Lycorine Induces Mitochondria-Dependent Apoptosis in Hepatoblastoma HepG2 Cells Through ROCK1 Activation

Lycorine, a naturally occurring compound extracted from the Amaryllidaceae plant family, has been reported to exhibit antitumor activity in various cancer cell types. In the present study, we investigated the molecular mechanisms underlying lycorine-induced apoptosis in hepatoblastoma HepG2 cells. We found that lycorine induced mitochondria-dependent apoptosis in HepG2 cells accompanied by mitochondrial permeability transition pore (mPTP) opening, mitochondrial membrane potential (MMP) loss, adenosine triphosphate (ATP) depletion, Ca2+ and cytochrome c (Cyto C) release, as well as caspase activation. Furthermore, we found Rho associated coiled-coil containing protein kinase 1 (ROCK1) cleavage/activation played a critical role in lycorine-induced mitochondrial apoptosis. In addition, the ROCK inhibitor Y-27632 was employed, and we found that co-treatment with Y-27632 attenuated lycorine-induced mitochondrial injury and cell apoptosis. Meanwhile, an in vivo study revealed that lycorine inhibited tumor growth and induced apoptosis in a HepG2 xenograft mouse model in association with ROCK1 activation. Taken together, all these findings suggested that lycorine induced mitochondria-dependent apoptosis through ROCK1 activation in HepG2 cells, and this may be a theoretical basis for lycorine’s anticancer effects.


INTRODUCTION
Rho-associated protein kinase (ROCK), a well-characterized Rho effector, is reported to be involved in many cellular processes, including actin dynamics, cell adhesion and migration, apoptosis, proliferation, and metabolism (Leung et al., 1996;Matsui et al., 1996;Totsukawa et al., 2000;Shi and Wei, 2007). In recent years, high expression of ROCK1 has been reported in several human cancers and often correlated with poor survival (Liu, 2011). Moreover, studies have reported that the inhibition of ROCK1 increases survival in various animal disease models (Whatcott et al., 2017). Furthermore, ROCK1 has been developed as a potential therapeutic target for diseases like neurological disorders, cardiovascular diseases, and cancers (Akagi et al., 2014;Hartmann et al., 2015;Henderson et al., 2016).
Apoptosis, which occurs in the process of cellular ageing, plays an important role in the homeostasis and development in normal tissues (Lockshin and Williams, 1965). Moreover, induction of apoptosis during carcinogenesis is considered to effectively attenuate the progression of cancers (Kerr and Searle, 1972). Current studies have reported two main apoptotic pathways: the intrinsic or mitochondrial pathway and the extrinsic or death receptor pathway. As we all know, mitochondria, the primary energy source for cells, play a significant role in cell metabolism (Gogvadze et al., 2008). Cancer cells have a characteristic of high energy demand. Thus, inducing apoptosis through targeting the mitochondria seems to be a reasonable therapeutic strategy.
In recent years, substantial researches have shown that most commercially available antitumor drugs are derived from nature (Cragg and Newman, 2005;Newman and Cragg, 2012). Lycorine, a naturally occurring compound extracted from the Amaryllidaceae plant family, has been confirmed to exhibit antitumor activity in multiple cancer cell types (Lamoral-Theys et al., 2009;Cao et al., 2013;Zeng et al., 2017). Increasing evidence demonstrates that lycorine's antitumor effects are mediated by inducing cell cycle arrest and antiproliferation, as evidenced in various cancer cell lines in vitro and in tumor xenograft models in vivo (Li et al., 2012;Cao et al., 2013;Wang et al., 2017). Drugs that target the apoptotic signalling pathway could potentially restore sensitivity to chemotherapy and cause cancer cell death (Gimenez-Bonafe et al., 2009;Fulda, 2015). Although it has been reported that lycorine induced mitochondrial dysfunction in multiple myeloma ARH-77 cells (Luo et al., 2015), the exact molecular mechanisms still remain unclear.
In the present study, we found that lycorine induced mitochondria-dependent apoptosis in hepatoblastoma HepG2 cells. Furthermore, an in vivo study revealed that lycorine induced apoptosis and inhibited tumor growth in a HepG2 xenograft mouse model. Meanwhile, all of these effects were in association with ROCK1 activation. Therefore, these findings may provide novel insights into the application of lycorine in the therapy strategies of hepatoblastoma.

Cell Culture
HepG2 cells, SMMC7721 cells and BEL7402 cells were obtained from American Type Culture Collection (Manassas, VA, United States). Cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum. Cells were cultured in the presence of 5% CO 2 at 37°C in humidified chambers.

Cell Viability Assay
Cells (1 × 10 4 cells/well) were seeded into a 96-well plate and incubated at 37°C with 5% CO 2 overnight. Various concentrations of lycorine (0, 0.2, 0.5, 1, 2, 10, 20, 50, 100 μM) were added into each well the next day. After 48 h of incubation, 10 μl Cell Counting Kit 8 (CCK8, Dojindo, Japan) was added and cells were incubated for another 2 h. Viable cell numbers were valued by measurement of optical density (OD) at 450 nm with a microplate reader (Thermo, Flash, Waltham, MA, United States). The cell viability percentage was calculated as: viability percentage (%) = 100% × (absorption value of treatment group)/ (absorption value of control group). All experiments were performed in triplicate.

Flow Cytometry Assay
Cell apoptosis was measured using flow cytometry assay. After incubated with lycorine for 48 h, cells were collected and washed twice with cold PBS. Cells resuspended in 1× binding buffer were stained with FITC-annexin V and propidium iodide (PI) (BD, Biosciences, 556547). After incubation for 15 min in the dark, 200 µl 1× binding buffer was added and cells were analyzed using flow cytometry (FACScan, Becton Dickinson).

ATP Luminescence Assay
The cellular ATP levels were detected using a firefly luciferasebased ATP determination kit (Beyotime, S0026). Briefly, after incubated with lycorine for 48 h, cells were lysed and centrifuged, and the ATP detection working solution was added to the supernatant later. The relative ATP levels were reflected as the percentage of levels that were observed using a microplate reader (Thermo, Varioskan Flash).

Measurement of Mitochondrial Membrane Potential
The JC-1 kit (Beyotime, C2006) was used to measure the mitochondrial membrane potential (MMP). Cells were seeded in a 24-well plate or 96-well plate overnight, and later incubated with lycorine for 48 h. Cells were incubated with JC-1 reagent solution for 15 min at 37°C in the dark. Subsequently, cells were washed twice with JC-1 buffer solution. The fluorescence was observed using a fluorescence microscope (CKX31 OLYMPUS, Japan) or analyzed by a microplate reader (Thermo, Varioskan Flash) at 530 nm (green) and 590 nm (red).

Mitochondrial Permeability Transition Pore (mPTP) Opening Detection Assay
Cells were seeded in a 96-well plate and incubated with lycorine for 48 h. After that, cells were incubated with 5 µM calcein-AM and 0.5 mM CoCl 2 (cytosolic calcein quencher) for 15 min at 37°C. Then cells were washed twice with PBS and analyzed using a microplate reader (Thermo, Varioskan Flash) at an excitation wavelength of 488 nm and an emission wavelength of 525 nm.

Western Blot Assay
Cells were harvested and washed twice with cold PBS. Then, cells were pelleted and lysed using RIPA buffer. Mitochondria were extracted from cells according to the manufacturer's protocol (Beyotime, C3601). The protein concentrations were determined using a BCA protein assay kit (Beyotime, P0010); 15-60 μg of sample protein was separated by SDS-PAGE and transferred to PVDF membranes (Millipore, MA, USA). The membranes were blocked with 5% fat-free dry milk for 2 h. After washing with TBS-T for three times, membranes were cultured with primary antibodies at 4°C overnight. The following day, membranes were washed with TBS-T for three times after incubation with horseradish peroxidase-conjugated secondary antibodies at room temperature for 2 h. Protein bands were visualized using ECL agents according to the manufacturer's instructions (Millipore, MA, USA).

Immunofluorescence
Cells were seeded in a 24-well plate and incubated with lycorine for 48 h. Cells were fixed with 4% paraformaldehyde for 15 min, then permeabilized with 0.1% Triton X-100 for 5 min and blocked with 5% fat-free dry milk for 30 min. The cells were subsequently co-incubated with ANT-1 and Cyp-D at 4°C overnight. Cells were counterstained with DAPI (C1005, Beyotime) followed by incubation with Alexa Fluor 488 donkey anti-rabbit IgG (A1101, 1:300) and Alexa Fluor 594 donkey anti-mouse IgG (A31573, 1:300) at 37°C for 1 h the following day. Images were taken using LSM780 confocal laser scanning microscope (Zeiss, Germany).

Tumor Xenografts
Animal experiments were approved by the Army Medical University Institutional Animal Care. Nude mice (4 weeks old) were purchased form Vital River Laboratories (VRL, Beijing, China). HepG2 cells (2 × 10 6 cells/mouse) were resuspended in the mixture of serum-free DMEM and Matrigel and injected subcutaneously into the right flanks of each mouse. Mice were randomly classified into two groups (n = 5 per group). Three days after tumor inoculation, the treatment group received lycorine (5 mg/kg/day, intraperitoneally for 3 weeks), and the control group received an equal volume of vehicle (saline). In the following days, tumor size and mice body weights were measured each 7 days; tumor volume was determined by a vernier caliper and calculated by the formula: volume = (width 2 × length)/2. All animals were sacrificed after 3 weeks of drug exposure. Tumor tissues from representative mice were lysed and subjected to Western blot analysis and further examined by immunohistochemical analysis and hematoxylin and eosin (H&E) staining.

Statistical Analysis
Data are expressed as the mean ± SD. The statistical analysis was performed by Student's t-tests or one-way analysis of variance (ANOVA) with Tukey or Dunnett's test using GraphPad Prism 6.0 statistical analysis software. *P < 0.05, **P < 0.01, and ***P < 0.001 were considered as statistical significance.

Lycorine Induced Apoptosis in HepG2 Cells
The chemical structure of lycorine is shown in Figure 1A. First, we performed CCK8 assay to assess the effects of lycorine on cytotoxicity in HepG2 cells, SMMC7721 cells, and BEL7402 cells. As shown in Supplementary Figure 1, exposure of HepG2 cells, SMMC7721 cells, and BEL7402 cells to lycorine (0, 2, 20, 50, and 100 μM) for 24 h resulted in a significant inhibition of cell viability to different degrees. Among them, HepG2 cells were most sensitive to lycorine's exposure. Thus, HepG2 cells were chosen for the following experiments.

Lycorine Induced Mitochondrial Apoptosis in HepG2 Cells
Previous studies have reported that mitochondrial pathway plays a critical role in apoptosis and the loss of MMP is reported to in association with mitochondrial injury (Pokorny et al., 2014;Fulda, 2015). To further investigate whether lycorine induces HepG2 cells apoptosis through a mitochondria-dependent pathway, we detected the changes of MMP under lycorine treatment. JC-1, a mitochondrial MMP-sensitive dye, is often used to detect the changes in MMP. After staining with JC-1, untreated cells have strong red fluorescence (JC-1 aggregation) and weak green florescence (JC-1 monomer). Accompanied by the loss of MMP, JC-1 aggregation dissipates to monomers, leading to a shift from red to green. As shown in Figure 2A, lycorine treatment (0, 10, and 20 μM) resulted in a decrease in red fluorescence and an increase in green fluorescence, suggesting that lycorine resulted in the loss of MMP. Furthermore, we also found that lycorine treatment resulted in a significant decrease in the levels of MMP in a dose-dependent manner using a microplate reader (Figure 2B; ***P < 0.001). It has been reported that mitochondrial dysfunction is usually followed by ATP depletion (Skulachev, 1999;Singleterry et al., 2014). As shown in Figure 2C, we found that there was a significant decrease in the ATP levels under lycorine treatment (***P < 0.001). It is well known that during intracellular apoptosis, the loss of Δψm is usually accompanied by cytochrome c (Cyto C) release from the mitochondria into the cytosol (Srinivasan and Avadhani, 2012). Our Western blot analysis also revealed that lycorine promoted Cyto C release from the mitochondria into the cytosol ( Figure 2D). Collectively, these findings indicated that lycorine induced mitochondrial apoptosis in HepG2 cells.

Lycorine Induced mPTP Opening in HepG2 Cells
It is well-known that mitochondrial permeability transition pore (mPTP) opening plays an important role in mitochondriainduced pro-death function and eventually leads to cell apoptosis (Halestrap et al., 2002;Halestrap, 2006;Baines, 2009). In the present study, we performed calcein-AM staining combined with CoCl 2 to explore whether mPTP opening occurred in lycorine-induced mitochondrial apoptosis. Calcein AM, a colourless esterase substrate, possesses the ability to enter living cell membranes and form a very polar green fluorescent material. Then the fluorescence from cytosolic calcein is quenched by CoCl 2 , and a fluorescence detector is used to detect the intensity of fluorescence and estimate the opening degree of mPTP when the fluorescence from the mitochondrial calcein is maintained. As shown in  Figure 3A, lycorine treatment resulted in a significant reduction of relative calcein fluorescence (*P < 0.05, ***P < 0.001), and this can be blocked by cyclosporine A (CSA), a well-known inhibitor of mPTP ( Figure 3B; *P < 0.05). Studies have reported that mPTP is composed of at least three major proteins: voltage-dependent anion channel (VDAC) located on the outer mitochondrial membrane (OMM), adenine nucleotide translocator (ANT) located on the inner mitochondrial membrane (IMM), and cyclophilin D (CypD) located in the mitochondrial matrix. It has been reported that the binding of CypD and ANT-1 initiates the opening of mPTP (Juhaszova et al., 2008;Zhen et al., 2014). Therefore, we examined the effect of lycorine on the interaction between CypD and ANT-1. As shown in Figure 3C, our results indicated that lycorine treatment resulted in an increase in the association between CypD and ANT-1. Taken together, these findings suggested that lycorine induced mPTP opening in HepG2 cells.

ROCK1 Activation Played a Significant Role in Lycorine-Induced Apoptosis
Previous studies have shown that ROCK1 plays a major role in a variety of cellular activities (Sebbagh et al., 2001;Zheng et al., 2011). Furthermore, ROCK1 activity is involved in cytoskeletal reorganization and membrane blebbing during apoptosis. To determine whether ROCK1 activation is involved in lycorineinduced apoptosis, the ROCK1 expression was detected using Western blot assay. Our results revealed that HepG2 cells treated with various concentrations of lycorine (0, 1, 2.5, 5, 10 and 20 μM) for 48 h exhibited decreased expression of ROCK1 (160 kDa) and increased cleaved ROCK1 (30 kDa) in a concentrationdependent manner (Figure 4A). To further assess the role of ROCK1 in lycorine-induced apoptosis, we employed Y-27632, a ROCK inhibitor. Western blot analysis showed that pre-incubation with Y-27632 partly blocked lycorine-induced ROCK1 activation ( Figure 4B). JC-1 staining showed that co-treatment with Y-27632 significantly attenuated lycorine-induced MMP loss (Figure 4C, D; **P < 0.01). Cells pre-incubated with Y-27632 also attenuated lycorine-induced ATP depletion and Cyto C release (Figure 4E, F; ***P < 0.001). Moreover, the administration of Y-27632 significantly attenuated lycorine-mediated caspase activation and PARP cleavage ( Figure 4G). Flow cytometry demonstrated that pre-incubation with Y-27632 attenuated lycorine-induced cell apoptosis ( Figure  4H). Taken together, these findings demonstrated that ROCK1 activation played a significant role in lycorine-induced apoptosis.

Lycorine Inhibited Tumor Growth, Induced Apoptosis, and Activated ROCK1 in a HepG2 Xenograft Model
To determine whether lycorine exhibits antitumor activity in vivo, nude mice were inoculated with xenografts ( Figure  5A, B). Our results revealed that lycorine treatment resulted in a significant suppression of tumor growth ( Figure 5C; *P < 0.05, ***P < 0.001). However, there was no statistically significant changes in body weight (Figure 5D) or other signs of toxicity like impaired movement and posture, indigestion, diarrhea, areas of redness, swelling, or agitation when compared to control group. Furthermore, immunohistochemical analysis revealed that lycorine caused an increase in immunoreactivity for C-Caspase 3 and C-PARP ( Figure 5E). Otherwise, liver and kidney samples were also excised, sectioned, and analyzed by hematoxylin and eosin (H&E) staining. Our results showed that there is no morphological difference between lycorine-treated and control groups ( Figure 5F). To determine whether ROCK1 activation is involved in lycorine-induced apoptosis in vivo, Western blot assay was performed. Our results showed that lycorine induced ROCK1 activation in the HepG2 xenograft model (Figure 5G). Taken together, these findings demonstrated that lycorine inhibited tumor growth and induced apoptosis in a HepG2 xenograft mouse model in association with ROCK1 activation.

DISCUSSION
In the present study, we demonstrated that lycorine inhibited tumor growth of HepG2 xenografts in vivo and induced apoptosis in hepatoblastoma HepG2 cells through a mitochondria-dependent pathway. Furthermore, we found that ROCK1 activation plays a critical role in lycorine-induced mitochondrial apoptosis. Our study may provide a mechanistic FIGURE 3 | Lycorine induced mPTP opening in HepG2 cells. (A) After incubated with lycorine (0, 1, 2.5, 5, 10, and 20 μM) for 48 h, HepG2 cells were stained with calcein-AM and CoCl 2 ; calcein fluorescence was detected using microplate reader. (B) After preincubated with cyclosporine A (CSA, 5 μM) for 2 h, cells were treated with lycorine (20 μM) for 48 h, the calcein fluorescence was detected using microplate reader. (C) Cells were counterstained with CypD, ANT-1, and DAPI. Images were captured using confocal microscope. Scale bar: 10 μm. *P < 0.05 and ***P < 0.001 compared to control. basis for the therapeutic rationale to develop lycorine as a novel drug candidate to treat hepatoblastoma.
Inefficient apoptosis is a proverbial hallmark of cancer cells. Thus, it is of great significance for us to examine apoptotic signaling pathways to explore novel strategies for cancer treatment. In recent years, most studies focus on elucidating signaling pathways involved in lycorine-induced apoptosis. For instance, lycorine was shown to induce apoptosis in A549 cells via AMPK-mammalian targeting of the rapamycin (mTOR)-S6K signaling pathway (Zeng et al., 2017). Lycorine inhibits breast cancer growth and metastasis by inducing apoptosis and blocking the Src/FAK-involved pathway (Ying et al., 2017). Furthermore, it has been reported that lycorine FIGURE 4 | ROCK1 activation played a significant role in lycorine-induced apoptosis. (A) HepG2 cells were treated with lycorine (0, 1, 2.5, 5, 10, and 20 μM) for 48 h; Western blot assay was performed to detect the levels of ROCK1 (160 kDa) and cleaved ROCK1 (30 kDa). (B) After pre-incubation with Y-27632 (20 μM) for 2 h, cells were incubated with lycorine (20 μM) for 48 h, the expression of ROCK1 (160 kDa) and cleaved ROCK1 (30 kDa) were detected using Western blot assay. (C and D) JC-1 staining was used to determine the changes of MMP; images were captured using confocal microscope. Otherwise, the fluorescence was detected using microplate reader. (E) ATP levels were detected using an ATP determination kit. (F) Western blot assay was used to examine the levels of Cyto C in mitochondria (Mito) and cytosol (Cyto). (G) Western blot assay was used to determine the expression of C-Caspase 3 and C-PARP. (H) Cells were stained with annexin V-FITC/PI, and flow cytometry was used to determine the percentage of apoptotic cells. **P < 0.01 and ***P < 0.001 compared to control.
promoted autophagy and apoptosis via TCRP1/Akt/mTOR axis inactivation in human hepatocellular carcinoma (Yu et al., 2017). Mitochondria, an important regulatory organelle in the intrinsic apoptotic pathway, play a critical role in the cell apoptosis process. Cancer cells characteristically proliferate in a rapid manner and maintain viability under potentially toxic conditions. Thus, inducing apoptosis in cancer cells through a mitochondriadependent pathway appears to be an effective strategy to alter metabolic efficiencies and mitochondrial function. In our study, we found that lycorine induced mitochondiral apoptosis by mPTP opening with the following evidence. First, lycorine induced apoptosis, PAPR cleavage and caspase activation in HepG2 cells and (G) Western blot assay from representative tumor tissues of vehicle control and lycorine-treated mice detected the expression of ROCK1 (160 kDa) and cleaved ROCK1 (30 kDa) (*P < 0.05 and ***P < 0.001 compared to control). HepG2 xenograft mouse model. Second, lycorine decreased MMP in HepG2 cells in a dose-dependent manner. Moreover, lycorine facilitated ATP depletion and promoted cytochrome c (Cyto C) release from mitochondria into cytosol. Third, lycorine increased the association between CypD and ANT-1, resulted in mPTP opening and Ca 2+ release. Otherwise, pre-incubation cells with cyclosporine A (CSA), an mPTP opening inhibitor, significantly blocked lycorine-induced mPTP opening.
Rho-associated coiled coil-containing protein kinase (ROCK) is a key downstream effector of the small GTPase RhoA proteins. ROCK1, one isoform of ROCK (ROCK1 and ROCK2), is necessary for membrane blebbing and cytoskeletal reorganization during apoptosis (Coleman et al., 2001). In recent years, ROCK1-targeting treatment strategy has drawn much attention for its promising clinical application in cancer therapy, diabetes, stem cell biology, and so on (Chun et al., 2011;Castro et al., 2013). Moreover, ROCK1 is reported to be cleaved and activated by a variety of mechanical stimuli and biochemical mediators in the regulation of apoptosis, as evidenced in various cell lines and animal disease models (Liu et al., 2013;Zhang et al., 2016). In our study, we found ROCK activation was involved in lycorine-induced apoptosis based on the following evidence. First, lycorine treatment decreased the levels of ROCK1 (160 kDa) and increased the cleaved ROCK1 (30 kDa). Second, cells pre-incubated with Y-27632, a ROCK inhibitor which was widely used to evaluate the role of ROCK kinases in a variety of cells and animal models (Ohki et al., 2001;Sinnett-Smith et al., 2001), markedly blocked lycorineinduced mitochondrial injury and cell apoptosis. In addition, our in vivo studies revealed that lycorine significantly inhibited tumor growth in HepG2 xenografts in association with ROCK1 activation. Recently, it has been reported that lycorine represses Akt/mTOR signalling via decreasing the levels of TCRP1 protein, resulting in apoptotic and autophagic processes activation (Yu et al., 2017). Moreover, recent studies have reported that ROCK1 is an upstream regulatory molecule of Akt/mTOR signalling (Vo et al., 2011;Zheng et al., 2018). Our research further clarified the anti-tumor mechanism of lycorine, suggesting that lycorine-mediated ROCK1 activation and mitochondria-dependent apoptosis may be a novel therapeutic strategy for hepatoblastoma treatment.

CONCLUSION
In conclusion, our findings indicated that lycorine induced mitochondrial apoptosis of HepG2 cells in vitro and inhibited tumor growth in a HepG2 xenograft mouse model in vivo. Collectively, these findings suggest a hierarchy of events in lycorine-induced apoptosis in which ROCK1 activation represents the primary insult, leading to mPTP opeining, Ca 2+ release, promoting Cyto C released from mitochondria to cytosol, caspase activation and eventually resulted in apoptosis (Figure 6). All of these results may provide a novel mechanistic basis for the application of lycorine in hepatoblastoma treatment.

ETHICS STATEMENT
The university's institutional animal care and use committee approved all the animal studies.