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.
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.
embelin; XIAP; mitochondria; apoptosis; human hepatocellular carcinoma HepG2 cells
Nonsteroidal anti-inflammatory drugs (NSAIDs) are known to induce apoptosis in a variety of cancer cells, including colon, prostate, breast and leukemia. Among them, aspirin, a classical NSAID, shows promise in cancer therapy in certain types of cancers. We hypothesized that aspirin might affect the growth of liver cancer cells since liver is the principal site for aspirin metabolism. Therefore, we investigated the effects of aspirin on the HepG2 human hepatocellular carcinoma cell line in vitro and the HepG2 cell xenograft model in BALB/c nude mice. We found that treatment with aspirin inhibited cell growth and induced apoptosis involving both extrinsic and intrinsic pathways as measured by DNA ladder formation, alteration in the Bax/Bcl-2 ratio, activation of the caspase activities and related protein expressions. In vivo antitumor activity assay also showed that aspirin resulted in significant tumor growth inhibition compared to the control. Oral administration of aspirin (100 mg/kg/day) caused a significant reduction in the growth of HepG2 tumors in nude mice. These findings suggest that aspirin may be used as a promising anticancer agent against liver cancer.
aspirin; hepatocellular carcinoma; apoptosis; xenograft model
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.
Apoptosis; Bcl-2; Bax; Caspase-3; Phosphatidylethanolamine; Human hepatoma HepG2 cell
Dihydromyricetin (DHM) is a major active ingredient of flavonoids compounds. It exhibited anticancer activity and induced apoptosis in human hepatocellular carcinoma HepG2 cells according to our previous data. In this study, we investigated whether p53 is involved in DHM-triggered viability inhibition and apoptosis induction in cancer cells. MTT [3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide] assay was employed to evaluate the viability of HepG2 cells after DHM treatment. Meanwhile, p53 small interfering RNA (siRNA) was adopted to silence p53 expression. Protein level of p53 and Bax/Bcl-2 were evaluated by western blot analysis. Cell counting assay showed that DHM inhibited HepG2 cell growth effectively in a time- and dose-dependent manner. P53 expression was significantly increased after DHM treatment, whereas Bcl-2 was reduced potently. Furthermore, after co-treatment with Pifithrin-α (PFT-α, p53 inhibitor), Bcl-2 expression was reversed. The expression of Bax was no significant change, which was also observed after p53 silence. These findings defined and supported a novel function that DHM could induce human hepatocellular carcinoma HepG2 cells apoptosis by up-regulating Bax/Bcl-2 expression via p53 signal pathway.
The Litchi (Litchi chinensis) fruit products possess rich amounts of flavanoids and proanthocyanidins. Its pericarp has been shown to inhibit breast and liver cancer cell growth. However, the anticolorectal cancer effect of Litchi seed extract has not yet been reported. In this study, the effects of polyphenol-rich Litchi seed ethanol extract (LCSP) on the proliferation, cell cycle, and apoptosis of two colorectal cancer cell lines Colo320DM and SW480 were examined. The results demonstrated that LCSP significantly induced apoptotic cell death in a dose-dependent manner and arrested cell cycle in G2/M in colorectal carcinoma cells. LCSP also suppressed cyclins and elevated the Bax : Bcl-2 ratio and caspase 3 activity. This study provides in vitro evidence that LCSP serves as a potential chemopreventive agent for colorectal cancer.
Efficacy of chemotherapy in advanced stages of colorectal tumours is limited. The quinolone antibiotic ciprofloxacin was recently shown to inhibit growth and to induce apoptosis in human bladder carcinomas cells. We investigated the effect of ciprofloxacin on colon carcinoma lines in vitro. CC-531, SW-403 and HT-29 colon carcinoma and HepG2 hepatoma cells (control cells) were exposed to ciprofloxacin. Proliferation was assessed by bromodeoxyuridine-incorporation into DNA and apoptosis was measured by flow cytometry after propidium iodide or JC-1 staining. Expression of anti-apoptotic Bcl-2 and pro-apoptotic Bax was analyzed by semiquantitative Western blot analysis and activity of caspases 3, 8 and 9 by substrate-cleavage assays. Ciprofloxacin suppressed DNA synthesis of all colon carcinoma cells time- and dose-dependently, whereas the hepatoma cells remained unaffected. Apoptosis reached its maximum between 200 and 500 μg ml−1. This was accompanied by an upregulation of Bax and of the activity of caspases 3, 8 and 9, and paralleled by a decrease of the mitochondrial membrane potential. Ciprofloxacin decreases proliferation and induces apoptosis of colon carcinoma cells, possibly in part by blocking mitochondrial DNA synthesis. Therefore, qualification of ciprofloxacin as adjunctive agent for colorectal cancer should be evaluated.
British Journal of Cancer (2002) 86, 443–448. DOI: 10.1038/sj/bjc/6600079 www.bjcancer.com
© 2002 The Cancer Research Campaign
apoptosis; cell cycle; ciprofloxacin; colorectal cancer; proliferation; caspase
Norcantharidin, the demethylated analog of cantharidin derived from a traditional Chinese medicine, Mylabris, has been used in the treatment of anti-cancer effects. However, the detailed mechanisms underlying this process are generally unclear. The aim of this study was to investigate the mechanism of NCTD-induced apoptosis in HepG2 cells.
The cytotoxicity was measured by MTT assay for cellular viability and by flow cytometry. The mitochondrial membrane potential and reactive oxygen species production was evaluated by flow cytometry analysis. The role of caspase activities were assayed using caspase apoptosis detection kit . Western blot analysis was used to evaluate the level of Cyto-C, Bcl-2, Bax, Bid, caspase 3, -9, -8 and PARP expression
After treatment with NCTD, a decrease in the viability of HepG2 cells and increase in apoptosis were observed. NCTD-induced apoptosis was accompanied by an increase in ROS production, loss of mitochondrial membrane potential and release of cytochrome c(cyto-c) from the mitochondria to the cytosol and down-regulation of anti-apoptotic protein Bcl-2 levels with concurrent up-regulation in pro-apoptotic protein Bax levels. However, another pro-apoptotic molecule, Bid, showed no change in such same treatment. NCTD-increased activity of caspase 9,caspase 3 and the subsequent cleavage caspase substrate PARP were also observed. The expression levels of pro-caspase-8 were not changed after NCTD treatment.
These results indicate that NCTD induced cytotoxicity in HepG2 cells by apoptosis, which is mediated through ROS generation and mitochondrial pathway.
Carvacrol is one of the members of monoterpene phenol and is present in the volatile oils of Thymus vulgaris, Carum copticum, origanum and oregano. It is a safe food additive commonly used in our daily life, and few studies have indicated that carvacrol has anti-hepatocarcinogenic activities. The rationale of the study was to examine whether carvacrol affects apoptosis of human hepatoma HepG2 cells. In this study, we showed that carvacrol inhibited HepG2 cell growth by inducing apoptosis as evidenced by Hoechst 33258 stain and Flow cytometric (FCM) analysis. Incubation of HepG2 cells with carvacrol for 24 h induced apoptosis by the activation of caspase-3, cleavage of PARP and decreased Bcl-2 gene expression. These results demonstrated that a significant fraction of carvacrol treated cells died by an apoptotic pathway in HepG2 cells. Moreover, carvacrol selectively altered the phosphorylation state of members of the MAPK superfamily, decreasing phosphorylation of ERK1/2 significantly in a dose-dependent manner, and activated phosphorylation of p38 but not affecting JNK MAPK phosphorylation. These results suggest that carvacrol may induce apoptosis by direct activation of the mitochondrial pathway, and the mitogen-activated protein kinase pathway may play an important role in the antitumor effect of carvacrol. These results have identified, for the first time, the biological activity of carvacrol in HepG2 cells and should lead to further development of carvacrol for liver disease therapy.
Carvacrol; Hepatocellular carcinoma; Apoptosis; Mitochondrial pathway; Mitogen-activated protein kinase pathway
Benzothiazole derivatives are known for various biological activities, and their potency in cancer therapy has received considerable attention in recent years. YLT322, a novel synthesized benzothiazole derivative, exhibits potent anti-tumor activity via inducing apoptosis both in vitro and in vivo. In this study, we found that YLT322 showed growth inhibition against a broad spectrum of human cancer cells and induced apoptosis of HepG2 cells in a dose- and time-dependent manner. The occurrence of its apoptosis was associated with activation of caspases-3 and -9, but not caspase-8. YLT322 increased the expression of Bax, decreased the expression of Bcl-2, and induced the release of cytochrome c which activates the mitochondrial apoptotic pathway. The down-regulation of phosphorylated p42/44 MAPK and phosphorylated Akt was also observed. Moreover, YLT322 suppressed the growth of established tumors in xenograft models in mice without obvious side effects. Histological and immunohistochemical analyses revealed an increase in TUNEL and caspase-3-positive cells and a decrease in Ki67-positive cells upon YLT322. These results suggest that YLT322 may be a potential candidate for cancer therapy.
As we previously demonstrated in human hepatocellular carcinoma (HepG2) cells, ethanol at low concentration triggers the Fas apoptotic pathway. However, its role in other intracellular signaling pathways remains unknown. Therefore, the aim of the present study was to evaluate the role of low concentration of ethanol on different intracellular signaling pathways. For this purpose, HepG2 cells were treated with 1 mM ethanol for 10 min and the phosphorylation state of protein kinases was determined. In addition, the mRNA levels of transcription factors and genes associated with the Fas apoptotic pathway were determined. Our data demonstrated that ethanol-induced phosphorylation of protein kinases modulates both anti-apoptotic and pro-apoptotic mechanisms in HepG2 cells. Pro-apoptosis resulted mainly from the strong inhibition of the G-protein couple receptor signaling pathway. Moreover, the signal transduction initiated by ethanol-induced protein kinases phosphorylation lead to increased expression of the transcription factors with subsequent expression of genes associated with the Fas apoptotic pathway (Fas receptor, Fas ligand, FADD and caspase 8). These results indicate that low concentration of ethanol exert their effect by predominant activation of pro-apoptotic events that can be divided in two phases. An early phase characterized by a rapid transient effect on protein kinases phosphorylation, after 10 min exposure, with subsequent increased expression of transcription factors for up to 6 hr. This early phase is followed by a second phase associated with increased gene expression that began after 6 hr and persisted for more than 24 hr. This information provided a novel insight into the mechanisms of action of ethanol (1mM) in human hepatocellular carcinoma cells.
Ethanol; HepG2 cells; protein kinases; signal transduction; transcription factors; gene expression
AIM: To investigate whether Melatonin has synergistic effects with Doxorubicin in the growth-inhibition and apoptosis-induction of human hepatoma cell lines HepG2 and Bel-7402.
METHODS: The synergism of Melatonin and Doxorubicin inhibited the cell growth and induced cell apoptosis in human hepatoma cell lines HepG2 and Bel-7402. Cell viability was analyzed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay. Cell apoptosis was evaluated using TUNEL method and flow cytometry. Apoptosis-related protein Bax, Bcl-2 and caspase-3 expressions were measured by immunohistochemical staining.
RESULTS: Treatment with Melatonin (10-8-10-5 mol/L) alone had a dose-related inhibitory effect on cell proliferation but no cytotoxic effect on hepatoma cell lines HepG2 and Bel-7402. Interestingly, when combined with Doxorubicin, Melatonin significantly increased the effects of cell growth inhibition and cell apoptosis. Furthermore, TUNEL staining and flow cytometry revealed that cooperative apoptosis induction was associated with decreased expression of Bcl-2 as well as increased expression of Bax and Caspase3.
CONCLUSION: The synergism of Melatonin and Doxorubicin inhibits hepatoma cell growth and induces cell apoptosis.
Melatonin; Doxorubicin; Human hepatoma cell line; Apoptosis
β-catenin is a multifunctional protein that is involved in cellular structure and the Wnt/β-catenin signaling pathway. Wnt/β-catenin signaling is believed to be an inducer of cell proliferation in different tumors. However, in certain physiological contexts β-catenin also promotes apoptosis. High levels of β-catenin are found in a number of cancer cell types. Recent studies have shown that β-catenin may be correlated with carcinogenesis. Its effects and interaction with interferon (IFN)γ signaling in hepatocellular carcinoma (HCC) cells remains unknown. In the present study, high levels of β-catenin did not induce antiproliferative effects or apoptosis and did not lead to changes in the levels of caspases or activated STATs. However, high levels of β-catenin did cause positive p53 accumulation and Bcl-XL downregulation in HepG2 cells, a HCC cell line. When treated with IFNγ, apoptosis was induced more rapidly compared with cells with low β-catenin levels (P<0.05), whereas caspases 3, 8 and 9 were markedly activated. The caspase inhibitor Z-VAD-FMK and the STAT3 inhibitor blocked this IFNγ-induced apoptosis. Therefore, we report that high levels of β-catenin promote IFNγ-induced apoptosis in HCC in a caspase- and STAT3-dependent manner, and facilitate the activation of executor caspases, possibly via regulation of p53 and Bcl-XL levels. These findings may provide foundations for the development of new IFN-based therapies against liver cancer.
β-catenin; interferon-γ; apoptosis; hepatocellular carcinoma
The role of limonin as potent anti carcinogenic, apoptosis and chemotherapeutic agents has been supported by limited studies.
Materials and Methods:
In this study, limonin is identified as a potent anti proliferative agent against human hepatoma HepG2 cells based on the cell viability study, LDH leakage assay. Induction of apoptosis in HepG2 cells by limonin was evidenced by western blot analysis of Bax, Cyclin D1, Caspase 3 and Caspase9.
Since Wnt signalling is involved in the initiation and sustaining of hepatocellular carcinoma we studied differential expression of LRP5, LRP6 and DKK wnt players.
Limonin found to down regulate these players which forms a rationale for further investigation on effect on limonin in cancer therapy.
Apoptosis; caspases; hepatocellular carcinoma; limonin; Wnt; β-catenin
The effects of the expression of the human Bcl-2 family proteins Bax, Bak, Bcl-2, and Bcl-XL were examined in the fission yeast Schizosaccharomyces pombe and compared with Bax-induced cell death in mammalian cells. Expression of the proapoptotic proteins Bax and Bak conferred a lethal phenotype in this yeast, which was strongly suppressed by coexpression of the anti-apoptotic protein Bcl-XL. Bcl-2 also partially abrogated Bax-mediated cytotoxicity in S. pombe, whereas a mutant of Bcl-2 (Gly145Ala) that fails to heterodimerize with Bax or block apoptosis in mammalian cells was inactive. However, other features distinguished Bax- and Bak-induced death in S. pombe from animal cell apoptosis. Electron microscopic analysis of S. pombe cells dying in response to Bax or Bak expression demonstrated massive cytosolic vacuolization and multifocal nuclear chromatin condensation, thus distinguishing this form of cell death from the classical morphological features of apoptosis seen in animal cells. Unlike Bax-induced apoptosis in 293 cells that led to the induction of interleukin-1 beta-converting enzyme (ICE)/CED-3-like protease activity, Bax- and Bak-induced cell death in S. pombe was accompanied neither by internucleosomal DNA fragmentation nor by activation of proteases with specificities similar to the ICE/CED-3 family. In addition, the baculovirus protease inhibitor p35, which is a potent inhibitor of ICE/CED-3 family proteases and a blocker of apoptosis in animal cells, failed to prevent cell death induction by Bax or Bak in fission yeast, whereas p35 inhibited Bax-induced cell death in mammalian cells. Taken together, these findings suggest that Bcl-2 family proteins may retain an evolutionarily conserved ability to regulate cell survival and death but also indicate differences in the downstream events that are activated by overexpression of Bax or Bak in divergent cell types.
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.
Liver neoplasm; Chrysin; 5-allyl-7-gen-difluoromethylenechrysin; Apoptosis; Proxisome proliferator-activated receptor γ
The present study aimed to examine the antiproliferative potentiality of an extract derived from the medicinal plant ginger (Zingiber officinale) on growth of breast cancer cells. Ginger treatment suppressed the proliferation and colony formation in breast cancer cell lines, MCF-7 and MDA-MB-231. Meanwhile, it did not significantly affect viability of nontumorigenic normal mammary epithelial cell line (MCF-10A). Treatment of MCF-7 and MDA-MB-231 with ginger resulted in sequences of events marked by apoptosis, accompanied by loss of cell viability, chromatin condensation, DNA fragmentation, activation of caspase 3, and cleavage of poly(ADP-ribose) polymerase. At the molecular level, the apoptotic cell death mediated by ginger could be attributed in part to upregulation of Bax and downregulation of Bcl-2 proteins. Ginger treatment downregulated expression of prosurvival genes, such as NF-κB, Bcl-X, Mcl-1, and Survivin, and cell cycle-regulating proteins, including cyclin D1 and cyclin-dependent kinase-4 (CDK-4). On the other hand, it increased expression of CDK inhibitor, p21. It also inhibited the expression of the two prominent molecular targets of cancer, c-Myc and the human telomerase reverse transcriptase (hTERT). These findings suggested that the ginger may be a promising candidate for the treatment of breast carcinomas.
Interleukin-1β (IL-1β) is a pivotal proinflammatory cytokine. To investigate the mechanism of IL-1β-induced cell death in human malignant melanoma A375-S2 cells, MTT assay, photomicroscopical observation, DNA agarose gel electrophoresis, radioimmunoassay and Western blot analysis were carried out. IL-1β did not only induce nuclear condensation and DNA fragmentation, but also increased degradation of two substrates of caspase-3, poly ADP-ribose polymerase (PARP) and inhibitor of caspase-activated DNase (ICAD). Simultaneously, release of precursor of IL-1β (pro-IL-1β) and endogenous IL-1β production were involved in the apoptotic process. IL-1β enhanced the ratio of Bax/Bcl-2 and Bax/Bcl-xL expression and up-regulated apoptosis inducing factor (AIF) expression, which required the activation of downstream caspases. These results suggest that IL-1β induces endogenous IL-1β production, enhances cleavage of caspase downstream substrates and promotes mitochondria mediated apoptosis in A375-S2 cells.
Interleukin-1; Human melanoma Cells; Apoptosis; Caspases; Mitochondria
Synthetic retinoid fenretinide is one of the most promising clinically tested retinoids. Previously, we have shown that fenretinide induces apoptosis of Huh7 cells, but HepG2 cells are relatively resistant to fenretinide-induced apoptosis. The current study examines the interactive role of fenretinide and histone deacetylase inhibitors (HDACi) in inducing apoptosis of human hepatocellular carcinoma (HCC) cells and the underlying mechanism. Trichostatin A (TSA) and scriptaid can either enhance fenretinide-induced apoptosis in the fenretinide sensitive HCC cells (Huh7 and Hep3B) or sensitize the fenretinide resistant cells (HepG2) to become sensitive to the apoptotic effect of fenretinide in a cancer-cell specific manner. The sensitivity of cells to fenretinide-induced apoptosis was neither associated with ROS production nor anti-oxidant gene expression. However, the level of RARβ and Nur77 was important for inducing apoptosis. Upon fenretinide and HDACi treatment, the expression of RARβ and Nur77 were induced and co-localized in the cytosol. The induction of Nur77 protein level, but not the mRNA level, was RARβ-dependent. In addition, RARβ interacted with Nur77. Nur77 was essential for fenretinide- and HDACi-induced apoptosis of Huh7 cells. Induction of the expression, the interaction, and the nuclear export of RARβ and Nur77 mediate fenretinide and HDACi induced apoptosis. Our findings suggest that targeting Nur77 and RARβ simultaneously provides an effective way to induce HCC cell death.
translocation; nuclear receptor; retinoid; TSA; scriptaid
In the present study we found that double-stranded RNA-dependent protein kinase (PKR) regulates the protein expression level and the phosphorylation of Bcl-2 and exploits an anti-apoptotic role in human hepatocellular carcinoma cells (HepG2). Saturated free fatty acids (FFAs), e.g. palmitate, have been shown to induce cellular apoptosis in various types of cells by different mechanisms. We found palmitate down-regulates the activity of PKR, and thereby decreases the protein level of Bcl-2, mediated, in part, by the NF-κB transcription factor. In addition to the protein level of Bcl-2, the phosphorylation of Bcl-2 at different amino acid residues, such as Ser70 and Ser87, is also important in regulating cellular apoptosis. The decrease in the phosphorylation of Bcl-2 at Ser70 upon exposure to palmitate is mediated by PKR and possibly JNK, while the phosphorylation of Bcl-2 at Ser87 is not affected by palmitate or PKR. In summary, PKR mediates the regulation of the protein level and the phosphorylation status of Bcl-2, providing a novel mechanism of palmitate-induced apoptosis in HepG2 cells.
palmitate; apoptosis; liver cell; HepG2; PKR; Bcl-2; phosphorylation of Bcl-2; NF-kappaB; JNK
Silica nanoparticles have been discovered to exert cytotoxicity and induce apoptosis in normal human cells. However, until now, few studies have investigated the cytotoxicity of silica nanoparticles in tumor cells.
This study investigated the cytotoxicity of 7–50 nm silica nanoparticles in human HepG2 hepatoma cells, using normal human L-02 hepatocytes as a control. Cell nucleus morphology changes, cellular uptake, and expression of procaspase-9, p53, Bcl-2, and Bax, as well as the activity of caspase-3, and intracellular reactive oxygen species and glutathione levels in the silica nanoparticle-treated cells, were analyzed.
The antitumor activity of the silica nanoparticles was closely related to particle size, and the antiproliferation activity decreased in the order of 20 nm > 7 nm > 50 nm. The silica nanoparticles were also cytotoxic in a dose- and time-dependent manner. However, the silica nanoparticles showed only slight toxicity in the L-02 control cells, Moreover, in HepG2 cells, oxidative stress and apoptosis were induced after exposure to 7–20 nm silica nanoparticles. Expression of p53 and caspase-3 increased, and expression of Bcl-2 and procaspase-9 decreased in a dose-dependent manner, whereas the expression of Bax was not significantly changed.
A mitochondrial-dependent pathway triggered by oxidative stress mediated by reactive oxygen species may be involved in apoptosis induced by silica nanoparticles, and hence cytotoxicity in human HepG2 hepatic cancer cells.
silica nanoparticles; cytotoxicity; apoptosis; HepG2 cells; mitochondrial-dependent pathway; oxidative stress
Thymoquinone (TQ) and diosgenin (DG), the active ingredients obtained from black cumin (Nigella sativa) and fenugreek (Trigonella foenum graecum), respectively, exert potent bioactivity, including anticancer effects. This study investigated the antineoplastic activity of these agents against squamous cell carcinoma in vitro and sarcoma 180–induced tumors in vivo. TQ and DG inhibited cell proliferation and induced cytotoxicity in A431 and Hep2 cells. These agents induced apoptosis by increasing the sub-G1 population, LIVE/DEAD cytotoxicity, chromatin condensation, DNA laddering and TUNEL-positive cells significantly (P<0.05). Increased Bax/Bcl-2 ratio, activation of caspases and cleavage of poly ADP ribose polymerase were observed in treated cells. These drugs inhibited Akt and JNK phosphorylations, thus inhibiting cell proliferation while inducing apoptosis. In combination, TQ and DG had synergistic effects, resulting in cell viability as low as 10%. In a mouse xenograft model, a combination of TQ and DG significantly (P<0.05) reduced tumor volume, mass and increased apoptosis. TQ and DG, alone and in combination, inhibit cell proliferation and induce apoptosis in squamous cell carcinoma. The combination of TQ and DG is a potential antineoplastic therapy in this common skin cancer.
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising candidate for the treatment of cancer, because it preferentially induces apoptosis in numerous cancer cells with little or no effect on normal cells. 5,7-Dihydroxyflavone is a dietary flavonoid commonly found in many plants. Here we show that the combined treatment with 5,7-dihydroxyflavone and TRAIL at subtoxic concentrations induced strong apoptotic response in human hepatocarcinoma HepG2 cells, acute leukemia Jurkat T cells, and cervical carcinoma HeLa cells. We further investigated the mechanisms by which 5,7-dihydroxyflavone augments TRAIL-induced apoptosis in HepG2 cells. 5,7-Dihydroxyflavone up-regulated the expression of pro-apoptotic protein Bax, attenuated the expression of anti-apoptotic proteins Bcl-2, Mcl-1, and IAPs, and reduced the phosphorylation levels of Akt and STAT3, weakening the anti-apoptotic signals thus facilitating the process of apoptosis. Moreover, 5,7-dihydroxyflavone and TRAIL were well tolerated in mice, and the combination of 5,7-dihydroxyflavone and TRAIL reduced tumor burden in vivo in a HepG2 tumor xenograft model. Interestingly, 5,7-dihydroxyflavone-mediated sensitization to TRAIL-induced cell death was not observed in normal human hepatocytes L-O2. These results suggest that the 5,7-dihydroxyflavone in combination with TRAIL might be used for cancer prevention and/or therapy.
Hepatocellular carcinoma (HCC) is receiving increased attention. This study was designed to investigate the effect of selective Cyclooxygenase-2 (COX-2) inhibitor, nimesulide, on the expression of Smad4 in human hepatocellular carcinoma HepG2.
Materials and Methods:
HepG2 cells were incubated in various concentrations of nimesulide (25, 50, 100, 200, 400 μmol/L) to detect the effect of proliferation by MTS. The apoptosis of HepG2 was determined by TUNEL; fluorescence microscope was used to observe the expression of Smad4.
The result showed that nimesulide inhibited the proliferation of HepG2 cell in a concentrations-dependent manner, and promoted the karyopyknosis and fragmentation of HepG2 cell nucleus, induced its apoptosis, the number of fluorescence labeling of Smad4 in Nimesulide group was higher than control group (P<0.05).
Nimesulide inhibits the proliferation and promotes apoptosis of HepG2 by up-regulation of Smad4 in HepG2.
Hepatic carcinoma cell; Nimesulide; Smad4
Melatonin induces apoptosis in many different cancer cell lines, including hepatocellular carcinoma cells. However, the responsible pathways have not been clearly elucidated. A member of the forkhead transcription factors' family, FoxO3a, has been implicated in the expression of the proapoptotic protein Bim (a Bcl-2-interacting mediator of cell death). In this study, we used human HepG2 liver cancer cells as an in vitro model to investigate whether melatonin treatment induces Bim through regulation by the transcription factor FoxO3a.
Cytotoxicity of melatonin was compared in HepG2 hepatoblastoma cells and primary human hepatocytes. Proapoptotic Bim expression was analysed by reverse transcriptase–polymerase chain reaction and western blot. Reporter gene assays and chromatin immunoprecipitation assays were performed to analyse whether FoxO3a transactivates the Bim promoter. Small interfering RNA (siRNA) was used to study the role of FoxO3a in Bim expression. Immunofluorescence was performed to analyse FoxO3a localisation in HepG2 cells.
Melatonin treatment induces apoptosis in HepG2 cells, but not in primary human hepatocytes. The proapoptotic effect was mediated by increased expression of the BH3-only protein Bim. During melatonin treatment, we observed increased transcriptional activity of the forkhead-responsive element and could demonstrate that FoxO3a binds to a specific sequence within the Bim promoter. Furthermore, melatonin reduced phosphorylation of FoxO3a at Thr32 and Ser253, and induced its increased nuclear localisation. Moreover, silencing experiments with FoxO3a siRNA prevented Bim upregulation.
This study shows that melatonin can induce apoptosis in HepG2 hepatocarcinoma cells through the upregulation of proapoptotic Bim mediated by nuclear translocation and activation of the transcription factor FoxO3a.
FoxO3a; Bim; hepatocellular carcinoma; melatonin