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1.  Diosgenin Induces Apoptosis in HepG2 Cells through Generation of Reactive Oxygen Species and Mitochondrial Pathway 
Diosgenin, a naturally occurring steroid saponin found abundantly in legumes and yams, is a precursor of various synthetic steroidal drugs. Diosgenin is studied for the mechanism of its action in apoptotic pathway in human hepatocellular carcinoma cells. Based on DAPI staining, diosgenin-treated cells manifested nuclear shrinkage, condensation, and fragmentation. Treatment of HepG2 cells with 40 μM diosgenin resulted in activation of the caspase-3, -8, -9 and cleavage of poly-ADP-ribose polymerase (PARP) and the release of cytochrome c. In the upstream, diosgenin increased the expression of Bax, decreased the expression of Bid and Bcl-2, and augmented the Bax/Bcl-2 ratio. Diosgenin-induced, dose-dependent induction of apoptosis was accompanied by sustained phosphorylation of JNK, p38 MAPK and apoptosis signal-regulating kinase (ASK)-1, as well as generation of the ROS. NAC administration, a scavenger of ROS, reversed diosgene-induced cell death. These results suggest that diosgenin-induced apoptosis in HepG2 cells through Bcl-2 protein family-mediated mitochndria/caspase-3-dependent pathway. Also, diosgenin strongly generated ROS and this oxidative stress might induce apoptosis through activation of ASK1, which are critical upstream signals for JNK/p38 MAPK activation in HepG2 cancer cells.
doi:10.1155/2012/981675
PMCID: PMC3375183  PMID: 22719792
2.  Saponins isolated from Asparagus induce apoptosis in human hepatoma cell line HepG2 through a mitochondrial-mediated pathway 
Ji, Y. | Ji, C. | Yue, L. | Xu, H.
Current Oncology  2012;19(Suppl 2):eS1-eS9.
Objective
Many scientific studies have shown that Asparagus officinalis has an antitumour effect and enhances human immunity, but the active components and the antitumour mechanisms are unclear. We investigated the effects of saponins isolated from Asparagus on proliferation and apoptosis in the human hepatoma cell line HepG2.
Methods
HepG2 cells were treated with varying concentrations of Asparagus saponins at various times. Using mtt and flow cytometry assays, we evaluated the effects of Asparagus saponins on the growth and apoptosis of HepG2 cells. Transmission electron microscopy was used to observe the morphology of cell apoptosis. Confocal laser scanning microscopy was used to analyze intracellular calcium ion concentration, mitochondrial permeability transition pore (mptp), and mitochondrial membrane potential (mmp). Spectrophotometry was applied to quantify the activity of caspase-9 and caspase-3. Flow cytometry was used to investigate the levels of reactive oxygen species (ros) and pH, and the expressions of Bcl2, Bax, CytC, and caspase-3, in HepG2 cells.
Results
Asparagus saponins inhibited the growth of HepG2 cells in a dose-dependent manner. The median inhibitory concentration (IC50) was 101.15 mg/L at 72 hours. The apoptosis morphology at 72 hours of treatment was obvious, showing cell protuberance, concentrated cytoplasm, and apoptotic bodies. The apoptotic rates at 72 hours were 30.9%, 51.7%, and 62.1% (for saponin concentrations of 50 mg/L, 100 mg/L, 200 mg/L). Treatment with Asparagus saponins for 24 hours increased the intracellular level of ros and Ca2+, lowered the pH, activated intracellular mptp, and decreased mmp in a dose-dependent manner. Treatment also increased the activity of caspase-9 and caspase-3, downregulated the expression of Bcl2, upregulated the expression of Bax, and induced release of CytC and activation of caspase-3.
Conclusions
Asparagus saponins induce apoptosis in HepG2 cells through a mitochondrial-mediated and caspase-dependent pathway, suggesting that they may be a potent agent for the treatment of hepatocellular carcinoma.
doi:10.3747/co.19.1139
PMCID: PMC3413253  PMID: 22876162
Asparagus; saponins; hepatocellular carcinoma; apoptosis; mitochondrial pathway; caspase; laser scanning confocal microscope; flow cytometry
3.  Abrus agglutinin suppresses human hepatocellular carcinoma in vitro and in vivo by inducing caspase-mediated cell death 
Acta Pharmacologica Sinica  2014;35(6):814-824.
Aim:
Abrus agglutinin (AGG) from the seeds of Indian medicinal plant Abrus precatorius belongs to the class II ribosome inactivating protein family. In this study we investigated the anticancer effects of AGG against human hepatocellular carcinoma in vitro and in vivo.
Methods:
Cell proliferation, DNA fragmentation, Annexin V binding, immunocytofluorescence, Western blotting, caspase activity assays and luciferase assays were performed to evaluate AGG in human liver cancer cells HepG2. Immunohistochemical staining and TUNEL expression were studied in tumor samples of HepG2-xenografted nude mice.
Results:
AGG induced apoptosis in HepG2 cells in a dose- and time-dependent manner. AGG-treated HepG2 cells demonstrated an increase in caspase 3/7, 8 and 9 activities and a sharp decrease in the Bcl-2/Bax ratio, indicating activation of a caspase cascade. Co-treatment of HepG2 cells with AGG and a caspase inhibitor or treatment of AGG in Bax knockout HepG2 cells decreased the caspase 3/7 activity in comparison to HepG2 cells exposed only to AGG. Moreover, AGG decreased the expression of Hsp90 and suppressed Akt phosphorylation and NF-κB expression in HepG2 cells. Finally, AGG treatment significantly reduced tumor growth in nude mice bearing HepG2 xenografts, increased TUNEL expression and decreased CD-31 and Ki-67 expression compared to levels observed in the untreated control mice bearing HepG2 cells.
Conclusion:
AGG inhibits the growth and progression of HepG2 cells by inducing caspase-mediated cell death. The agglutinin could be an alternative natural remedy for the treatment of human hepatocellular carcinomas.
doi:10.1038/aps.2014.15
PMCID: PMC4086388  PMID: 24793310
anticancer drug; Abrus agglutinin; hepatocellular carcinoma; apoptosis; caspase; Akt; Hsp90; NF-κB; HepG2 xenografts
4.  Elm tree bark extract inhibits HepG2 hepatic cancer cell growth via pro-apoptotic activity 
Journal of Veterinary Science  2012;13(1):7-13.
Control of inflammation is widely accepted as an important strategy for cancer chemoprevention. Anti-inflammatory effects of bark extracts of elm tree (BEE) have been amply reported. Therefore, BEE may be a good candidate cancer chemopreventive agent. Considering the high incidence of hepatic cancer and limited therapeutic approaches for treating this disease, it is important to develop liver cancer-specific chemopreventive agents. To evaluate the chemopreventive potential of BEE, we investigated the growth inhibition effect of BEE on the HepG2 human hepatocellular carcinoma cell line. We performed a cell counting kit-8 assay to determine cell viability, and 4,6-diamino-2-phenylindole staining and flow cytometry to measure apoptotic cell death. Finally, the expression levels of pro- and anti-apoptotic proteins were measured. BEE inhibited the growth of HepG2 cells and induced apoptosis in a dose-dependent manner. Pro-apoptotic activity was promoted via the mitochondrial pathway of apoptosis, as demonstrated by the activation of pro-apoptotic proteins Bax, caspase-9, caspase-3, and poly (ADP-ribose) polymerase as well as the down-regulation of the anti-apoptotic protein Bcl-2. These results suggest that BEE may have potential use in hepatic cancer chemoprevention by suppressing cancer cell growth via pro-apoptotic activity.
doi:10.4142/jvs.2012.13.1.7
PMCID: PMC3317460  PMID: 22437530
apoptosis; elm tree extract; HepG2 cell; Ulmus davidiana
5.  Perifosine induces cell cycle arrest and apoptosis in human hepatocellular carcinoma cell lines by blockade of Akt phosphorylation 
Cytotechnology  2010;62(5):449-460.
Hepatocellular carcinoma (HCC) is one of the most common solid cancers, representing the third cause of cancer-related death among cirrhotic patients. Treatment of advanced HCC has become a very active area of research. Perifosine, a new synthetic alkylphospholipid Akt inhibitor, has shown anti-tumor activity by inhibition of Akt phosphorylation. In this study, the effect of perifosine on the cell proliferation and apoptosis in hepatoma cells has been investigated. Cell growth inhibition was detected by MTT assay, cell cycle was analyzed by flow cytometry, AnnexinV-FITC apoptosis detection kit was used to detect cell apoptosis, and protein expression was examined by Western blotting analysis. Our present studies showed that Akt phosphorylation was inhibited by perifosine in HepG2 and Bel-7402 human hepatocellular carcinoma cells. Perifosine inhibited the growth of HepG2 cells and Bel-7402 cells in a dose-dependent manner, and arrested cell cycle progression at the G2 phase. Apoptosis induction became more effective with increasing perifosine concentration. The caspase cascade and its downstream effectors, Poly (ADP-ribose) polymerase (PARP), were also activated simultaneously upon perifosine treatment. The proapoptotic effect of perifosine was in part depending on regulation of the phosphorylation level of ERK and JNK. Perifosine cotreatment substantially increased cytotoxic effects of cisplatin in HepG2 cells. Down-regulating the expression of Bcl-2 and up-regulating the level of Bax may be the potential mechanism for this synergistic effect. Our findings suggest that the small molecule Akt inhibitor perifosine shows substantial anti-tumor activity in human hepatoma cancer cell lines, and is a good candidate for treatment combinations with classical cytostatic compounds in hepatocellular carcinoma.
doi:10.1007/s10616-010-9299-4
PMCID: PMC2993858  PMID: 20842425
Hepatocellular carcinoma; Perifosine; PI3 K/Akt; Apoptosis; Caspase; Bcl-2
6.  Pro-apoptotic effects of tectorigenin on human hepatocellular carcinoma HepG2 cells 
AIM: To investigate the effects of tectorigenin on human hepatocellular carcinoma (HCC) HepG2 cells.
METHODS: Tectorigenin, one of the main components of rhizome of Iris tectorum, was prepared by simple methods, such as extraction, filtration, concentration, precipitation and recrystallization. HepG2 cells were incubated with tectorigenin at different concentrations, and their viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Apoptosis was detected by morphological observation of nuclear change, agarose gel electrophoresis of DNA ladder, and flow cytometry with Hoechst 33342, Annexin V-EGFP and propidium iodide staining. Generation of reactive oxygen species was quantified using DCFH-DA. Intracellular Ca2+ was monitored by Fura 2-AM. Mitochondrial membrane potential was monitored using Rhodamine 123. Release of cytochrome c from mitochondria to cytosol was detected by Western blotting. Activities of caspase-3, -8 and -9 were investigated by Caspase Activity Assay Kit.
RESULTS: The viability of HepG2 cells treated by tectorigenin decreased in a concentration- and time-dependent manner. The concentration that reduced the number of viable HepG2 cells by 50% (IC50) after 12, 24 and 48 h of incubation was 35.72 mg/L, 21.19 mg/L and 11.06 mg/L, respectively. However, treatment with tectorigenin at 20 mg/L resulted in a very slight cytotoxicity to L02 cells after incubation for 12, 24 or 48 h. Tectorigenin at a concentration of 20 mg/L greatly inhibited the viability of HepG2 cells and induced the condensation of chromatin and fragmentation of nuclei. Tectorigenin induced apoptosis of HepG2 cells in a time- and dose-dependent manner. Compared with the viability rate, induction of apoptosis was the main mechanism of the anti-proliferation effect of tectorigenin in HepG2 cells. Furthermore, tectorigenin-induced apoptosis of HepG2 cells was associated with the generation of reactive oxygen species, increased intracellular [Ca2+]i, loss of mitochondrial membrane potential, translocation of cytochrome c, and activation of caspase-9 and -3.
CONCLUSION: Tectorigenin induces apoptosis of HepG2 cells mainly via mitochondrial-mediated pathway, and produces a slight cytotoxicity to L02 cells.
doi:10.3748/wjg.v18.i15.1753
PMCID: PMC3332288  PMID: 22553399
Tectorigenin; Iris tectorum maxim; Apoptosis; Hepatocellular carcinoma; HepG2; Mitochondria; Liver cancer
7.  Antitumor effect of matrine in human hepatoma G2 cells by inducing apoptosis and autophagy 
AIM: To study the antitumor effect of matrine in human hepatoma G2 (HepG2) cells and its molecular mechanism involved in antineoplastic activities.
METHODS: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to detect viability of HepG2 cells. The effect of matrine on cell cycle was detected by flow cytometry. Annexin-V-FITC/PI double staining assay was used to detect cellular apoptosis. Cellular morphological changes were observed under an inverted phase contrast microscope. Transmission electron microscopy was performed to further examine ultrastructural structure of the cells treated with matrine. Monodansylcadaverine (MDC) staining was used to detect autophagy. Whether autophagy is blocked by 3-methyladenine (3-MA), an autophagy inhibitor, was evaluated. Expression levels of Bax and Beclin 1 in HepG2 cells were measured by real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR).
RESULTS: Matrine significantly inhibited the proliferation of HepG2 cells in a dose- and time-dependent manner, and induced G1-phase cell cycle arrest and apoptosis of HepG2 cells in a dose-dependent manner. The total apoptosis rate was 0.14% for HepG2 cells not treated with matrine. In contrast, the apoptosis rate was 28.91%, 34.36% and 38.80%, respectively, for HepG2 cells treated with matrine at the concentration of 0.5, 1.0 and 2.0 mg/mL. The remarkable morphological changes were observed under an inverted phase contrast microscope. Abundant cytoplasmic vacuoles with varying sizes were observed in HepG2 cells treated with matrine. Furthermore, vacuolization in cytoplasm progressively became larger and denser when the concentration of matrine was increased. Electron microscopy demonstrated formation of abundant autophagic vacuoles in HepG2 cells after matrine treatment. When the specific autophagic inhibitor, 3-MA, was applied, the number of autophagic vacuoles greatly decreased. MDC staining showed that the fluorescent density was higher and the number of MDC-labeled particles in HepG2 cells was greater in matrine treatment group than in control group. Fewer autophagic vacuoles were observed in the combined 3-MA and matrine treatment group when 3-MA was added before matrine treatment, indicating that both autophagy and apoptosis are activated when matrine-induced death of hepatoma G2 cells occurs. Real-time quantitative RT-PCR revealed that the expression levels of Bax gene, an apoptosis-related molecule, and Beclin 1 gene which plays a key role in autophagy were higher in matrine treatment group than in control group, indicating that Beclin 1 is involved in matrine-induced autophagy and the pro-apoptotic mechanism of matrine may be related to its upregulation of Bax expression.
CONCLUSION: Matrine has potent antitumor activities in HepG2 cells and may be used as a novel effective reagent in treatment of hepatocellular carcinoma.
doi:10.3748/wjg.v16.i34.4281
PMCID: PMC2937108  PMID: 20818811
Matrine; Autophagy; Apoptosis; Bax; Beclin 1; Hepatocellular carcinoma
8.  8-bromo-7-methoxychrysin-induced apoptosis of hepatocellular carcinoma cells involves ROS and JNK 
AIM: To investigate whether the apoptotic activities of 8-bromo-7-methoxychrysin (BrMC) involve reactive oxygen species (ROS) generation and c-Jun N-terminal kinase (JNK) activation in human hepatocellular carcinoma cells (HCC).
METHODS: HepG2, Bel-7402 and L-02 cell lines were cultured in vitro and the apoptotic effects of BrMC were evaluated by flow cytometry (FCM) after propidium iodide (PI) staining, caspase-3 activity using enzyme-linked immunosorbent assay (ELISA), and DNA agarose gel electrophoresis. ROS production was evaluated by FCM after dichlorodihydrofluorescein diacetate (DCHF-DA) probe labeling. The phosphorylation level of JNK and c-Jun protein was analyzed by Western blotting.
RESULTS: FCM after PI staining showed a dose-dependent increase in the percentage of the sub-G1 cell population (P < 0.05), reaching 39.0% ± 2.8% of HepG2 cells after 48 h of treatment with BrMC at 10 μmol/L. The potency of BrMC to HepG2 and Bel-7402 (32.1% ± 2.6%) cells was found to be more effective than the lead compound, chrysin (16.2% ± 1.6% for HepG2 cells and 11.0% ± 1.3% for Bel-7402 cell) at 40 μmol/L and similar to 5-flurouracil (33.0% ± 2.1% for HepG2 cells and 29.3% ± 2.3% for Bel-7402 cells) at 10 μmol/L. BrMC had little effect on human embryo liver L-02 cells, with the percentage of sub-G1 cell population 5.4% ± 1.8%. Treatment of HepG2 cells with BrMC for 48 h also increased the levels of active caspase-3, in a concentration-dependent manner. z-DEVD-fmk, a caspase-3-specific inhibitor, prevented the activation of caspase-3. Treatment with BrMC at 10 μmol/L for 48 h resulted in the formation of a DNA ladder. Treatment of cells with BrMC (10 μmol/L) increased mean fluorescence intensity of DCHF-DA in HepG2 cells from 7.2 ± 1.12 at 0 h to 79.8 ± 3.9 at 3 h and 89.7 ± 4.7 at 6 h. BrMC did not affect ROS generation in L-02 cells. BrMC treatment failed to induce cell death and caspase-3 activation in HepG2 cells pretreated with N-acetylcysteine (10 mmol/L). In addition, in HepG2 cells treated with BrMC (2.5, 5.0, 10.0 μmol/L) for 12 h, JNK activation was observed. Peak JNK activation occurred at 12 h post-treatment and this activation persisted for up to 24 h. The expression of phosphorylated JNK and c-Jun protein after 12 h with BrMC-treated cells was inhibited by N-acetylcysteine and SP600125 pre-treatment, but GW9662 had no effect. SP600125 substantially reduced BrMC-induced cell death and caspase-3 activation of HepG2 cells. N-acetylcysteine and GW9662 also attenuated induction of cell death and caspase-3 activation in HepG2 cells treated with BrMC.
CONCLUSION: BrMC induces apoptosis of HCC cells by ROS generation and sustained JNK activation.
doi:10.3748/wjg.v16.i27.3385
PMCID: PMC2904884  PMID: 20632440
Hepatocellular carcinoma; 8-bromo-7-methoxychysin; Chrysin; Reactive oxygen species; Jun N-terminal kinase
9.  Induction of apoptosis in human liver carcinoma HepG2 cell line by 5-allyl-7-gen-difluoromethylenechrysin 
AIM: To investigate the effect of 5-allyl-7-gen-difluoromethylenechrysin (ADFMChR) on apoptosis of human liver carcinoma HepG2 cell line and the molecular mechanisms involved.
METHODS: HepG2 cells and L-02 cells were cultured in vitro and the inhibitory effect of ADFMChR on their proliferation was measured by MTT assay. The apoptosis of HepG2 cells was determined by flow cytometry (FCM) using propidium iodide (PI) fluorescence staining. DNA ladder bands were observed by DNA agarose gel electrophoresis. The influence of ADFMChR on the proxisome proliferator-activated receptor γ (PPARγ), NF-κB, Bcl-2 and Bax protein expression of HepG2 cells were analyzed by Western blotting.
RESULTS: MTT assay showed that ADFMChR significantly inhibited proliferation of HepG2 cells in a dose-dependent manner, with little effect on growth of L-02 cells, and when IC50 was measured as 8.45 μmol/L and 191.55 μmol/L respectively, the potency of ADFMChR to HepG2 cells, was found to be similar to 5-fluorouracil (5-FU, IC50 was 9.27 μmol/L). The selective index of ADFMChR cytotoxicity to HepG2 cells was 22.67 (191.55/8.45), higher than 5-FU (SI was 7.05 (65.37/9.27). FCM with PI staining demonstrated that the apoptosis rates of HepG2 cells treated with 3.0, 10.0 and 30.0 μmol/L ADFMChR for 48 h were 5.79%, 9.29% and 37.8%, respectively, and were significantly higher when treated with 30.0 μmol/L ADFMChR than when treated with 30.0 μmol/L ChR (16.0%) (P < 0.05) and were similar to those obtained with 30.0 μmol/L 5-FU (41.0%). DNA agarose gel electrophoresis showed that treatment of HepG2 cells with 10.0 μmol/L ADFMChR for 48 h and 72 h resulted in typical DNA ladders which could be reversed by 10.00 μmol/L GW9662, a blocker of PPARγ. Western blotting analysis revealed that after 24 h of treatment with 3.0, 10.0, 30.0 μmol/L ADFMChR, PPARγ and Bax protein expression in HepG2 cells increased but Bcl-2 and NF-κB expression decreased; however, pre-incubation with 10.0 μmol/L GW9662 could efficiently antagonize and weaken the regulatory effect of 3.0, 30.0 μmol/L ADFMChR on PPARγ and NF-κB protein expression in HepG2 cells.
CONCLUSION: ADFMChR induces apoptosis of HepG2 cell lines by activating PPARγ, inhibiting protein expression of Bcl-2 and NF-κB, and increasing Bax expression.
doi:10.3748/wjg.15.2234
PMCID: PMC2682238  PMID: 19437563
Liver neoplasm; Chrysin; 5-allyl-7-gen-difluoromethylenechrysin; Apoptosis; Proxisome proliferator-activated receptor γ
10.  Embelin-induced apoptosis of HepG2 human hepatocellular carcinoma cells and blockade of HepG2 cells in the G2/M phase via the mitochondrial pathway 
Embelin is a small-molecule inhibitor extracted from plants of the Myrsinaceae family demonstrating specific inhibition of the X-linked inhibitor of apoptosis protein (XIAP) to affect the proliferation and apoptosis of various types of tumor cells. However, the mechanism of action for this effect remains unclear. The purpose of the present study was to investigate the role of the mitochondrial pathway in embelin-induced HepG2 human hepatocellular carcinoma cell apoptosis and the effect of embelin on the cell cycle. HepG2 human hepatocellular carcinoma cells were treated with different doses of embelin. The MTT method was used to determine cell viability, and flow cytometry was used to assess the rate of apoptosis and the changes in mitochondrial membrane potential; the cell cycle was also analyzed. Western blot analysis was performed to determine the expression levels of the apoptosis-associated proteins Bax, Bcl-2 and the caspase family. The results revealed that embelin induced the apoptosis of the HepG2 cells in a dose- and time-dependent manner. In addition, embelin caused changes in mitochondrial membrane potential. Flow cytometric analysis demonstrated that embelin caused blockade of the HepG2 cells in the G2/M phase of the cell cycle.
doi:10.3892/etm.2012.637
PMCID: PMC3501403  PMID: 23170120
embelin; XIAP; mitochondria; apoptosis; human hepatocellular carcinoma HepG2 cells
11.  TIP30 regulates apoptosis-related genes in its apoptotic signal transduction pathway 
AIM: To investigate the role of TIP30 in apoptotic signal pathway in hepatoblastoma cells and to provide a basis for TIP30 as a gene therapy candidate in the regression of hepatoblastoma cells.
METHODS: Apoptosis of human hepatoblastoma cell lines HepG2 (p53 wild), Hep3B (p53 null) and PLC/RPF/5 (p53 mutant) infected with Ad-TIP30 (bearing a wild type human Tip30 gene) were analyzed and p53, Bax and Bcl-xl expression levels were compared among these cells. MTT assay, DNA fragmentation, in situ 3’ end labeling of DNA, annexin-V FITC staining were used to detect cell death and apoptosis in cells at various time intervals subsequent to infection, and to determine whether TIP30 had an effect on the expression levels of some apoptosis-related gene products such as Bax, p53 and Bcl-xl. A similar time course experiment was performed by Western blotting.
RESULTS: In MTT assay, the viability of HepG2 cells decreased significantly from 99.7% to 10% and displayed more massive cell death within 5-8 d than Hep3B and PLC/RPF/5 cells, with their viability decreased from 97.8% to 44.3% and 98.1% to 50.4%, respectively. In annexin-V FITC assay, the percentage of apoptosis cells in HepG2 cells was two to three-fold higher than that in control cells (infected with Ad-GFP), two-fold higher than that in Hep3B cells and 1.4-fold higher than that in PLC/RPF/5 cells 36 h after infection, respectively. Moreover, in HepG2 cells, the p53 began to increase 6-8 h after infection, reaching a maximum level between 8 and 12 h after infection and then dropped. Bax showed a similar increase in the cells as p53 reached the maximum at 8-12 h and subsequently decreased. Interestingly, Bcl-xl protein levels were down regulated during 24 to 36 h after Ad-TIP30 infection. In contrast, ectopic expression of TIP30 in Hep3B and PLC/RPF/5 cells had no effect on the regulation of Bax expression, but had an effect on Bcl-xl levels. In comparison with HepG2 cells, these data suggested that up-regulation of p53 levels by TIP30 might be a pre-requisite for Bax and Bax/Bcl-xl ratio increase. We hypothesized that TIP30 might regulate Bax gene partly through p53, which sensitizes cells to apoptosis by involving a p53 apoptosis signal transduction pathway.
CONCLUSION: TIP30 plays an important role in predisposing hepatoblastoma cells to apoptosis through regulating expression levels of these genes. Ad-TIP30 carrying exogenous TIP30-anti-tumor genes may be regarded as a potential candidate for the treatment of hepatocellular carcinoma.
doi:10.3748/wjg.v11.i2.221
PMCID: PMC4205406  PMID: 15633220
Hepatoblastoma; TIP30; Apoptosis; Signal transduction
12.  Cratoxylum formosum (Jack) Dyer ssp. pruniflorum (Kurz) Gogel. (Hóng yá mù) extract induces apoptosis in human hepatocellular carcinoma HepG2 cells through caspase-dependent pathways 
Chinese Medicine  2014;9:12.
Background
Cratoxylum formosum (Jack) Dyer ssp. pruniflorum (Kurz) Gogel. (Hóng yá mù) (CF) has been used for treatment of fever, cough, and peptic ulcer. Previously, a 50% ethanol-water extract from twigs of CF was shown highly selective in cytotoxicity against cancer cells. This study aims to investigate the molecular mechanisms underlying the apoptosis-inducing effect of CF.
Methods
The cytotoxicity of CF was evaluated in the human hepatocellular carcinoma (HCC) HepG2 cell line in comparison with a non-cancerous African green monkey kidney epithelial cell line (Vero) by a neutral red assay. The apoptosis induction mechanisms were investigated through nuclear morphological changes, DNA fragmentation, mitochondrial membrane potential alterations, and caspase enzyme activities.
Results
CF selectively induced HepG2 cell death compared with non-cancerous Vero cells. A 1.5-fold higher apoptotic effect compared with melphalan was induced by 120 μg/mL of the 50% ethanol-water extract of CF. The apoptotic cell death in HepG2 cells occurred via extrinsic and intrinsic caspase-dependent pathways in dose- and time-dependent manners by significantly increasing the activities of caspase 3/7, 8, and 9, decreasing the mitochondrial membrane potential, and causing apoptotic body formation and DNA fragmentation.
Conclusions
CF extract induced a caspase-dependent apoptosis in HepG2 cells.
doi:10.1186/1749-8546-9-12
PMCID: PMC3985586  PMID: 24708784
13.  Zinc oxide nanoparticles selectively induce apoptosis in human cancer cells through reactive oxygen species 
Background
Zinc oxide nanoparticles (ZnO NPs) have received much attention for their implications in cancer therapy. It has been reported that ZnO NPs induce selective killing of cancer cells. However, the underlying molecular mechanisms behind the anticancer response of ZnO NPs remain unclear.
Methods and results
We investigated the cytotoxicity of ZnO NPs against three types of cancer cells (human hepatocellular carcinoma HepG2, human lung adenocarcinoma A549, and human bronchial epithelial BEAS-2B) and two primary rat cells (astrocytes and hepatocytes). Results showed that ZnO NPs exert distinct effects on mammalian cell viability via killing of all three types of cancer cells while posing no impact on normal rat astrocytes and hepatocytes. The toxicity mechanisms of ZnO NPs were further investigated using human liver cancer HepG2 cells. Both the mRNA and protein levels of tumor suppressor gene p53 and apoptotic gene bax were upregulated while the antiapoptotic gene bcl-2 was downregulated in ZnO NP-treated HepG2 cells. ZnO NPs were also found to induce activity of caspase-3 enzyme, DNA fragmentation, reactive oxygen species generation, and oxidative stress in HepG2 cells.
Conclusion
Overall, our data demonstrated that ZnO NPs selectively induce apoptosis in cancer cells, which is likely to be mediated by reactive oxygen species via p53 pathway, through which most of the anticancer drugs trigger apoptosis. This study provides preliminary guidance for the development of liver cancer therapy using ZnO NPs.
doi:10.2147/IJN.S29129
PMCID: PMC3289443  PMID: 22393286
ZnO nanoparticles; cancer therapy; p53; apoptosis; ROS
14.  Retinal ischemia-induced apoptosis is associated with alteration in Bax and Bcl-xL expression rather than modifications in Bak and Bcl-2 
Molecular Vision  2009;15:2101-2110.
Purpose
Apoptosis is known to play a key role in cell death after retinal ischemia. However, little is known about the kinetics of the signaling pathways involved and their contribution to this process. The aim of this study was to determine whether changes in the expression of molecules in the mitochondrial apoptotic pathway might explain the progression of retinal damage following ischemia/reperfusion.
Methods
Retinal ischemia was induced by elevating intraocular pressure in the vitreous cavity to 150 mmHg for a period of 60 min. At time 0, 3 h (early phase), and 24 h (late phase) after reperfusion, the retinas were harvested and modifications in the expression of Bax, Bak, Bcl-2, and Bcl-xL as well as caspase-3 and −7, were examined by qPCR and, in some cases, by western blot.
Results
qPCR analysis performed at the early phase after ischemia revealed a time dependent decrease in Bax, Bak, and Bcl-xL and no alteration in Bcl-2 mRNA expression in response to retinal ischemia. At the protein level, proapoptotic Bax and Bak were not modulated while Bcl-2 and Bcl-xL were significantly upregulated. At this stage, the Bax per Bcl-2 and Bax:Bcl-xL ratios were not modified. At the late phase of recovery, Bax and Bcl-xL mRNAs were downregulated while Bak was increased. Increased Bax:Bcl-2 and Bax:Bcl-xL ratios at both the mRNA and protein levels were observed 24 h after the ischemic insult. Analysis of caspases associated with mitochondria-mediated apoptosis revealed a specific increase in the expression of caspase-3 in the ischemic retinas 24 h after reperfusion, and a decrease in the expression of caspase-7.
Conclusions
This study revealed that Bcl-2-related family members were differently regulated in the early and late phases after an ischemic insult. We showed that the Bax:Bcl-2 and Bax:Bcl-xL balances were not affected in the initial phases, but the Bax:Bcl-xL ratio shifted toward apoptosis during the late phase of recovery. This shift was reinforced by caspase-3 upregulation.
PMCID: PMC2765238  PMID: 19862336
15.  Hot water extract of Chlorella vulgaris induced DNA damage and apoptosis 
Clinics  2010;65(12):1371-1377.
OBJECTIVES:
The aim of this study was to determine the antiproliferative and apoptotic effects of hot water extracts of Chlorella vulgaris on hepatoma cell line HepG2.
INTRODUCTION:
The search for food and spices that can induce apoptosis in cancer cells has been a major study interest in the last decade. Chlorella vulgaris, a unicellular green algae, has been reported to have antioxidant and anti‐cancer properties. However, its chemopreventive effects in inhibiting the growth of cancer cells have not been studied in great detail.
METHODS:
HepG2 liver cancer cells and WRL68 normal liver cells were treated with various concentrations (0‐4 mg/ml) of hot water extract of C. vulgaris after 24 hours incubation. Apoptosis rate was evaluated by TUNEL assay while DNA damage was assessed by Comet assay. Apoptosis proteins were evaluated by Western blot analysis.
RESULTS:
Chlorella vulgaris decreased the number of viable HepG2 cells in a dose dependent manner (p < 0.05), with an IC50 of 1.6 mg/ml. DNA damage as measured by Comet assay was increased in HepG2 cells at all concentrations of Chlorella vulgaris tested. Evaluation of apoptosis by TUNEL assay showed that Chlorella vulgaris induced a higher apoptotic rate (70%) in HepG2 cells compared to normal liver cells, WRL68 (15%). Western blot analysis showed increased expression of pro‐ apoptotic proteins P53, Bax and caspase‐3 in the HepG2 cells compared to normal liver cells WRL68, and decreased expression of the anti‐apoptotic protein Bcl‐2.
CONCLUSIONS:
Chlorella vulgaris may have anti‐cancer effects by inducing apoptosis signaling cascades via an increased expression of P53, Bax and caspase‐3 proteins and through a reduction of Bcl‐2 protein, which subsequently lead to increased DNA damage and apoptosis.
doi:10.1590/S1807-59322010001200023
PMCID: PMC3020351  PMID: 21340229
HepG2; Chlorella vulgaris; DNA damage; chemopreventive; apoptosis
16.  Effects of Terminalia arjuna bark extract on apoptosis of human hepatoma cell line HepG2 
AIM: To investigate the effects of Terminalia arjuna (T. arjuna) extract on human hepatoma cell line (HepG2) and its possible role in induction of apoptosis.
METHODS: Human hepatoma cells were treated with different concentrations of ethanolic extract of T. arjuna and its cytotoxicity effect was measured by trypan blue exclusion method and lactate dehydrogenase leakage assay. Apoptosis was analyzed by light and fluorescence microscopic methods, and DNA fragmentation. The mechanism of apoptosis was studied with expression of p53 and caspase-3 proteins. Glutathione (GSH) content was also measured in HepG2 cells after T. arjuna treatment.
RESULTS: T. arjuna inhibited the proliferation of HepG2 cells in a concentration-dependent manner. Apoptotic morphology was observed in HepG2 cells treated with T. arjuna at the concentrations of 60 and 100 mg/L. DNA fragmentation, accumulation of p53 and cleavage of procaspase-3 protein were observed in HepG2 cells after the treatment with T. arjuna. The depletion of GSH was observed in HepG2 cells treated with T. arjuna.
CONCLUSION: T. arjuna induced cytotoxicity in HepG2 cells in vitro. Apoptosis of HepG2 cells may be due to the DNA damage and expression of apoptotic proteins. Depletion of GSH may be involved in the induction of apoptosis of HepG2 cells.
doi:10.3748/wjg.v12.i7.1018
PMCID: PMC4087891  PMID: 16534840
Human hepatoma cell line; Terminalia arjuna; Apoptosis; p53; Caspase-3; GSH
17.  Molecular mechanisms of apoptosis induced by Scorpio water extract in human hepatoma HepG2 cells 
AIM: To clarify the mechanism underlying the anti-mutagenic and anti-cancer activities of Scorpio water extract (SWE).
METHODS: Human hepatoma HepG2 cells were incubated with various concentrations of SWE. After 24-h incubation, cytotoxicity and apoptosis evaluations were determined by MTT and DNA fragmentation assay, respectively. After treatment with SWE, mitochondrial membrane potential (MMP) was determined by measuring the retention of the dye 3,3’-dihexyloxacarbocyanine (DiOC6(3)) and the protein expression including cytochrome C and poly-(ADP-ribose) polymerase (PARP) were measured by Western blotting. Caspase-3 and -9 enzyme activities were measured using specific fluorescence dyes such as Ac-DEVD-AFC and Ac-LEHD-AFC.
RESULTS: We found that treatment with SWE induced apoptosis as confirmed by discontinuous DNA fragmentation in cultured human hepatoma HepG2 cells. Our investigation also showed that SWE-induced apoptosis of HepG2 cells were associated with intracellular events including disruption of MMP, increased translocation of cytochrome C from mitochondria to cytosol, activation of caspase-3, and PARP. Pre-treatment of N-acetyl-Asp-Glu-Val-Asp-CHO (Ac-DEVD-CHO), a caspase-3 specific inhibitor, or cyclosporin A (CsA), an inhibitor of MMP disruption, completely abolished SWE-induced DNA fragmentation.
CONCLUSION: These results suggest that SWE possibly causes mitochondrial damage, leading to cytochrome C release into cytosol and activation of caspases resulting in PARP cleavage and execution of apoptotic cell death in HepG2 cells. These results further suggest that Scorpio may be a valuable agent of therapeutic intervention of human hepatomas.
doi:10.3748/wjg.v11.i7.943
PMCID: PMC4250782  PMID: 15742393
Scorpio; Human hepatoma HepG2 cell; Apoptosis
18.  Exogenous phosphatidylethanolamine induces apoptosis of human hepatoma HepG2 cells via the bcl-2/bax pathway 
AIM: To investigate the signaling pathways implicated in phosphatidylethanolamine (PE)-induced apoptosis of human hepatoma HepG2 cells.
METHODS: Inhibitory effects of PE on human hepatoma HepG2 cells were detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cell cycle, apoptosis and mitochondrial transmembrane potential (ΔΨm) were analyzed by flow cytometry. Immunocytochemical assay and Western blotting were used to examine Bcl-2, Bax and caspase-3 protein levels in HepG2 cells treated with PE.
RESULTS: PE inhibited the growth of HepG2 cells in a dose- and time- dependent manner. It did not affect the cell cycle, but induced apoptosis. PE significantly decreased δΨm at 0.25, 0.5 and 1 mmol/L, respectively, suggesting that PE induces cell apoptosis by decreasing the mitochondrial transmembrane potential. The Bcl-2 expression level induced by different concentrations of PE was lower than that in control groups. However, the Bax expression level induced by PE was higher than that in the control group. Meanwhile, PE increased the caspase-3 expression in a dose- and time-dependent manner.
CONCLUSION: Exogenous PE induces apoptosis of human hepatoma HepG2 cells via the bcl-2/bax pathway.
doi:10.3748/wjg.15.1751
PMCID: PMC2668781  PMID: 19360919
Apoptosis; Bcl-2; Bax; Caspase-3; Phosphatidylethanolamine; Human hepatoma HepG2 cell
19.  MicroRNA-206 overexpression promotes apoptosis, induces cell cycle arrest and inhibits the migration of human hepatocellular carcinoma HepG2 cells 
MicroRNA-206 (miR-206) is known to regulate cell proliferation and migration and is involved in various types of cancer. However, the role of miR-206 in human hepatocellular carcinoma (HHC) has not been previously reported. In the present study, the expression of Notch3 in HCC and adjacent non-neoplastic tissue was immunohistochemically assessed on formalin-fixed, paraffin-embedded sections. miR-206 mimics were transiently transfected into HepG2 cells using Lipofectamine™ 2000. Subsequently, we evaluated the role of miR-206 in cell proliferation, apoptosis, cell cycle arrest and migration by MTS assay, Hoechst 33342 staining, Annexin V-FITC/PI assay, flow cytometry and wound healing assay. Using quantitative reverse transcription polymerase chain reaction (qRT-PCR) and western blot analysis, we detected the expression of Notch3, Bax, Bcl-2, Hes1, p57 and matrix metalloproteinase (MMP)-9 at the mRNA and protein level, respectively. In addition, we measured the expression of miR-206 at the mRNA level and that of caspase-3 at the protein level. After miR-206 was upregulated in HepG2 cells, Notch3, Hes1, Bcl-2 and MMP-9 were downregulated both at the mRNA and protein level, whereas p57 and Bax were upregulated. Cleaved caspase-3 protein expression was also markedly increased. Cell proliferation was significantly attenuated and apoptosis was markedly increased. Furthermore, miR-206 overexpression induced cell cycle arrest and inhibited the migration of HepG2 cells. Taken together, our results uggest that miR-206 is a potential regulator of apoptosis, the cell cycle and migration in HepG2 cells and that it has the potential for use in the targeted therapy of HCC and is a novel tumor suppressor.
doi:10.3892/ijmm.2014.1800
PMCID: PMC4094593  PMID: 24919811
hepatocellular carcinoma; microRNA-206; apoptosis; cell cycle arrest; migration; tumor suppressor
20.  Study of Epigenetic Properties of Poly(HexaMethylene Biguanide) Hydrochloride (PHMB) 
Poly(HexaMethylene Biguanide) hydrochloride (PHMB) CAS No. [32289-58-0] is a particularly effective member of the biguanides antiseptic chemical group, and has been in use since the early fifties in numerous applications. It has been proposed that PHMB be classified as a category 3 carcinogen although PHMB is not genotoxic. It has been hypothesized that PHMB may have epigenetic properties effects, including non-genotoxic modifications of DNA bases, DNA methylation and mitogenic cytokine production. These properties have been assessed in vitro using 3 cell types: Caco-2 cells (from a human colon adenocarcinoma) with a non-functional p53 gene. (∆p53: mut p53), N2-A (Neuro-2A cells, mouse neural cells), the brain being a possible target organ in rodents and HepG2 cells (human hepatocellular carcinoma) with functional p53 gene. From the concentration 1 μg/mL up to 20 μg/mL of PHMB, no effect was observed, either growth stimulation or inhibition. Viability testing using neutral red led to an IC 50 of 20–25 μg/mL after treatment with PHMB for 3 h, whereas the MTT test led to IC50 values of 80 μg/mL, 160 μg/mL and 160 μg/mL respectively for HepG2 cells, Neuro-2A cells and Caco-2 cells. PHMB does not induce significant oxidative stress (production of MDA or lipoperoxidation, nor does it induce hydroxylation of DNA (8-OH-dG) and/or its hypermethylation (m5dC), the latter being strongly implicated in DNA replication and regulation and cell division. PHMB does not induce significant production of mitogenic cytokines such as TNF-α (tumor necrosis factor), interleukins (IL-1 alpha), and the transcription factor nuclear factor kappa B (NF-κB) which can cause either apoptosis or stimulate the growth of transformed cells or tumors. Instead, from concentrations of 20 to 100 μg/mL, PHMB kills cells of all types in less than 3 h. The expression of genes involved in the mechanisms of cell death induced by PHMB, including p53, the pro apoptotic gene bax and others, the anti-apoptotic bcl-2 and caspase-3 has been evaluated by RT-PCR. Finally, the status of GAP-junctions (GJIC) in the presence of PHMB has been determined and appeared to not be significantly affected. Taken together the data show that in vitro PHMB does not exhibit clear and remarkable epigenetic properties except a slight increase of some cytokines and transcription factor at higher concentrations at which cell lysis occurs rapidly.
doi:10.3390/ijerph110808069
PMCID: PMC4143850  PMID: 25111876
Poly(Hexamethylene Biguanide) hydrochloride (PHMB); epigenetic effects; cytokines production
21.  Modulation of apoptosis in human hepatocellular carcinoma (HepG2 cells) by a standardized herbal decoction of Nigella sativa seeds, Hemidesmus indicus roots and Smilax glabra rhizomes with anti- hepatocarcinogenic effects 
Background
A standardized poly-herbal decoction of Nigella sativa seeds, Hemidesmus indicus roots and Smilax glabra rhizomes used traditionally in Sri Lanka for cancer therapy has been demonstrated previously, to have anti-hepatocarcinogenic potential. Cytotoxicity, antioxidant activity, anti-inflammatory activity, and up regulation of p53 and p21 activities are considered to be some of the possible mechanisms through which the above decoction may mediate its anti-hepatocarcinogenic action. The main aim of the present study was to determine whether apoptosis is also a major mechanism by which the decoction mediates its anti-hepatocarcinogenic action.
Methods
Evaluation of apoptosis in HepG2 cells was carried out by (a) microscopic observations of cell morphology, (b) DNA fragmentation analysis, (c) activities of caspase 3 and 9, as well as by (d) analysis of the expression of pro-apoptotic (Bax) and anti-apoptotic (Bcl-2) proteins associated with cell death.
Results
The results demonstrated that in HepG2 cells, the decoction can induce (a) DNA fragmentation and (b) characteristic morphological changes associated with apoptosis (nuclear condensation, membrane blebbing, nuclear fragmentation and apoptotic bodies). The decoction could also, in a time and dose dependent manner, up regulate the expression of the pro-apoptotic gene Bax and down regulate expression of anti-apoptotic Bcl-2 gene (as evident from RT-PCR analysis, immunohistochemistry and western blotting). Further, the decoction significantly (p < .001) enhanced the activities of caspase-3 and caspase-9 in a time and dose dependent manner.
Conclusions
Overall findings provide confirmatory evidence to demonstrate that the decoction may mediate its reported anti-hepatocarcinogenic effect, at least in part, through modulation of apoptosis.
doi:10.1186/1472-6882-12-25
PMCID: PMC3364896  PMID: 22458551
22.  Apoptosis is Induced in Cancer Cells via the Mitochondrial Pathway by the Novel Xylocydine-Derived Compound JRS-15 
The novel compound JRS-15 was obtained through the chemical modification of xylocydine. JRS-15 exhibited much stronger cytotoxic and pro-apoptotic activity than its parent compound in various cancer cell lines, with IC50 values in HeLa, HepG2, SK-HEP-1, PC-3M and A549 cells ranging from 12.42 to 28.25 μM. In addition, it is more potent for killing cancer than non-cancerous cells. Mechanistic studies showed that JRS-15 treatment arrested cell cycle at the G1/S phase, which further triggered the translocation of Bax and Bak to the mitochondria, resulting in mitochondrial membrane potential (MMP) depolarization and the subsequent release of cytochrome c and the second mitochondria-derived activator of caspase (Smac). The sequential activation of caspase-9 and caspase-3/7 and the cleavage of poly (ADP-ribose) polymerase (PARP) were observed following these mitochondrial events. Caspase-8, an initiator caspase that is required to activate the membrane receptor-mediated extrinsic apoptosis pathway was not activated in JRS-15-treated cells. Further analysis showed that the levels of the anti-apoptotic proteins Bcl-xL and XIAP were significantly reduced upon JRS-15 treatment. Furthermore, the caspase-9 inhibitor z-LEHD-fmk, the pan-caspase inhibitor z-VAD-fmk, and Bcl-xL or XIAP overexpression all effectively prevented JRS-15-induced apoptosis. Taken together, these results indicate that JRS-15 induces cancer cell apoptosis by regulating multiple apoptosis-related proteins, and this compound may therefore be a good candidate reagent for anticancer therapy.
doi:10.3390/ijms14010850
PMCID: PMC3565295  PMID: 23344045
apoptosis; Bax; Bak; Bcl-xL; cell cycle; cytochrome c; JRS-15; Smac; XIAP
23.  Hepatocyte-specific deletion of the anti-apoptotic protein Mcl-1 triggers proliferation and hepatocarcinogenesis in mice 
Hepatology (Baltimore, Md.)  2010;51(4):1226-1236.
Regulation of hepatocellular apoptosis is crucial for liver homeostasis. Increased sensitivity of hepatocytes towards apoptosis results in chronic liver injury, whereas apoptosis resistance is linked to hepatocarcinogenesis and non-responsiveness to therapy-induced cell death. Recently, we have demonstrated an essential role of the anti-apoptotic Bcl-2 family member Myeloid cell leukemia-1 (Mcl-1) in hepatocyte survival. In mice lacking Mcl-1 specifically in hepatocytes (Mcl-1Δhep) spontaneous apoptosis caused severe liver damage. Here, we demonstrate that chronically increased apoptosis of hepatocytes coincides with strong hepatocyte proliferation resulting in hepatocellular carcinoma (HCC). Liver cell tumor formation was observed in >50% of Mcl-1Δhep mice already by the age of 8 months, whereas 12 month-old wild-type and heterozygous Mcl-1flox/wt mice lacked tumors. Tumors revealed a heterogenous spectrum ranging from small dysplastic nodules to HCC. The neoplastic nature of the tumors was confirmed by histology, expression of the HCC marker glutamine synthetase and chromosomal aberrations. Liver carcinogenesis in Mcl-1Δhep mice was paralleled by markedly increased levels of survivin, an important regulator of mitosis which is selectively overexpressed in common human cancers.
Conclusion
The present study provides in vivo evidence that increased apoptosis of hepatocytes not only impairs liver homeostasis but is also accompanied by hepatocyte proliferation and hepatocarcinogenesis. Our findings might have implications for understanding apoptosis-related human liver diseases.
The survival of multicellular organisms depends on the maintenance of tissue homeostasis. Under physiological conditions apoptosis contributes to liver homeostasis by removing damaged hepatocytes. Proliferation, growth and programmed hepatocyte cell death are highly coordinated and tightly controlled events in the normal liver (1).
On the one hand, increased apoptosis sensitivity contributes to liver injury. On the other hand, defective apoptosis was demonstrated to lead to excessive hepatocellular survival and has emerged as a major mechanism by which pre-malignant hepatocytes obtain a competitive advantage over normal liver cells (2). Various molecular alterations have been characterized causing an imbalance in the regulation of apoptosis. Among these are alterations in p53 signalling, expression of death receptors, growth factors and mitochondrial integrity (3). Decreased activity of pro-apoptotic signalling as well as increased activity of anti-apoptotic events are associated with HCC development and progression (4).
Among the main cellular changes that trigger apoptosis of hepatocytes is the permeabilization of the outer mitochondrial membrane followed by the release of pro-apoptotic factors (5). The Bcl-2 protein family plays a pivotal role for mitochondrial integrity and the selective interactions between pro- and anti-apoptotic family members regulate mitochondrial activation (6). Bcl-2 family members are similar within the Bcl-2 homology regions (BH1-BH4) and can be divided in pro- and anti-apoptotic Bcl-2 proteins.
Pro-apoptotic Bcl-2 proteins comprise (1) multi-domain members, which lack the BH4 domain (e.g. Bax, Bak), and (2) BH3-only proteins, which lack BH1, 2 and 4 domains (e.g. Bid, Noxa, Puma). BH3-only proteins initiate the mitochondrial signalling cascade by sensing cellular damage (7). After activation, BH3-only proteins are released to neutralise anti-apoptotic Bcl-2 proteins. Subsequently, Bax and Bak trigger mitochondrial membrane leakage and the release of mitochondrial proteins, including cytochrome c, Smac/DIABLO (second mitochondria-derived activator of caspases/direct IAP-binding protein with low pI) and apoptosis-inducing factor (AIF). Smac/DIABLO proteins inactivate the IAP (inhibitors of apoptosis proteins) family, which consists of IAP1/2, BRUCE, NAIP, ILP2, ML-IAP, survivin and XIAP. XIAP is a direct caspase inhibitor. Other IAPs including survivin have several functions apart from caspase inhibition, eg, triggering of ubiquitination processes (8). Anti-apoptotic Bcl-2 family members (eg, Bcl-2, Bcl-xL and Mcl-1), interact with Bax and Bak to inhibit the activation of mitochondria (7).
Both Bcl-xL and Mcl-1 have been identified as major anti-apoptotic Bcl-2 proteins in the liver (9-11). Liver homeostasis is severely disturbed in Mcl-1Δhep mice (10, 11). Spontaneous hepatocyte apoptosis was observed in livers of Mcl-1Δhep mice in profound liver cell damage and increased susceptibility of hepatocytes towards pro-apoptotic stimuli (10). In addition, Mcl-1 has been shown to be highly expressed in a subset of human HCC, contributing to apoptosis resistance of cancer cells (12, 13). Thus, abrogation of the pro-survival function of Mcl-1 (1) either by diminishing its levels or (2) by inactivating its function, have shown promising results with regards to treatment of HCC (12, 13).
In this study, we show that liver-specific depletion of Mcl-1 increases hepatocyte apoptosis, induces hepatocellular proliferation and causes HCC in the absence of overt inflammation.
doi:10.1002/hep.23479
PMCID: PMC2936921  PMID: 20099303
liver; hepatocellular carcinoma; apoptosis; Bcl-2 proteins; survivin
24.  Ethanolic Extract of Agaricus blazei Fermentation Product Inhibits the Growth and Invasion of Human Hepatoma HA22T/VGH and SK-Hep-1 Cells 
Hepatoma is a leading cause of death in the world. SK-Hep-1 and HA22T/VGH cells are poorly differentiated human hepatocellular carcinoma cell lines with invasive and migratory abilities. Agaricus blazei (AB) is a mushroom with many biological effects and active ingredients, and the ethanolic extract of AB fermentation product (AB-pE) was demonstrated to inhibit the growth of hepatoma Hep3B and HepG2 cells in our previous study. In this study, we further investigated the anticancer and anti-invasive abctivities of the AB-pE. Results showed that the AB-pE inhibited the growth of SK-Hep1 and HA22T/VGH cells (with IC50 values of 26.8 and 28.7 μg/mL, respectively) and led cells toward apoptosis after 48 h of treatment. Activation of caspase-3 by AB-pE (12.5~200 μg/mL) in a dose-dependent manner was observed in both cell lines using fluorescence microscopy and flow cytometry. The apoptosis triggered by the AB-pE was regulated by the increased expression of Bax, the activation of caspase-3, caspase-9, and PARP, and the decreased expression of Bcl-2. Additionally, the AB-pE showed the potential ability to inhibit invasion of SK-Hep1 and HA22T/VGH cells according to the results of a Matrigel invasion assay. Our results suggested that the AB-pE may be a further developed for its potential against hepatoma due to its antiproliferative (via apoptosis) and anti-invasive activities in hepatoma cells.
PMCID: PMC3942917  PMID: 24716127
Agaricus blazei; Anti-hepatoma activity; Antitumor invasion activity; SK-Hep-1 cells; HA22T/VGH cells
25.  Ethanol extracts of Cinnamomum kanehirai Hayata leaves induce apoptosis in human hepatoma cell through caspase-3 cascade 
OncoTargets and therapy  2014;8:99-109.
Inducing apoptosis to susceptible cells is the major mechanism of most cytotoxic anticancer drugs in current use. Cinnamomum kanehirai Hayata (Lauraceae), a unique and native tree of Taiwan, is the major host for the medicinal fungus Antrodia cinnamomea which exhibits anti-cancer activity. Because of the scarcity of A. cinnamomea, C. kanehirai Hayata instead, is used as fork medicine in liver cancer. Here we observed the C. kanehirai Hayata ethanol extract could inhibit the cellular viability of both HepG2 and HA22T/VGH human hepatoma cell lines in a dose- and time-dependent manner. We found the mode of cell death was apoptosis according to cell morphological changes by Liu’s stain, oligonucleosomal DNA fragmentation by gel electrophoresis, externalization of phosphotidyl serine by detecting Annexin V and hypoploid population by cell cycle analysis. Our results showed that the extracts caused cleavage of caspase-3 and increased enzyme activity of caspase-8 and caspase-9. Caspase 3 inhibitor partially reversed the viability inhibition by the extract. Furthermore, the up-regulation of Bax and down-regulation of Bcl-2 were also noted by the extract treatment. In conclusion, C. kanehirai Hayata ethanol extract induced intrinsic pathway of apoptosis through caspase-3 cascade in human hepatoma HA22T/VGH and HepG2 cells, which might shed new light on hepatoma therapy.
doi:10.2147/OTT.S68765
PMCID: PMC4317145
Antrodia cinnamomea; hepatoma; apoptosis; anticancer

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