Green tea constituent (−)-Epigallocatechin-3-gallate inhibits hep G2 cell proliferation and induces apoptosis through p53-dependent and fas-mediated pathways

(-)-Epigallocatechin-3-gallate (EGCG) is a polyphenolic compound found in green tea. It has been reported to possess a wide range of pharmacological properties, and is one of the most promising chemopreventive agents for cancer. To provide a better understanding of the preventive effect of EGCG on liver cancer, we examined EGCG for its effect on proliferation and cell cycle progression in a human liver cancer cell line, Hep G2. The results showed that EGCG inhibited the proliferation of Hep G2 by inducing apoptosis and blocking cell cycle progression in the G1 phase. ELISA showed that EGCG significantly increased the expression of p53 and p21/WAF1 protein, and this contributed to cell cycle arrest. An enhancement in Fas/APO-1 and its two form ligands, membrane-bound Fas ligand (mFasL) and soluble Fas ligand (sFasL), as well as Bax protein, was responsible for the apoptotic effect induced by EGCG. Taken together, our study suggests that the induction of p53 and the activity of the Fas/FasL apoptotic system play major roles in the antiproliferative activity of EGCG in Hep G2 cells.     

The antiproliferative activity of aloe-emodin is through p53-dependent and p21-dependent apoptotic pathway in human hepatoma cell lines

The aim of this study is to investigate the anticancer effect of aloe-emodin in two human liver cancer cell lines, Hep G2 and Hep 3B. We observed that aloe-emodin inhibited cell proliferation and induced apoptosis in both examined cell lines, but with different the antiproliferative mechanisms. In Hep G2 cells, aloe-emodin induced p53 expression and was accompanied by induction of p21 expression that was associated with a cell cycle arrest in G1 phase. In addition, aloe-emodin had a marked increase in Fas/APO1 receptor and Bax expression. In contrast, with p53-deficient Hep 3B cells, the inhibition of cell proliferation of aloe-emodin was mediated through a p21-dependent manner that did not cause cell cycle arrest or increase the level of Fas/APO1 receptor, but rather promoted aloe-emodin induced apoptosis by enhancing expression of Bax. These findings suggest that aloe-emodin may be useful in liver cancer prevention.     

Resveratrol- induced apoptosis is mediated by p53-dependent pathway in Hep G2 cells

Resveratrol, a phytoalexin found in many plants, has been reported to possess a wide range of pharmacological properties and is one of the promising chemopreventive agents for cancer. Here, we examined the antiproliferation effect of resveratrol in two human liver cancer cell lines, Hep G2 and Hep 3B. Our results showed that resveratrol inhibited cell growth in p53-positive Hep G2 cells only. This anticancer effect was a result of cellular apoptotic death induced by resveratrol via the p53-dependent pathway. Here we demonstrated that the resveratrol-treated cells were arrested in G1 phase and were associated with the increase of p21 expression. In addition, we also illustrated that the resveratrol-treated cells had enhanced Bax expression but they were not involved in Fas/APO-1 apoptotic signal pathway. In contrast, the p53-negative Hep 3B cells treated with resveratrol did not show the antiproliferation effect neither did they show significant changes in p21 nor Fas/APO-1 levels. In summary, our study demonstrated that the resveratrol effectively inhibited cell growth and induced programmed cell death in Hepatoma cells on a molecular basis. Furthermore, these results implied that resveratrol might also be a new potent chemopreventive drug candidate for liver cancer as it played an important role to trigger p53-mediated molecules involved in the mechanism of p53-dependent apoptotic signal pathway.     

The proliferative inhibition and apoptotic mechanism of Saikosaponin D in human non-small cell lung cancer A549 cells

Saikosaponin D is a saponin extract derived from several species of Bupleurum (Umbelliferae), which is used for the treatment of various liver diseases in traditional Chinese medicine. In this study, we report that Saikosaponin D inhibits the cell growth of human lung cancer cell line A549 and provide a molecular understanding of this effect. The results showed that Saikosaponin D inhibited the proliferation of A549 by inducing apoptosis and blocking cell cycle progression in the G1 phase. ELISA assay showed that Saikosaponin D significantly increased the expression of p53 and p21/WAF1 protein, contributing to cell cycle arrest. An enhancement in Fas/APO-1 and its two form ligands, membrane-bound Fas ligand (mFasL) and soluble Fas ligand (sFasL), as well as Bax protein, was responsible for the apoptotic effect induced by Saikosaponin D. Taken together, our study suggests that the induction of p53 and activity of the Fas/FasL apoptotic system may participate in the antiproliferative activity of Saikosaponin D in A549 cells.     

Tetrazolium violet inhibits cell growth and induces cell death in C127 mouse breast tumor cells

Tetrazolium violet (TV), a tetrazolium salt, has been applied in several fields, including estimating respiration rate, as a redox indicator of microbial growth, and for estimating the number of viable animal cells. It has recently been found that TV is capable of inducing apoptosis in rat glioblastoma cells by way of an elusive mechanism. In this study, we demonstrated that TV also induced apoptosis in mouse breast tumor C127 cells as evidenced by nucleus condensation and nucleus fragmentation. Our data showed that TV caused activation of caspase-3 and caspase-8, but not caspase-9. An enhancement in Fas and its two ligands, membrane-bound Fas ligand (mFasL) and soluble Fas ligand (sFasL), might be responsible for the apoptotic effect induced by TV. Also, the results first reported that TV not only inhibited C127 cells proliferation but also blocked cell cycle progression in the G1 and G2 phase, determined by MTT assay and flow cytometry analysis. Immunofluorescence assay demonstrated that TV significantly increased the expression of p53 protein, which caused cell cycle arrest. Taken together, p53, Fas/FasL, and the caspase apoptotic system may participate in the antiproliferative activity of TV in C127 cells.     

Anti-Fas antibody-induced apoptosis in human colorectal carcinoma cell lines: role of the p53 gene

The cell surface Fas antigen transducts an apoptotic signal by its crosslinking with Fas ligand or anti-Fas antibody in a variety of human cultured cells. In this study, we examined the expression of Fas antigen and its mediation of apoptosis in six human colorectal carcinoma cell lines. A flow cytometric analysis revealed that LoVo, DLD-1, WiDr and SW837 cell lines showed higher expression levels of Fas antigen, in contrast to lower expression in COLO201 and COLO320DM. Interferon- enhanced the expression of Fas antigen in all of the cell lines examined. Both Fas ligand and Fas-associated phosphatase-1 (FAP-1) were expressed only in COLO320DM. Anti-Fas antibody induced apoptosis in LoVo carrying wild-type p53 gene, but not in the other five cell lines carrying mutated p53 gene. The transfection of wild-type p53 gene using an adenovirous vector upregulated P53 protein in WiDr and SW837 cells, both of which showed, however, no increase in apoptotic cells by anti-Fas antibody treatment. These results indicated that (1) Fas antigen was variably expressed, regardless of the p53 gene status and (2) the susceptibility to anti-Fas antibody-mediated apoptosis did not correlate to Fas, Fas ligand or FAP-1 expression levels. Therefore, we conclude that wild-type P53 expression might not necessarily be essential for Fas-mediated apoptosis in human colorectal carcinoma cell lines.     

Induction of apoptosis in p53-deficient human hepatoma cell line by wild-type p53 gene transduction: inhibition by antioxidant

We investigated the role of wild-type (wt)-p53 as an inducer of apoptotic cell death in human hepatoma cell lines. Following the retrovirus-mediated transduction of the wt-p53 gene, Hep3B cells lacking the endogenous p53 expression began to die through apoptosis in 4 h. They showed a maximal apoptotic death at 12 h, whereas HepG2 cells expressing endogenous p53 did not. However, the transduction of the wt-p53 gene elicited growth suppression of both Hep3B and HepG2 cells. P21(WAF1/CIP1), a p53-inducible cell cycle inhibitor, was induced, not only in Hep3B cells undergoing apoptosis, but also in HepG2 cells. The kinetics of the p21(WAF1/CIP1) induction, DNA fragmentation, and growth suppression of the Hep3B cells showed that DNA fragmentation and growth suppression progressed rapidly following p21(WAF1/CIP1) accumulation. N-acetyl-cysteine or glutathione, potent antioxidants, strongly inhibited the DNA fragmentation, but did not reduce the elevated level of p21(WAF1/CIP1). These findings suggested that p21(WAF1/CIP1) was not a critical mediator for the execution of p53-mediated apoptosis, although it contributed to the growth inhibition of cells undergoing apoptosis. Furthermore, p53-mediated apoptosis could be repressed by antioxidants.     

Mechanism of Arctigenin-Induced Specific Cytotoxicity against Human Hepatocellular Carcinoma Cell Lines: Hep G2 and SMMC7721

Arctigenin (ARG) has been previously reported to exert high biological activities including anti-inflammatory, antiviral and anticancer. In this study, the anti-tumor mechanism of ARG towards human hepatocellular carcinoma (HCC) was firstly investigated. We demonstrated that ARG could induce apoptosis in Hep G2 and SMMC7721 cells but not in normal hepatic cells, and its apoptotic effect on Hep G2 was stronger than that on SMMC7721. Furthermore, the following study showed that ARG treatment led to a loss in the mitochondrial out membrane potential, up-regulation of Bax, down-regulation of Bcl-2, a release of cytochrome c, caspase-9 and caspase-3 activation and a cleavage of poly (ADP-ribose) polymerase in both Hep G2 and SMMC7721 cells, suggesting ARG-induced apoptosis was associated with the mitochondria mediated pathway. Moreover, the activation of caspase-8 and the increased expression levels of Fas/FasL and TNF-α revealed that the Fas/FasL-related pathway was also involved in this process. Additionally, ARG induced apoptosis was accompanied by a deactivation of PI3K/p-Akt pathway, an accumulation of p53 protein and an inhibition of NF-κB nuclear translocation especially in Hep G2 cells, which might be the reason that Hep G2 was more sensitive than SMMC7721 cells to ARG treatment.     

rBTI通过上调PTEN的表达抑制Hep G2细胞增殖

磷酸酶及张力蛋白的同源基因(PTEN) 是一种抑癌基因,可以调控细胞的增殖,与癌症的发生和发展息息相关。本研究采用MTT法和流式细胞术分别检测了重组荞麦胰蛋白酶抑制剂(rBTI)对人肝癌细胞株Hep G2细胞的增殖以及周期的影响。免疫荧光及Western印迹法检测了PTEN和p PTEN的亚细胞定位及蛋白表达的变化。采用qRT-PCR及Western印迹法检测了周期相关蛋白的表达。旨在探究PTEN和p PTEN在rBTI抑制Hep G2细胞增殖和周期阻滞中的作用。结果表明,rBTI能显著抑制Hep G2细胞增殖,将细胞周期阻滞在G0/G1期,并呈时间和剂量依赖性;rBTI作用于Hep G2后,可显著上调PTEN和p-PTEN的表达。同时发现,p-PTEN主要分布于细胞核中,能与核仁发生共定位;周期相关蛋白检测表明,细胞内p53、p21转录水平和蛋白水平均增加。综上所述,rBTI通过上调PTEN的表达,使得细胞周期阻滞于G0/G1期,进而抑制Hep G2细胞的增殖。     

Targeted p21(WAF1/CIP1) Activation by RNAa Inhibits Hepatocellular Carcinoma Cells

RNA activation (RNAa) is a mechanism of gene activation triggered by promoter-targeted small double-stranded RNA (dsRNA), also known as small activating RNA (saRNA). p21(WAF1/CIP1) (p21) is a putative tumor suppressor gene due to its role as a key negative regulator of the cell cycle and cell proliferation. It is frequently downregulated in cancer including hepatocellular carcinoma (HCC), but is rarely mutated or deleted, making it an ideal target for RNAa-based overexpression to restore its tumor suppressor function. In the present study, we investigated the antigrowth effects of p21 RNAa in HCC cells. Transfection of a p21 saRNA (dsP21-322) into HepG2 and Hep3B cells significantly induced the expression of p21 at both the mRNA and protein levels, and inhibited cell proliferation and survival. Further analysis of dsP21-322 transfected cells revealed that dsP21-322 arrested the cell cycle at the G(0)/G(1) phase in HepG2 cells but at G(2)/M phase in Hep3B cells which lack functional p53 and Rb genes, and induced both early and late stage apoptosis by activating caspase 3 in both cell lines. These results demonstrated that RNAa of p21 has in vitro antigrowth effects on HCC cells via impeding cell cycle progression and inducing apoptotic cell death. This study suggests that targeted activation of p21 by RNAa may be explored as a novel therapy for the treatment of HCC.     

Fas is involved in the p53-dependent apoptotic response to ionizing radiation in mouse testis

Apoptosis induced in male germ cells following ionizing radiation is dependent on functional p53 (Trp53) being present. We sought to determine whether Fas (Tnfrsf6/CD95/APO-1), an apoptotic factor, is involved in this p53-dependent germ cell death. In p53 knock-out mice exposed to 5 Gy of x-radiation, germ cells were protected from cell death, as assessed by counting apoptotic seminiferous tubules 12 h following radiation. Similarly, spermatid head counts in p53 knock-out mice remained near normal 29 days after exposure to 0.5 Gy of radiation, whereas wild-type animals had a more than twofold reduction in spermatid head counts. Fas mRNA expression remained at pretreatment levels in p53 knock-out mice; however, Fas increased in a time-dependent manner in wild-type mice following exposure to 5 Gy of radiation, indicating that radiation-induced Fas expression is p53-dependent. The functional significance of Fas involvement was demonstrated when lpr(cg) mice, having a nonfunctional Fas receptor, were exposed to 5 Gy of radiation; the number of apoptotic seminiferous tubules 12 h following radiation was significantly reduced compared to that of wild-type mice. Additionally, lpr(cg) mice exposed to 0.5 Gy of radiation had increased spermatid head counts 29 days following radiation compared to wild-type mice. Interestingly, gld mice with a non-functional Fas ligand (Tnfsf6/FasL/CD95L) were as sensitive to radiation as wild-type animals, and levels of FasL mRNA were not affected by radiation treatment. These results indicate that apoptosis and up-regulation of Fas following radiation are both p53-dependent events. Although Fas is necessary, in part, for radiation-induced p53-dependent apoptosis, FasL is not.     

Induction of Fas-mediated extrinsic apoptosis, p21WAF1-related G2/M cell cycle arrest and ROS generation by costunolide in estrogen receptor-negative breast cancer cells, MDA-MB-231

Costunolide (C(15)H(20)O(2)) is a sesquiterpene lactone that was isolated from many herbal medicines and it has diverse effects according to previous reports. However, the anti-cancer effects and the mechanism of actions are still unknown in breast cancer. In this study, we first observed that costunolide inhibits cell growth in a dose-and time-dependent manner. To examine the mechanism by which costunolide inhibits cell growth, we checked the effect of costunolide on apoptosis and the cell cycle. Costunolide induced apoptosis through the extrinsic pathway, including the activation of Fas, caspase-8, caspase-3, and degradation of PARP. However, did not have the same effect on the intrinsic pathway as revealed by analysis of mitochondrial membrane potential (Δψm) with JC-1 dye and expression of Bcl2 and Bax proteins level. Furthermore, costunolide induced cell cycle arrest in the G2/M phase via decrease in Cdc2, cyclin B1 and increase in p21WAF1 expression, independent of p53 pathway in p53-mutant MDA-MB-231 cells and increases Cdc2-p21WAF1 binding. In addition, costunolide had a slight induced effect on ROS generation. Among the mechanisms of p21WAF1 induction examined, costunolide-induced increase in p21WAF1 expression was related with protein stability and ROS generation. Through this study we confirm that costunolide induces G2/M cell cycle arrest and apoptotic cell death via extrinsic pathway in MDA-MB-231 cells suggesting that it could be a promising anticancer drug especially for ER-negative breast cancer.     

Cell death induced by the Fas/Fas ligand pathway and its role in pathology.

Engagement of the cell death surface receptor Fas by Fas ligand (FasL) results in apoptotic cell death, mediated by caspase activation. Cell death mediated via Fas/FasL interaction is important for homeostasis of cells in the immune system and for maintaining immune-privileged sites in the body. Killing via the Fas/FasL pathway also constitutes an important pathway of killing for cytotoxic T cells. Fas ligand is induced in activated T cells, resulting in activation-induced cell death by the Fas/FasL pathway. Recently it has been shown that the Fas receptor can also be up-regulated following a lesion to the cell, particularly that induced by DNA-damaging agents. This can then result in killing of the cell by a Fas/FasL-dependent pathway. Up-regulation of Fas receptor following DNA damage appears to be p53 dependent.     

p53 Retards cell-growth and suppresses etoposide-induced apoptosis in Pin1-deficient mouse embryonic fibroblasts

We studied the effects of Pin1, a regulatory molecule of the oncosuppressor p53, on both cell cycle arrest and apoptosis by treating primary mouse embryonic fibroblasts (MEFs) with etoposide. Etoposide induced G1 arrest in both wild-type and Pin1 null (pin1(-/-)) MEFs, and G2/M arrest and apoptotic cell death in MEFs lacking either p53 only (p53(-/-)) or both Pin1 and p53 (pin1(-/-)p53(-/-)). Both pin1(-/-) and pin1(-/-)p53(-/-) MEFs were enhanced the release of cytochrome c from the mitochondria, which might induce apoptosis. In response to etoposide treatment, apoptotic cell death was displayed in pin1(-/-)p53(-/-) MEFs but not in pin1(-/-) MEFs. These results suggest that p53 retards growth and suppresses etoposide-induced apoptosis in pin1(-/-) MEFs.     

Emodin-induced apoptosis through p53-dependent pathway in human hepatoma cells

Most of the commonly used cytotoxic anticancer drugs have been shown to induce apoptosis in susceptible cells. However, the signaling pathway of their apoptotic effects remains undefined. In this study, the cytotoxic effect of emodin on various human hepatoma cell lines was investigated. Results demonstrated that emodin exhibited strongly suppressing effect on HepG2/C3A, PLC/PRF/5, and SK-HEP-1 cells, with the IC(50) value of 42.5, 46.6, and 53.1 microM, respectively. Furthermore, emodin induced apoptosis in HepG2/C3A cells was clearly verified by the appearance of DNA fragmentation and sub-G(1) accumulation. Besides, HepG2/C3A cells were found to be arrested in G(2)/M phase after the cells were treated with 60 microM emodin for 48 h. Moreover, significant increase in the levels of apoptosis-related signals such as p53 (419.3 pg/ml), p21 (437.4 units/ml), Fas (6.6 units/ml), and caspase-3 (35.4 pmol/min) were observed in emodin treated HepG2/C3A cells. Taken together, emodin displays effective inhibitory effects on the growth of various human hepatoma cell lines and stimulates the expression of p53 and p21 that resulted in the cell cycle arrest of HepG2/C3A cells at G(2)/M phase. Results also suggest that emodin-induced apoptosis in HepG2/C3A cells were mediated through the activation of p53, p21, Fas/APO-1, and caspase-3. It implies that emodin could be a useful chemotherapeutical agent for treatment of hepatocellular carcinoma (HCC).     

Different levels of p53 induced either apoptosis or cell cycle arrest in a doxycycline-regulated hepatocellular carcinoma cell line <Emphasis Type="Italic">in vitro</Emphasis>

Induction of p53 gene expression in cancer cells can lead to both cell cycle arrest and apoptosis. To clarify whether the level of p53 expression determines the apoptotic response of hepatocellullar carcinoma (HCC) cells, we assessed the effect of various levels of expression of p53 gene on a p53-deficient HCC cell line, Hep3B, utilizing a doxycycline (Dox)-regulated inducible p53 expression system. Our results showed that apoptosis was induced in HCC cells with high levels of p53 expression. However, lower level of p53 expression induced only cell cycle arrest but not apoptosis. Bax expression was up-regulated following high levels of p53 expression, while bcl-2 expression was not altered by the level of p53 expression. Moreover, p21 expression was observed in both high and low expression of p53. These results suggest the level of p53 expression could determine if the HCC cells would go into cell cycle arrest or apoptosis. Bax may participate, at least in part, in inducing p53-dependent apoptosis and the induction of p21 alone was able to cause cell cycle arrest but not apoptosis.     

Different cell cycle mechanisms between UV-induced and X-ray-induced apoptosis in WiDr colorectal carcinoma cells

Although DNA-damaging agents such as ultraviolet (UV) and X-ray can induce apoptosis, the difference in the apoptotic mechanism is not clearly understood. In the present study, we investigated the effects of these two genotoxic agents on the induction of DNA damage and subsequent apoptotic cell death from the viewpoint of cell cycle regulation by using WiDr cells. Transient G1 arrest was observed after UV exposure, whereas G2 but not G1 arrest was induced after X-ray irradiation. UV-exposure could induce G1 arrest in both mutant-type (mt-p53) and wild-type p53 (wt-p53) cells, but obvious G1 arrest was not observed in the cells lacking in p53 expression. An increase in the DNA fragmentation was observed at S phase in UV-irradiated cells and at G2 phase in X-irradiated cells, respectively. UV-irradiated cells showed an increase production of p53 protein and accumulation of p21 protein. On the contrary, both p53 and p21 proteins remained at a low level in X-irradiated cells. Treatment with aphidicolin, an S phase blocking agent, prolonged cell cycle arrest and reduced the rate of apoptotic cell death in both UV-irradiated and X-irradiated cells. From these results, it is suggested that UV-induced apoptosis occurs mainly at S phase and is regulated by increased production of p53 and p21 proteins, while X-ray-induced apoptosis occurs after G2 blockade and may be independent of p53.     

Insights into cancer therapeutic design based on p53 and TRAIL receptor signaling.

Knowledge of the emerging pathways of cell death downstream of the p53 tumor suppressor and the TRAIL death-inducing ligand is suggesting ways to improve therapeutic design in cancer. In contrast to its unique G1 cell cycle arresting mechanism that is maintained by p21(WAF1), there are signals transduced by p53 to multiple apoptotic effectors perhaps due to the importance of apoptosis in suppressing tumors. There is evidence for cytoplasmic as well as mitochondrial activation of caspases downstream of p53, although in some cell lineages the signal ultimately involves the mitochondria. The TRAIL signaling pathway appears promising for therapeutic development despite sharing some similarities with the toxic Fas and TNF pathways, in terms of effector molecules and downstream signals. One of the key findings is the tissue specificity of cell death responses, a feature that could be exploited in strategies to widen the therapeutic window of combination cancer therapies. Efforts continue to develop p53-targeted cancer therapy, and novel clues to enhance or block specific effectors may improve therapeutic design.