Hydroxycamptothecin-induced apoptosis in hepatoma SMMC-7721 cells and the role of mitochondrial pathway

The camptothecin (CPT) derivative hydroxycamptothecin (HCPT) containing 10-hydroxy represents one of the most potent topoisomerase I inhibitors described. This anticancer agent, currently undergoing clinical trials on gastric tumours, has been shown more active and less toxic than conventional camptothecins. To shed light on the mechanism of action of HCPT at the cellular level, we examined cell growth, apoptosis, changes of mitochondrial membrane potential, cytochrome c and AIF translocation in cancer cells by exposing these cells to HCPT for indicated time. The effect of HCPT on cell proliferation was measured by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromid) assay and apoptosis was measured using flow cytometry, fluorescence microscopy and electron microscopy. Changes of mitochondrial membrane potential were monitored by fluorescence microscope. Western blot analysis was used to evaluate the release of mitochondrial cytochrome c and AIF; On the other hand, translocation of cytochrome c and AIF from mitochondria to cytosol during apoptosis were confirmed by confocal microscopy. HCPT could noticeably inhibit the proliferation of SMMC-7721cells and the IC(50) dose was about 0.22muM; SMMC-7721 cells treated with HCPT showed typical characteristics of apoptosis rather than necrotic including phosphatidylserine (PS) exposed from the inner to the outer leaflet of the plasma membrane, abnormal cell morphology, chromatin condensation and nuclear fragmentation; On the other hand, during process of cell apoptosis, mitochondrial transmembrane potential was reduced; Compared with the control group, the mRNA and protein expression of cytochrome c and AIF in treated and untreated SMMC-7721 cells were not significantly changed (not shown). However, when cells were treated with HCPT, the massive translocation of cytochrome c and AIF to the nucleus was evident. Our results indicate that HCPT can inhibit proliferation and induce apoptosis of human hepatoma SMMC-7721 cells. Mitochondrial pathway of apoptosis, especially for cytochrome c and AIF translocation, may play an important role in apoptosis induced by HCPT.     

Overexpression of DRG2 increases G2/M phase cells and decreases sensitivity to nocodazole-induced apoptosis

DRG2, a member of the DRG subfamily in the GTP-binding protein superfamily, was identified as a repressed gene product in fibroblasts transformed by SV40. The significance of this down-regulation and the cellular role of DRG2 has not been understood in the past. To investigate the function of DRG2 we made a Jurkat cell line, Jurkat-LNCX2-DRG2, stably transfected with pLNCX2-DRG2 to overexpress human DRG2. Cell cycle distribution analysis revealed an increased accumulation of G(2)/M phase cells in Jurkat-LNCX2-DRG2 cells, indicating a retardation of cell-cycle progression. In addition, an overexpression of DRG2 reduced the sensitivity of Jurkat cells to the mitotic poison nocodazole. Our data suggest that overexpression of DRG2 in Jurkat cells affects genes regulating cell-cycle arrest and apoptosis, and that these molecular changes may be important in the growth or differentiation of cells.     

Abrogation of G2/M arrest sensitizes curcumin-resistant hepatoma cells to apoptosis

In this study, we showed that curcumin treatment resulted in activation of Chk1-mediated G2 checkpoint, which was associated with the induction of G2/M arrest and the resistance of cancer cells to curcumin-induced apoptosis. Further investigation revealed that inhibition of Chk1 significantly abrogated G2/M arrest and sensitized curcumin-resistant cells to apoptosis via upregulation of Bad and in turn the loss of mitochondrial membrane potential. These results indicate that Chk1-mediated G2/M arrest may serve as a mechanism for curcumin resistance and Chk1 represents a potential target for the reversal of this resistance. Our findings should be helpful for clinical application of curcumin.     

c-Myc influences olaquindox-induced apoptosis in human hepatoma G2 cells

Olaquindox, a synthetic antimicrobial compound, was banned as feed additives in the U.S. and the EU. In China, the use of olaquindox is banned in poultry and aquaculture feed, restricted in livestock feed for growth promotion. Olaquindox's safety is the object of increasing attention. The present study was undertaken to investigate whether and how olaquindox elevates expression of c-Myc, which influences olaquindox-induced apoptosis in HepG2 cells. For a better understanding of c-Myc's role in susceptibility of human hepatoma G2 cells to olaquindox-induced apoptosis, two vectors (the pSilencer-cmyc(Si-cmyc) and the control vector) were transfected to HepG2 cells. The cells were pretreated with Si-cmyc, which expressed only 35-65% c-Myc protein levels compared to those of the parental cells and the control cells. We examined effects of olaquindox on reactive oxygen species (ROS) production in these c-Myc low-expressing cells, and on apoptosis. Our data revealed that ROS production induced by olaquindox treatment was partially blocked by Si-cmyc transfection and partly inhibited olaquindox-induced apoptosis through decreased ROS generation. Further data showed that olaquindox induced decreased ROS by Si-cmyc transfection through decreased cytochrome c release to cytosol, which inhibited apoptosis of the cells. These results suggest that c-Myc might be important during olaquindox-induced apoptosis in human hepatoma G2 cells.     

Flavonoids from tartary buckwheat induce G2/M cell cycle arrest and apoptosis in human hepatoma HepG2 cells

The cytotoxicity and antioxidant activity on human hepa toma cell line HepG2 of three flavonoids homogenous com pounds from tartary buckwheat seeds and bran, namely quercetin, isoquercetin, and rutin, were investigated. The total antioxidant competency detection results indicated that the antioxidant capacity of quercetin was the strongest in a biological response system. A [3-（4,5-dimethylthiazol-2-yl）-2,5diphenyltetrazolium bromide] assay showed that quercetin exhibited the strongest cytotoxic effects against the HepG2 cell line. Flow cytometric analysis indicated that quercetin significantly increased the production of reactive oxygen species, and led to the G2/M phase arrest accom panied by an increase of apoptotic cell death after 48 h of incubation. Quercetininduced cell apoptosis was shown to involve p53 and p21 upregulation, Cyclin D1, Cdk2, and Cdk7 downregulation. These results suggested that the in duction of G2/M arrest, apoptosis, and cell death by quer cetin may associate with increased expression of p53 and p21, decrease of Cyclin D1, Cdk2, and Cdk7 levels, and generation of reactive oxygen species in cells. This study will help to better understand and fully utilize medicinal resources of plant flavonoids.     

Mycoepoxydiene, a fungal polyketide, induces cell cycle arrest at the G2/M phase and apoptosis in HeLa cells

Mycoepoxydiene (MED) is a polyketide isolated from a marine fungus associated with mangrove forests. It contains an oxygen-bridged cyclooctadiene core and an α,β-unsaturated δ-lactone moiety. MED induced the reorganization of cytoskeleton in actively growing HeLa cells by promoting formation of actin stress fiber and inhibiting polymerization of tubulin. MED could induce cell cycle arrest at G2/M in HeLa cells. MED-associated apoptosis was characterized by the formation of fragmented nuclei, PARP cleavage, cytochrome c release, activation of caspase-3, and an increased proportion of sub-G1 cells. Additionally, MED activated MAPK pathways. Interestingly, the time of JNK, p38, and Bcl-2 activation did not correlate with the release of cytochrome c. This study is the first report demonstrating the action mechanism of MED against tumor cell growth. These results provide the potential of MED as a novel low toxic antitumor agent.     

抑制survivin表达增强人肝癌HepG2细胞对高线性能量转移射线的辐射敏感性

探讨了肿瘤细胞中survivin的表达对高线性能量转移(LET)射线辐射敏感性的影响.根据Gen Bank提供的survivin序列,合成特异性survivin-siRNA寡核苷酸,转染人肝癌HepG2细胞,抑制survivin的表达.发现siRNA转染后诱导了HepG2细胞G2/M期阻滞,增加了自发性和辐射诱导的细胞凋亡.在高线性能量转移(LET)碳离子辐照后,siRNA转染细胞的克隆存活率明显下降.这些结果表明survivin表达是HepG2细胞产生对高LET射线辐射抗性的关键因素.     

壳寡糖对肝癌细胞SMMC-7721中Bcl-2和Caspase-3表达的影响

目的检测壳寡糖对人肝癌SMMC-7721细胞的抑制效果及对凋亡调控蛋白Bcl-2和Caspase-3的影响。方法采用噻唑蓝（MTT）法检测不同浓度壳寡糖对肝癌细胞SMMC-7721细胞增殖的抑制作用,并利用荧光Hoechst33258染色法检测细胞凋亡状况。最后通过免疫细胞化学方法研究壳寡糖对肝癌细胞SMMC-7721中Bcl-2和Caspase-3表达的影响。结果壳寡糖能够抑制SMMC-7721细胞增殖,并且促进SMMC-7721细胞的凋亡,并且壳寡糖能够上调促凋亡蛋白Caspase-3的表达和降低抑制凋亡蛋白Bcl-2的表达。结论壳寡糖对人肝癌SMMC-7721细胞的增殖有抑制作用,此作用可能是通过促进Caspase-3的表达和抑制Bcl-2的表达来实现的。     

HMBA对人肝癌SMMC—7721细胞周期相关基因表达的影响

本文研究HMBA对人肝癌SMMC－7721细胞周期G0／G1期阻滞相关基因表达的影响。免疫细胞化学和核酸原位杂交检测结果显示,HMBA可明显上调p21^WAF1/CIP1、p16蛋白表达并增强p21^WAF1/CIP1基因转录,同时对CDK4、Cyclin D1蛋白表达以及c-myc基因转录均具有明显的下调作用。结果表明,HMBA可通过增强p21^WAF1/CIP1、p16基因表达而抑制Cyclin D1-CDK4活性,最终导致细胞进入S期所需的c-myc等基因转录活性下降,从而将细胞周期阻滞于G0／G1期,诱导人肝癌细胞分化。     

Biochemical and morphological characteristics of selenite-induced apoptosis in human hepatoma hep G2 cells

Selenium is a cellular growth inhibitor in many mammary tumor cells. To comprehend the mechanism for the selenium-induced cell death, we examined the effects of sodium selenite, which has been one of the most extensively investigated selenium compounds, in human hepatoma Hep G2 cells. Cell viability gradually decreased after treatment with sodium selenite within the concentration range of 10–50 μM. Low (10 μM) selenite has shown a high-percentage laddering pattern compared to the high (25 μM) cytotoxic selenium concentration in agarose gel electrophoresis. G₂M-phase enrichment was also concentration dependent. The most consistent transmission electron microscopic finding was the existence of large lysosomes. Based on these data, we hypothesize that sodium selenite predominantly shows its apoptotic effect over hydrogen selenite accumulation.     

Calcium/calmodulin-dependent phosphorylation and activation of human Cdc25-C at the G2/M phase transition in HeLa cells

The human tyrosine phosphatase (p54(cdc25-c)) is activated by phosphorylation at mitosis entry. The phosphorylated p54(cdc25-c) in turn activates the p34-cyclin B protein kinase and triggers mitosis. Although the active p34-cyclin B protein kinase can itself phosphorylate and activate p54(cdc25-c), we have investigated the possibility that other kinases may initially trigger the phosphorylation and activation of p54(cdc25-c). We have examined the effects of the calcium/calmodulin-dependent protein kinase (CaM kinase II) on p54(cdc25-c). Our in vitro experiments show that CaM kinase II can phosphorylate p54(cdc25-c) and increase its phosphatase activity by 2.5-3-fold. Treatment of a synchronous population of HeLa cells with KN-93 (a water-soluble inhibitor of CaM kinase II) or the microinjection of AC3-I (a specific peptide inhibitor of CaM kinase II) results in a cell cycle block in G2 phase. In the KN-93-arrested cells, p54(cdc25-c) is not phosphorylated, p34(cdc2) remains tyrosine phosphorylated, and there is no increase in histone H1 kinase activity. Our data suggest that a calcium-calmodulin-dependent step may be involved in the initial activation of p54(cdc25-c).     

Characterization of the cellular response during apoptosis induction in cadmium-treated hep G2 human hepatoma cells

Cadmium is a toxic transition heavy metal of continuing occupational and environmental concern, with a wide variety of adverse effects on regulation of gene expression and cellular signal transduction pathways. Injury to cells by cadmium leads to a complex series of events that can culminate in the death of the cell. It has been reported that cadmium induces apoptosis in many cell lines. However, the morphological characteristics leading to apoptosis or subsequent regeneration in cells exposed to cadmium have not been clarified. We evaluated whether human hepatoma cells maintained in culture undergo apoptosis when exposed to cadmium. Cytotoxic activity of cadmium on Hep G2 cells determined using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay. A DNA ladder assay was performed by electrophoresis. Cell cycle analysis was quantified by flow cytometry. Nuclear morphology was studied by fluorescence microscopy after staining with propidium iodide and Hoechst 33342. Morphologic alterations in culture hepatocytes treated with CdCl₂ were observed by transmission electron microscopy. We have demonstrated that apoptosis is a major mode of elimination of damaged HepG2 cells in cadmium toxicity and it precedes necrosis.     

Regulation of Moloney murine leukemia virus replication in chronically infected cells arrested at the G0/G1 phase.

The replication of Moloney murine leukemia virus (MMuLV) in chronically infected mouse cells arrested at the G0/G1 phase of the cell cycle by different procedures was investigated. MMuLV production was inhibited in glutamine- and isoleucine (Gln-Ile)-deprived G0/G1 cells. In contrast, butyric acid treatment, which efficiently arrested the cells at the G0/G1 phase of the cell cycle, did not inhibit MMuLV production. Furthermore, the inhibition of MMuLV production caused by either Gln-Ile deprivation or by interferon (IFN) treatment was overcome by butyric acid treatment. Thus, the replication of MMuLV could be dissociated from cell proliferation. The inhibition of MMuLV production in Gln-Ile-deprived cell cultures was compared to the inhibitory effect of IFN, which is known to affect budding and release of the virus. Rates of MMuLV protein synthesis were not affected in both the IFN-treated and Gln-Ile-deprived cells. However, processing of the viral polyprotein Pre65gag into p30 was blocked in the Gln-Ile-deprived cells. Furthermore, whereas in IFN-treated cells, MMuLV accumulated on the cell surface and could be released upon treatment with trypsin, in Gln-Ile-deprived cells, no virions were released by such treatment. These results indicate that in cells arrested by Gln-Ile deprivation, MMuLV is inhibited at a posttranslation step. This step appears to precede the anti-MMuLV block induced by IFN.     

Curcumin induces apoptosis through mitochondrial hyperpolarization and mtDNA damage in human hepatoma G2 cells

Curcumin, a major pigment of turmeric, is a natural antioxidant possessing a variety of pharmacological activities and therapeutic properties. But its mechanisms are unknown. In our previous study, we found that a 2-h exposure to curcumin induced DNA damage to both the mitochondrial DNA (mtDNA) and the nuclear DNA (nDNA) in HepG2 cells and that mtDNA damage was more extensive than nDNA damage. Therefore, experiments were initiated to evaluate the role of mtDNA damage in curcumin-induced apoptosis. The results demonstrated that HepG2 cells challenged with curcumin for 1 h showed a transient elevation of the mitochondrial membrane potential (DeltaPsim), followed by cytochrome c release into the cytosol and disruption of DeltaPsim after 6 h exposure to curcumin. Apoptosis was detected by Hoechst 33342 and annexin V/PI assay after 10 h treatment. Interestingly, the expression of Bcl-2 remained unchanged. A resistance to apoptosis for the corresponding rho0 counterparts confirmed a critical dependency for mitochondria during the induction of apoptosis in HepG2 cells mediated by curcumin. The effects of PEG-SOD in protecting against curcumin-induced cytotoxicity suggest that curcumin-induced cytotoxicity is directly dependent on superoxide anion O2- production. These data suggest that mitochondrial hyperpolarization is a prerequisite for curcumin-induced apoptosis and that mtDNA damage is the initial event triggering a chain of events leading to apoptosis in HepG2 cells.