Anti-Cancer Effects of Pristimerin and the Mechanisms: A Critical Review

As a quinonemethide triterpenoid extracted from species of the Celastraceae and Hippocrateaceae, pristimerin has been shown potent anti-cancer effects. Specifically, it was found that pristimerin can affect many tumor-related processes, such as apoptosis, autophagy, migration and invasion, vasculogenesis, and drug resistance. Various molecular targets or signaling pathways are also involved, such as cyclins, reactive oxygen species (ROS), microRNA, nuclear factor kappa B (NF-κB), mitogen-activated protein kinase (MAPK), and PI3K/AKT/mammalian target of rapamycin (mTOR) pathways. In this review, we will focus on the research about pristimerin-induced anti-cancer activities to achieve a deeper understanding of the targets and mechanisms, which offer evidences suggesting that pristimerin can be a potent anti-cancer drug.


PRISTIMERIN: BROAD-SPECTRUM ANTI-CANCER EFFECT
Cancer is a complicated disease, which starts with a normal change through the activation of proto-oncogenes or the suppression of tumor suppressor genes (Elmore, 2007). These alterations result in diversed and interactive changes at the level of cellular processes which are involved in the regulation of proliferation, differentiation, apoptosis, migration, and tissue homeostasis. Finally, biological properties for cancer cells are acquired, including infinite proliferation potential, independent exogenous growth factors, and resistance to death signals (Brattain et al., 1994;Dent and Aranda-Anzaldo, 2019;Petho et al., 2019;Shen et al., 2019).
In view of the potent anti-cancer effect in a broad spectrum (cancer cell lines and molecular targets), it possesses a great potential for pristimerin to develop as a multiple-target anticancer drug.

Growth Inhibition
Pristimerin induces a potent effect of growth inhibition within wide range types of human tumors; the cytotoxicity of pristimerin in different cancer cell lines is summarized in Table 1.

Apoptosis Induction
Apoptosis is a kind of programmed cell death, whose activation is regulated by a series of genes, in the purpose of eliminating redundant, damaged, even infected cells to maintain homeostasis (Ke et al., 2016). Anti-cancer agents killing tumor cells by the induction of apoptosis is generally studied (Wu et al., 2017;Xiao et al., 2018;Qi et al., 2019). Two main subtypes of apoptosis have been divided into the intrinsic mitochondrial pathway and the extrinsic death receptor pathway (Elmore, 2007).
Pristimerin-induced apoptotic effects were mainly due to mitochondrial dysfunction, activation of both extrinsic and intrinsic caspases, and cleavage of poly ADP-ribose polymerase (PARP). It has been reported that pristimerin can induce caspase-dependent apoptosis in human glioma cancer cells , pancreatic cancer cells (Deeb et al., 2014b), and hepatoma cancer cells . Pristimerin-induced inhibition of Bcl-2 (as well as Bcl-2 mRNA) is sufficient to promote mitochondrial permeability transition and release of cytochrome c mediated by Bax and Bak without the inhibition of Bcl-xL in pancreatic cancer cells (Deeb et al., 2014b). On the other hand, caspase inhibitor failed to antagonize the effects of pristimerin, indicating that the lethal effect of pristimerin may not be caspase-dependent in human glioma U251 and U87 cells (Zhao et al., 2016).
The apoptotic effect of pristimerin is related to Bcl-2, and it mediates down-regulation of Bcl-2 through reactive oxygen species (ROS)-dependent ubiquitin-proteasomal degradation pathway in human prostate cancer LNCaP and PC-3 cells (Liu et al., 2013). ROS-induced apoptosis by pritimerin was also reported in hepatocellular carcinoma HepG2 cells, involving EGFR and Akt proteins (Guo et al., 2013). In colorectal carcinoma cells, the associated induction of JNK activation and MMP loss was observed (Yousef et al., 2016b), similar with the results in cervical cancer cells (Byun et al., 2009).
In human colon cancer cells, pristimerin caused cell cycle arrest and apoptosis through cyclin-CDK, mitochondrial dysfunction, and caspase-dependent mechanisms. Besides, the inhibition of DNA synthesis in HL-60 was also associated with pristimerin-induced apoptosis (Costa et al., 2008).
Pristimerin-induced apoptosis could be mediated by microRNA (miRNA). miRNAs exert a post-transcriptional gene silencing effect through binding to target mRNA and endonucleolytic cleavage of the mRNA by protein argonaute-2 (AGO2) (Kobayashi and Tomari, 2016). It was reported that pristimerin induced apoptosis through inhibiting AGO2 and PTPN1 expression via miR-542-5p in glioma cancer cells U373 . Synergization with cisplatin, pristimerin led to apoptosis via inhibiting the miR-23a, regulating PTEN/ Akt signaling-related PTEN and the phosphorylation of Akt and GSK3β in lung carcinoma NCI-H446 and A549 cells (Zhang et al., 2019).

Autophagy Induction
As another programmed necrosis, autophagy is a homeostatic cellular self-digestive process. Autophagy triggered by various cellular stress plays vital role in cell death, providing novel target for developing anti-cancer drug (Mizushima et al., 2008;Ravanan et al., 2017). LC3-II promotes the expansion and maturation of autophagy, which is considered as signal of autophagy activation. Pristimerin-induced autophagy was reported in human breast cancer MDA-MB-231 (Cevatemre et al., 2018;Lee et al., 2018) and MCF-7 cells (Cevatemre et al., 2018). As evidenced by the increase of p62 and LC3-II with an unfolded protein response (UPR), pristimerin induced an incompleted autophagy through Wnt signaling. Although endoplasmic reticulum (ER) stress is also a trigger of autophagy (Smith and  Wilkinson, 2017), it was not concluded whether the observed ER stress by pristimerin induced autophagy (Cevatemre et al., 2018). Additionally, a combination treatment of pristimerin and paclitaxel strengthened the extracellular signal-related kinase (ERK)-dependent autophagic cell death, with increase of p62 degradation and beclin1 expression (Lee et al., 2018).
On the contrary, pristimerin suppressed autophagy, downregulating LC3BII and beclin1 to sensitize the apoptosis caused by cisplatin in lung carcinoma A549 and NCI-H446 cells (Zhang et al., 2019).

Inhibition of Metastasis, Migration, Invasion, Angiogensis, and Cancer Stem Cell
The cancer metastases include a series of process, such as the completion of a complex succession of cell-biological event, cancer cell invasion, migration, and forming metastatic colonization in clinic (Valastyan and Weinberg, 2011). Pristimerin was reported to inhibit migration and invasion via targeting G protein signaling 4 (RGS4) in breast cancer MDA-MB-231 cells (Mu et al., 2012a) and HER2 in human breast carcinoma SKBR3 cells (Lee et al., 2013). Furthermore, mammalian target of rapamycin (mTOR) may be associated with its upstream Akt in pristimerin-induced inhibition of migration and invasion in colorectal cancer HCT-116 cells (Yousef et al., 2016b). Pristimerin suppressed the invasion of human prostate cancer PC-3 cells through inhibition of epithelial-to-mesenchymal transition (EMT), which was confirmed by the EMT-related markers (Chaffer et al., 2016), including N-cadherin, fibronectin, vimentin and ZEB1 (Zuo et al., 2015). MMP2 and MMP9, which are important proteins regulating invasion and metastasis, were decreased by pristimerin in esophageal cancer EC9706 and EC109 cells in a dose-dependent manner, resulting in inhibition of migration and invasion (Tu et al., 2018).
To supply nutrients and clear metabolic wastes, novel capillary blood vessels grow from pre-existing vasculature, which is called angiogenesis. However, aberrant angiogenesis plays a key role in cancer development (Valastyan and Weinberg, 2011). Thus, anti-angiogenic therapy is promising and under development . Pristimerin was reported to in vivo inhibit the neovascularization of chicken chorioallantoic membrane (CAM) and vessel ex vivo sprout in rat aortic ring assay, through a vascular endothelial growth factor (VEGF)-dependent mechanism (Mu et al., 2012b). Also, the decreased-VEGF by pristimerin was reported through the inhibition of HIF-1α via the SPHK-1 signaling pathway in hypoxic prostate cancer PC-3 cells (Lee et al., 2016). In addition, pristimerin-induced cancer stem cell toxicity was observed in breast cancer stem cells (Cevatemre et al., 2018) and esophageal squamous cell carcinoma (ESCC) (Tu et al., 2018).

Reversal of Drug Resistance
Multi-drug resistance (MDR) is defined as the resistance of cancer cells not limited to a specific chemotherapeutic drug through different structures and mechanisms of action . ABCB1 (P-glycoprotein, Pgp) is recognized as putative drug transporter, which is encoded by the ABCB1 gene, one of (ATP)binding cassette (ABC) transporter family (Dewanjee et al., 2017). Pristimerin may overcome ABCB1-mediated chemotherapeutic drug resistance through disturbing the stability of ABCB1 independent of its mRNA expression in human oral epidermoid carcinoma cells KBv200 (Yan et al., 2017). In addition, with inhibition of NF-κB and Bcr-Abl, pristimerin is effective in vitro and in vivo against imatinib-resistant chronic myelogenous leukemia cells . Additionally, Akt signaling was related to the reversal of MDR in multidrug-resistant MCF-7/ ADR breast cancer cells (Xie et al., 2016).

Synergization With Chemotherapeutic Drugs
Drug combination for cancer treatment has been well established to strengthen the anti-tumor action in varied aspects (Ho and Cheung, 2014;Andre et al., 2018), including therapeutic drug combination with natural product (Efferth, 2017;Sanchez et al., 2019). Pristimerin was reported to synergize with paclitaxel in human breast cancer cells (Lee et al., 2018), with 5-fluorouracil (5-FU) in esophageal ESCC (Tu et al., 2018). In cervical cancer cells, combination with taxol could induce cell death through ROS-mediated mitochondrial dysfunction (Eum et al., 2011). In NCI-H446 and A549 lung carcinoma cells, combination with cisplatin could induce cell apoptosis through inhibiting the miRNA-23a and Akt/GSK3β signaling pathway (Zhang et al., 2019). In pancreatic cancer cells, pristimerin could potentiate the cytotoxic effect of gemcitabine with the possible mechanism being the inhibition of gemcitabine-induced NF-κB activation (Wang et al., 2012).

In Vivo Anti-Tumor Activities
Pristimerin was widely reported its in vivo anti-tumor activities, which is summarized in Table 2.
Pristimerin was associated with the N-terminal threonine of the β5 subunit through its conjugated ketone carbon C 6 , exerting a chymotrypsin-like activity (Yang et al., 2010), which is also associated with RGS4 (Mu et al., 2012a).
Pristimerin can inhibit Bcl-2, finally induced mitochondrial cell death via an ROS-dependent ubiquitin-proteasomal degradation pathway (Liu et al., 2013). Pristimerin combination with taxol caused mitochondrial apoptosis due to ROS generation and direct proteasome inhibition (Eum et al., 2011). In addition, pristimerininduced inhibition of proteosome and IKK phosphorylation of IκB together led to UPR and suppression of NF-κB activity and cyclin D2 expression in myeloma cells H929 and U266 (Tiedemann et al., 2009).

Telomerase
Telomere is a ribonucleoprotein complex located in the end of chromosomes, maintaining telomere length homeostasis to keep chromosomal stability (Wang and Feigon, 2017). Due to the differences in telomere homeostasis between cancer and normal cells, targeting telomerase may be a promising approach to find effective and safe anti-cancer treatments (Armstrong and Tomita, 2017).
Pristimerin can inhibit telomerase activity in human prostate cancer LNCaP and PC-3 cells . The mechanism is related to inhibition of human telomerase reverse transcriptase (hTERT) and its mRNA expression, which codes the catalytic subunit of the telomerase. At the same time, knocking-down of hTERT strengthened the effects of pristimerin. Furthermore, hTERT regulatory proteins c-Myc, Sp1, p-STAT3, and p-Akt were inhibited in a dose-dependent manner .

NF-κB Pathway
NF-κB family transcription factors are crucial regulators of cell survival and inflammatory processes (Napetschnig and Wu, 2013). The inactive NF-κBs are isolated from nucleus by inhibitor of NF-κB (IκB) proteins. When activated IKK (IκB kinase) makes a proteasomal degradation of IκB, the subsequent process will occur, including the release of NF-κB, translocation of NF-κB nuclear and activation of gene transcription. NF-κB can be activated by both intracellular and extracellular stimuli, including cytokines (TNFα, IL-1β), bacterial, and viral products (LPS) (Xia et al., 2014).
NF-κB-regulated anti-apoptotic Bcl-2, Bcl-xL, c-IAPl, and surviving in human ovarian carcinoma cells , Cox-2 and VEGF in human pancreatic cancer cells (Deeb et al., 2014b). NF-κB pathway may link anti-tumor activity of pristimerin and its anti-inflammatory properties (Park and Kim, 2018). Pristimerin suppressed the translocation of NF-κB nuclear; however, there was no change of the total NF-κB protein in pancreatic cancer (Wang et al., 2012). In contrast, pristimerin inhibited both genetic expression and activation of NF-кB protein with suppression of p65 mRNA in human colorectal cancer cells (Yousef et al., 2018). TNFα-induced NF-κB activation was observed by the downstream MMP9, cyclin D1, and c-Myc in ESCC cells (Tu et al., 2018). When combined with pristimerin, the inactivation of Bcr-Abl by imatinib did not interfere with the TNFα-induced NF-κB activation, which implicated that NF-κB inactivation and Bcr-Abl inhibition may be parallel mechanisms of pristimerin-induced activity in human chronic myelogenous leukemia cells . G1 phase arrest was also associated with NF-κB pathway in human pancreatic cancer cells (Wang et al., 2012), as well as proteosome in human myeloma cells (Tiedemann et al., 2009). Moreover, pristimerin inhibited expression of miR-542-5p targeting PTPN1, which encodes protein tyrosine phosphatase 1B (PTP1B) related to NF-κB pathway .

Wnt/β-Catenin Pathway
Wnt proteins are key mediators in a series of important cellular process. The abnormal activation of Wnt/β-catenin pathway can cause a wide range of diseases including cancers (Krishnamurthy and Kurzrock, 2018;Pedone and Marucci, 2019). Pristimerin was reported to suppress Wnt/β-catenin pathway through targeting and inhibiting the expression of LRP6 and its phosphorylation, which may contribute to autophagy in human breast cancer MCF-7 cells (Cevatemre et al., 2018).

CONCLUSIONS AND PERSPECTIVE
Plants, particularly medicinal herbs, have become increasingly popular due to their potent therapeutic effects. Pristimerin, a quininemethide triterpenoid compound isolated from species of the Celastraceae and Hippocrateaceae families, has displayed biological and pharmacological activities, particularly inhibiting cancer. This review summarizes the reported results on anticancer activities and related mechanisms of pristimerin.
Pristimerin has shown anti-cancer potency in vivo ( Table 2) and in vitro ( Table 3) via specific mechanisms (Figure 2). Like many other chemotherapeutic drugs, pristimerin exerts cytotoxicity largely related to apoptosis, while the mechanism of autophagy is merely reported. The cross-talk of apoptosis and autophagy mediated by pristimerin is still remained to be explored. So far, the mechanism study of pristimerin has little reported on lung cancer, epigenetic regulation, and combination with immunotherapy. Furthermore, pristimerin has been reported to have poor selective toxicity in some cancer cells or compared with its derivatives (Costa et al., 2008;Wei et al., 2014). Comprehensive evaluation of pristimerin toxicity is yet to be carried out (as well as clinical trials). In summary, pristimerin possesses potent anti-cancer effect and further study will bring about novel drug development based on pristimerin. Down-regulated Bcl-2 through an ROS-dependent ubiquitin-proteasomal degradation pathway (Liu et al., 2013) Prevented survivin via the ubiquitin-proteasome pathway  Inhibited hTERT expression via the inhibition of SP1, c-Myc, STAT3, and B/Akt  Breast cancer SKBR3 Down-regulated HER2, decreased fatty acid synthase (Lee et al., 2013)

DATA AVAILABILITY
All datasets analyzed for this study are included in the manuscript and the supplementary files.

AUTHOR CONTRIBUTIONS
JZ and HC conceived this review; JL and YY wrote the article. HS, YL, and CS revised the article.