun</surname>
<given-names>L.</given-names>
</name>
<name>
<surname>Shah</surname>
<given-names>V. H.</given-names>
</name>
<name>
<surname>Malhi</surname>
<given-names>H.</given-names>
</name>
<etal/>
</person-group> (<year>2021</year>). <article-title>Coordinated signaling of activating transcription factor 6&#x3b1; and inositol-requiring enzyme 1&#x3b1; regulates hepatic stellate cell-mediated fibrogenesis in mice</article-title>. <source>Am. J. Physiol. Gastrointest. Liver Physiol.</source> <volume>320</volume> (<issue>5</issue>), <fpage>G864</fpage>&#x2013;<lpage>G879</lpage>. <pub-id pub-id-type="doi">10.1152/ajpgi.00453.2020</pub-id>
</citation>
</ref>
<ref id="B44">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Yang</surname>
<given-names>Z.</given-names>
</name>
<name>
<surname>Teng</surname>
<given-names>Y.</given-names>
</name>
<name>
<surname>Lin</surname>
<given-names>M.</given-names>
</name>
<name>
<surname>Peng</surname>
<given-names>Y.</given-names>
</name>
<name>
<surname>Du</surname>
<given-names>Y.</given-names>
</name>
<name>
<surname>Sun</surname>
<given-names>Q.</given-names>
</name>
<etal/>
</person-group> (<year>2024</year>). <article-title>Reinforced immunogenic endoplasmic reticulum stress and oxidative stress via an orchestrated nanophotoinducer to boost cancer photoimmunotherapy</article-title>. <source>ACS Nano</source> <volume>18</volume>, <fpage>7267</fpage>&#x2013;<lpage>7286</lpage>. <pub-id pub-id-type="doi">10.1021/acsnano.3c13143</pub-id>
</citation>
</ref>
<ref id="B45">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Yoshida</surname>
<given-names>H.</given-names>
</name>
<name>
<surname>Matsui</surname>
<given-names>T.</given-names>
</name>
<name>
<surname>Yamamoto</surname>
<given-names>A.</given-names>
</name>
<name>
<surname>Okada</surname>
<given-names>T.</given-names>
</name>
<name>
<surname>Mori</surname>
<given-names>K.</given-names>
</name>
</person-group> (<year>2001</year>). <article-title>XBP1 mRNA is induced by ATF6 and spliced by IRE1 in response to ER stress to produce a highly active transcription factor</article-title>. <source>Cell</source> <volume>107</volume> (<issue>7</issue>), <fpage>881</fpage>&#x2013;<lpage>891</lpage>. <pub-id pub-id-type="doi">10.1016/s0092-8674(01)00611-0</pub-id>
</citation>
</ref>
<ref id="B46">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Zhang</surname>
<given-names>R.</given-names>
</name>
<name>
<surname>Karijolich</surname>
<given-names>J.</given-names>
</name>
</person-group> (<year>2024</year>). <article-title>RNA recognition by PKR during DNA virus infection</article-title>. <source>J. Med. Virol.</source> <volume>96</volume> (<issue>2</issue>), <fpage>e29424</fpage>. <pub-id pub-id-type="doi">10.1002/jmv.29424</pub-id>
</citation>
</ref>
<ref id="B47">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Zhao</surname>
<given-names>C.</given-names>
</name>
<name>
<surname>Deng</surname>
<given-names>H.</given-names>
</name>
<name>
<surname>Chen</surname>
<given-names>X.</given-names>
</name>
</person-group> (<year>2022</year>). <article-title>Harnessing immune response using reactive oxygen Species-Generating/Eliminating inorganic biomaterials for disease treatment</article-title>. <source>Adv. Drug Deliv. Rev.</source> <volume>188</volume>, <fpage>114456</fpage>. <pub-id pub-id-type="doi">10.1016/j.addr.2022.114456</pub-id>
</citation>
</ref>
<ref id="B48">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Zuo</surname>
<given-names>D.</given-names>
</name>
<name>
<surname>Zhou</surname>
<given-names>Z.</given-names>
</name>
<name>
<surname>Wang</surname>
<given-names>H.</given-names>
</name>
<name>
<surname>Zhang</surname>
<given-names>T.</given-names>
</name>
<name>
<surname>Zang</surname>
<given-names>J.</given-names>
</name>
<name>
<surname>Yin</surname>
<given-names>F.</given-names>
</name>
<etal/>
</person-group> (<year>2017</year>). <article-title>Alternol, a natural compound, exerts an anti-tumour effect on osteosarcoma by modulating of STAT3 and ROS/MAPK signalling pathways</article-title>. <source>J. Cell Mol. Med.</source> <volume>21</volume> (<issue>2</issue>), <fpage>208</fpage>&#x2013;<lpage>221</lpage>. <pub-id pub-id-type="doi">10.1111/jcmm.12957</pub-id>
</citation>
</ref>
</ref-list>
</back>
</article>