The effects of antimycin A on endothelial cells in cell death,reactive oxygen species and GSH levels

Antimycin A (AMA) inhibits mitochondrial electron transport chain between cytochrome b and c. Here, we evaluated the effects of AMA on the growth and death of endothelial cells (ECs) in relation to reactive oxygen species (ROS) and glutathione (GSH) levels. AMA inhibited the growth of calf pulmonary artery endothelial cells (CPAEC) and human umbilical vein endothelial cells (HUVEC). AMA also induced apoptosis in both ECs which was accompanied by the loss of mitochondrial membrane potential (MMP; ΔΨ_m). HUVEC were more sensitive to AMA than CPAEC. AMA increased ROS level in CPAEC but decreased the levels in HUVEC. Z-VAD (pan-caspase inhibitor) mildly prevented apoptosis in AMA-treated ECs without the significant changes of ROS. N-acetyl-cysteine (NAC; a well-known antioxidant) decreased ROS levels in AMA-treated ECs. NAC reduced CPAEC death by AMA but enhanced HUVEC death by it. Furthermore, AMA increased GSH depleted cell numbers in ECs. Buthionine sulfoximine (BSO; an inhibitor of GSH synthesis), showing a pro-apoptotic effect on AMA-treated HUVEC, significantly increased GSH depleted cell number but it did not affect cell death and GSH depletion in AMA-treated CPAEC. In conclusion, AMA inhibited the growth of ECs via caspase-dependent apoptosis. ROS level change by AMA was partially related to CPAEC death, but did not affect HUVEC death. The change of GSH contents by AMA influenced the death of ECs.     

Propyl gallate inhibits the growth of calf pulmonary arterial endothelial cells via glutathione depletion

Propyl gallate (PG) as a synthetic antioxidant exerts a variety of effects on tissue and cell functions. Here, we evaluated the effects of PG on the growth and death of endothelial cells (ECs), especially calf pulmonary artery endothelial cells (CPAEC) in relation to reactive oxygen species (ROS) and glutathione (GSH). PG dose-dependently inhibited the growth of CPAEC and human umbilical vein endothelial cells (HUVEC) at 24 h. PG induced cell death in CPAEC, which was accompanied by the loss of mitochondrial membrane potential (MMP; ΔΨ_m). PG generally increased ROS level in CPAEC but not in HUVEC. PG also dose-dependently increased GSH depleted cells in both ECs. The treatment with antioxidant of N-acetyl-cysteine (NAC) or ascorbate acid (AA) prevented CPAEC growth inhibition and death by PG, which was accompanied by the attenuation of GSH depletion but not by the reduction of ROS level. In conclusion, PG induced growth inhibition and death of ECs, especially CPAEC via GSH depletion.     

Plumbagin-induced apoptosis in lymphocytes is mediated through increased reactive oxygen species production, upregulation of Fas, and activation of the caspase cascade

Extracts from plants containing plumbagin (PLB) continue to be used as a treatment of a number of chronic immunologically-based diseases. However, most of these claims are supported only by anecdotal evidence with few scientific reports describing the mechanism of action or the efficacy of plumbagin in the suppression of the immune response. In the current study, we tested the hypothesis that plumbagin-induced suppression of the immune response was mediated through the induction of apoptosis. Splenocytes from C57BL/6 mice cultured in the presence of 0.5 µM or greater concentrations of PLB significantly reduced proliferative responses to mitogens, including anti-CD3 mAbs, concanavalin A (Con A), lipopolysaccharide (LPS) and staphylococcal enterotoxin B (SEB) in vitro. Exposure of naïve and activated splenocytes to PLB led to a significant increase in the levels of apoptosis. In addition, PLB treatment led to a significant increase in the levels of reactive oxygen species (ROS) in naïve and activated splenocytes. Furthermore, treatment with the ROS scavenger, N-acetylcysteine (NAC), prevented PLB-induced apoptosis, suggesting a role of ROS in PLB-induced apoptosis. PLB-induced apoptosis led to ROS-mediated activation of both the extrinsic and intrinsic apoptotic pathways. In addition, plumbagin led to increased expression of Fas. Finally, treatment of mice with PLB (5 mg/kg) led to thymic and splenic atrophy as well as a significant suppression of the response to SEB and dinitroflourobenzene (DNFB) in vivo. Together, these results suggest that plumbagin has significant immunosuppressive properties which are mediated by generation of ROS, upregulation of Fas, and the induction of apoptosis.     

Propyl gallate decreases the proliferation of Calu-6 and A549 lung cancer cells via affecting reactive oxygen species and glutathione levels

Propyl gallate (3,4,5-trihydroxybenzoic acid propyl ester, PG) has an anti-proliferative effect in various cells. In this study, Calu-6 and A549 lung cancer cells were used to examine the anti-proliferative effect of PG in relation to reactive oxygen species (ROS) and glutathione (GSH) levels. PG (100–1,600 μM) dose-dependently inhibited the proliferation of Calu-6 and A549 cells at 24 h, and PG at 800–1,600 μM strongly induced cell death in both cell lines. PG (800–1,600 μM) increased cellular metabolism in Calu-6 but not A549 cells at 4 h. PG either increased or decreased ROS levels, including O₂˙⁻ and ˙OH, depending on the incubation doses and times of 1 or 24 h. Even these effects differed between Calu-6 and A549 cell types. PG reduced the activity of superoxide dismutase (SOD) in Calu-6 cells, and it augmented the activity of catalase in A549 cells. PG dose-dependently increased the number of GSH depleted cells in both Calu-6 and A549 cells at 24 h. In addition, PG decreased GSH levels in both lung cancer cells at 1 h. Furthermore, diethyldithiocarbamate (DDC; an inhibitor of SOD) and 3-amino-1,2,4-triazole (AT; an inhibitor of catalase) differently affected cellular metabolism, ROS and GSH levels in PG-treated and PG-untreated Calu-6 and A549 cells at 1 h. In conclusion, PG dose-dependently decreased the proliferation of Calu-6 and A549 lung cancer cells, which was related to changes in ROS levels and the depletion of GSH.     

Enhanced cell death effects of MAP kinase inhibitors in propyl gallate-treated lung cancer cells are related to increased ROS levels and GSH depletion

Propyl gallate (PG) has an anti-growth effect in lung cancer cells. The present study investigated the effects of mitogen-activated protein kinase (MAPK; MEK, JNK, and p38) inhibitors on PG-treated Calu-6 and A549 lung cancer cells in relation to cell death as well as reactive oxygen species (ROS) and glutathione (GSH) levels. PG induced cell death in both Calu-6 and A549 lung cancer cells at 24 h, which was accompanied by loss of mitochondrial membrane potential (MMP; ΔΨ_m). All of the tested MAPK inhibitors increased cell death in both PG-treated lung cancer cell lines. In particular, MEK inhibitor strongly enhanced cell death and MMP (ΔΨ_m) loss in PG-treated Calu-6 cells and p38 inhibitor had the same effects in A549 cells as well. PG increased ROS levels and caused GSH depletion in both cell lines at 24 h. MAPK inhibitors increased O₂^•- levels and GSH depletion in PG-treated Calu-6 cells, and JNK and p38 inhibitors increased ROS levels and GSH depletion in PG-treated A549 cells. In conclusion, MAPK inhibitors increased cell death in PG-treated Calu-6 and A549 lung cancer cells. Enhanced cell death and GSH depletion in Calu-6 cells caused by the MEK inhibitor were related to increased O₂^•- levels, and the effects of the p38 inhibitor in A549 cells were correlated with increased general ROS levels.     

Structurally related antitumor effects of flavanones in vitro and in vivo: involvement of caspase 3 activation, p21 gene expression, and reactive oxygen species production

Flavonoids exist extensively in plants and Chinese herbs, and several biological effects of flavonoids have been demonstrated. The antitumor effects in colorectal carcinoma cells (HT29, COLO205, and COLO320HSR) of eight flavanones including flavanone, 2'-OH flavanone, 4'-OH flavanone, 6-OH flavanone, 7-OH flavanone, naringenin, nargin, and taxifolin were investigated. Results of the MTT assay indicate that 2'-OH flavanone showed the most potent cytotoxic effect on these three cells, and cell death induced by 2'-OH flavanone was via the occurrence of DNA ladders, apoptotic bodies, and hypodiploid cells, all characteristics of apoptosis. Induction of caspase 3 protein processing and enzyme activity associated with cleavage of poly(ADP-ribose) polymerase (PARP) was identified in 2'-OH flavanone-treated cells, and a peptidyl inhibitor (Ac-DEVD-FMK) of caspase 3 attenuated the cytotoxicity of 2'-OH flavanone in COLO205 and HT-29 cells. Elevation of p21 (but not p53) and a decrease in Mcl-1 protein were found in 2'-OH flavanone-treated COLO205 and HT-29 cells. Elevation of intracellular reactive oxygen species (ROS) was detected in 2'-OH flavanone-treated cells by the 2',7'-dichlorodihydrofluorescein diacetate (DCHF-DA) assay, and ROS scavengers including 4,5-dihydro-1,3-benzene disulfonic acid (tiron), catalase, superoxide dismutase (SOD), and pyrrolidine dithiocarbamate (PDTC) suppressed the 2'-OH flavanone-induced cytotoxic effect. Subcutaneous injection of COLO205 induced tumor formation in nude mice, and 2'-OH flavanone showed a significant inhibitory effect on tumor formation. The appearance of apoptotic cells with H&E staining, and an increase in p21, but not p53, protein by immunohistochemistry were observed in tumor tissues under 2'-OH flavanone treatment. Primary tumor cells (COLO205-X) derived from a tumor specimen elicited by COLO205 were established, and 2'-OH flavanone showed an significant apoptotic effect in COLO205-X cells in accordance with the appearance of DNA ladders, caspase 3 protein processing, PARP protein cleavage, and increasing p21 protein. These results revealed in vitro, ex vivo, and in vivo antitumor activities of 2'-OH flavanone via apoptosis induction, and indicates that 2'-OH flavanone is an active compound worthy of development for cancer chemotherapy.     

灵芝多糖肽对人脐静脉内皮细胞氧化损伤的保护作用

目的研究灵芝多糖肽对人脐静脉内皮细胞(HU-VECs)氧化损伤的保护作用。方法原代培养人脐静脉内皮细胞,CD31免疫荧光法鉴定细胞。以叔丁基氢过氧化物(tBOOH)为氧化剂损伤细胞,造成氧化损伤模型,培养液中加入不同浓度灵芝多糖(6.125、12.5、25、50、100mg·mL-1),以CCK-8法检测细胞存活率;Hoechst333258检测细胞氧化损伤引起的凋亡;比色法检测Caspase-3活性变化;电镜检测细胞及细胞器形态学改变。结果灵芝多糖(6.125、12.5、25、50、100mg·mL-1)可减轻叔丁基氢过氧化物对HUVECs的氧化损伤,CCK-8检测灵芝多糖给药组,HUVEC细胞存活率增加。Hoechst33258检测给药组细胞凋亡数量较损伤组减少。比色法检测损伤组Caspase-3活性明显增高,给药组较损伤组减少。电镜可见灵芝多糖给药组减轻细胞器的氧化损伤,减少细胞凋亡。结论灵芝多糖肽(GLPP)对人脐静脉内皮细胞氧化损伤具有保护作用。     

Induction of apoptosis and autophagy by sodium selenite in A549 human lung carcinoma cells through generation of reactive oxygen species

Selenium in the form of sodium selenite has been reported to exert anti-tumor effects in several cancer cell types by inducing autophagic cell death and apoptosis mediated by reactive oxygen species (ROS). However, the exact molecular pathways underlying these effects have not been fully established. The present study used A549 human lung carcinoma cells for further investigation of the anti-cancer mechanism of sodium selenite. We showed that sodium selenite modulated both the extrinsic and intrinsic apoptotic pathways, which were interconnected by Bid truncation. We used z-VAD-fmk, a pan-caspase inhibitor, to demonstrate that sodium selenite-induced apoptosis was dependent on the activation of caspases. Sodium selenite also increased autophagy, as indicated by an increase in microtubule-associated protein light chain-3 (LC3) puncta, accumulation of LC3II, and elevation of autophagic flux. Pretreatment with bafilomycin A1 enhanced sodium selenite-induced apoptosis, indicating that sodium selenite-induced autophagy functioned as a survival mechanism. Sodium selenite treatment also resulted in generation of ROS, which abrogated mitochondrial membrane potential (MMP) and regulated both apoptosis and autophagy. Phospho-nuclear factor erythroid 2-related factor 2 (p-Nrf2) showed a ROS-dependent translocation to the nucleus, which suggested that Nrf2 might increase cell survival by suppressing ROS accumulation and apoptosis mediated by oxidative stress. Sodium selenite treatment of A549 cells therefore appeared to trigger both apoptosis and cytoprotective autophagy, which were both mediated by ROS. The data suggest that regulation of ROS generation and autophagy can be a potential strategy for treating lung cancer that is resistant to pro-apoptotic therapeutics.     

Different effects of adenylyl cyclase activators and phosphodiesterases inhibitors on cervical cancer (HeLa) and breast cancer (MCF-7) cells proliferation

Breast and cervical cancers are the most common cancers in Iran and worldwide. Hormonal stimulation of cyclic adenosine mono phosphate (cAMP) and the cAMP-dependent protein kinase PKA regulates cell growth by different mechanism. cAMP can stimulate cell growth in many cell types while inhibiting cell growth in others. In some cell lines have been shown that the proliferation of tumor cells is reduced by increasing cAMP in cells. In this study, we evaluate growth arrest of selective PDE3 and non-selective PDE inhibitors, which lead to increase level of cAMP in cervical (HeLa) and breast cancer (MCF7) cell lines have been studied. Cells were incubated with different concentrations of selective, non-selective PDE inhibitors, beta adrenergic receptor agonist and direct stimulator of adenylyl cyclase. Cell viability was quantitated by MTT assay. Apoptotic cells were determined using PI staining of DNA fragmentation by flow cytometry (sub-G1 peak). Result showed that selective PDE inhibitors decreased cell viability in HeLa and MCF-7 cells as a time-dependent manner. Non-selective inhibitor and beta-adrenergic receptor agonist also decrease cell viability but they are less powerful than selective PDE3 inhibitors. Forskolin had no effect in viability of cells. Analysis of DNA fragmentation by flow cytometry showed apoptosis involved in selective PDE3 inhibitors induced toxicity in HeLa cell. Thus, the growth inhibitory effects of selective PDE3 inhibitors are more effective than non-selective inhibitor. Further studies are needed to investigate the mechanism of action is on the field.     

The effect of quercetin on pro-apoptotic activity of cisplatin in HeLa cells

It is well known that some tumour cells are very resistant to chemotherapy-induced cell death which indicate poor prognosis for patients. Thus the aim of the present study was to investigate the effect of quercetin on pro-apoptotic activity of cisplatin in human cervix carcinoma cells (HeLa). Three variants of experiments were performed. In the first one cells were incubated with studied drugs separately for 8 and 24h. In the second, drugs were added to the culture medium simultaneously. In third cisplatin or quercetin addition was followed by subsequent quercetin or cisplatin treatment, respectively. We observed different apoptotic effects, dependent on the drug succession. Preincubation of cells with quercetin followed by cisplatin treatment appeared to be the most effective and was correlated with strong activation of caspase-3 and inhibition of both heat shock proteins (Hsp72) and multi-drug resistance proteins (MRP) levels. Our results indicate that quercetin pretreatment sensitizes HeLa cells to cisplatin-induced apoptosis in HeLa cells.     

Reactive oxygen species contribute to oridonin-induced apoptosis and autophagy in human cervical carcinoma HeLa cells

Aim:

To investigate the role of reactive oxygen species (ROS) in oridonin-induced apoptosis and autophagy in HeLa cells.

Methods:

The cell viability was measured using MTT assay. Morphological changes of apoptosis and autophagy were examined using Hoechst 33258 staining and monodansylcadaverine (MDC) staining, respectively. The mitochondrial membrane potential (ΔΨm) was measured using fluorescent dye rhodamine 123. DCF-induced fluorescence was used to measure the intracellular ROS level. Protein expression was examined using Western blot.

Results:

Treatment of HeLa cells with oridonin (20–160 μmol/L) inhibited the cell growth in time- and concentration-dependent manners. The cells treated with oridonin (80 μmol/L) for 24 h displayed marked DNA fragmentation and MDC-positive autophagosomes. In the presence of the specific autophagy inhibitor 3-MA (2 mmol/L), the oridonin-induced apoptosis was significantly enhanced. Treatment of HeLa cells with oridonin (20–120 μmol/L) induced intracellular ROS generation in a concentration-dependent manner. In the presence of the ROS scavenger NAC (5 mmol/L), the oridinin-induced ROS generation was markedly reduced. NAC (5 mmol/L) or non-thiol antioxidant catalase (1000 U/mL) significantly reduced the oridonin-induced inhibition of cell growth and apoptosis. Furthermore, oridonin significantly reduced ΔΨm, which was blocked by NAC. Oridonin markedly increased Bax expression in mitochondria, and decreased Bcl-2 expression in both the cytosol and mitochondria. Oridonin also markedly increased the phosphorylation of Bcl-2 in the cytosol. All the effects were blocked by NAC. Oridonin increased the levels of caspase-3 and caspase-8, and decreased the expression of pro-caspase 3 and pro-caspase 9, which were blocked by NAC.

Conclusion:

ROS plays a critical role in oridonin-induced apoptosis and autophagy.     

Reactive oxygen species accumulation contributes to gambogic acid-induced apoptosis in human hepatoma SMMC-7721 cells

It is reported that gambogic acid (GA), the main active compound of gamboge which is a dry resin extracted from Garcinia hanburyi tree, has potent antitumor activity both in vivo and in vitro. Activation of mitochondrial apoptotic pathway in cancer cells is one effective therapy for cancer treatment. In the present study, we focus on the effect of GA on induction of reactive oxygen species (ROS) accumulation and triggering the mitochondrial signaling pathway in human hepatoma SMMC-7721 cells. The results indicated that GA induced ROS accumulation and collapse of mitochondrial membrane potential in SMMC-7721 cells in a concentration-dependent manner and subsequently induced that release of Cytochrome c and apoptosis-inducing factor from mitochondria to cytosol, which inhibited ATP generation and induced apoptosis in the cells. Moreover, GA elevated the phosphorylation of c-Jun-N-terminal protein kinase (JNK) and p38, which was the downstream effect of ROS accumulation. Furthermore, N-acetylcysteine, a ROS production inhibitor, partly reversed the activation of JNK and p38 and the induction of apoptosis in GA-treated cells. Collectively, our study demonstrated that accumulation of ROS played an important role in GA-induced mitochondrial signaling pathway, which provided further theoretical support for the application of GA as a promising anticancer agent.     

Involvement of reactive oxygen species/c-Jun NH(2)-terminal kinase pathway in kotomolide A induces apoptosis in human breast cancer cells

The anticancer effects of kotomolide A (KTA), a new butanolide constituent isolated from the leaves of Cinnamomum kotoense (Lauraceae), on the two human breast cancer cell lines MCF-7 and MDA-MB-231, were first investigated in our study. KTA exhibited selectively antiproliferative effects in cancer cell lines without showing any toxicity in normal mammary epithelial cells. Treatment of cancer cells with KTA to trigger G2/M phase arrest was associated with increased p21/WAF1 levels and reduced amounts of cyclin A, cyclin B1, cdc2 and cdc25C. KTA induced cancer cell death treatment by triggering mitochondrial and death receptor 5 (DR5) apoptotic pathways, but did not act on the Fas receptor. Exposure of MCF-7 and MDA-MB-231 cells to KTA resulted in cellular glutathione reduction and ROS generation, accompanied by JNK activation and apoptosis. Both antioxidants, NAC and catalase, significantly decreased apoptosis by inhibiting the phosphorylation of JNK and subsequently triggering DR5 cell death pathways. The reduction of JNK expression by siRNA decreased KTA-mediated Bim cleavage, DR5 upregulation and apoptosis. Furthermore, daily KTA i.p. injections in nude mice with MDA-MB-231 s.c. tumors resulted in a 50% decrease of mean tumor volume, compared with vehicle-treated controls. Taken together, the data show that cell death of breast cancer cells in response to KTA is dependent upon ROS generation and JNK activation, triggering intrinsic and extrinsic apoptotic pathways. The ROS/JNK pathway could be a useful target for novel approaches in breast cancer chemotherapy.     

Sulforaphane induces reactive oxygen species-mediated mitotic arrest and subsequent apoptosis in human bladder cancer 5637 cells

The present study was undertaken to determine whether sulforaphane-derived reactive oxygen species (ROS) might cause growth arrest and apoptosis in human bladder cancer 5637 cells. Our results show that the reduced viability of 5637 cells by sulforaphane is due to mitotic arrest, but not the G2 phase. The sulforaphane-induced mitotic arrest correlated with an induction of cyclin B1 and phosphorylation of Cdk1, as well as a concomitant increased complex between cyclin B1 and Cdk1. Sulforaphane-induced apoptosis was associated with the activation of caspase-8 and -9, the initiators caspases of the extrinsic and intrinsic apoptotic pathways, respectively, and activation of effector caspase-3 and cleavage of poly (ADP-ribose) polymerase. However, blockage of caspase activation inhibited apoptosis and abrogated growth inhibition in sulforaphane-treated 5637 cells. This study further investigated the roles of ROS with respect to mitotic arrest and the apoptotic effect of sulforaphane, and the maximum level of ROS accumulation was observed 3 h after sulforaphane treatment. However, a ROS scavenger, N-acetyl-L-cysteine, notably attenuated sulforaphane-mediated apoptosis as well as mitotic arrest. Overall, these results suggest that sulforaphane induces mitotic arrest and apoptosis of 5637 cells via a ROS-dependent pathway.     

Role of reactive oxygen species, glutathione and NF-kappaB in apoptosis induced by 3,4-methylenedioxymethamphetamine ("Ecstasy") on hepatic stellate cells

"Ecstasy" (3,4-methylenedioxymethamphetamine, MDMA), is a derivative of amphetamine with hepatotoxic effects that has been shown to induce apoptosis of cultured liver cells. In the present work, we studied the role played by oxidative stress in the apoptotic response caused by MDMA on a cell line of hepatic stellate cells (HSC). MDMA-treatment provoked oxidative stress determined as reactive oxygen species (ROS) accumulation and decrease of intracellular reduced glutathione levels. Pre-treatment with the antioxidant pyrrolidine dithiocarbamate blocked ROS production but did not prevent MDMA-induced apoptosis of HSC. The pro-oxidant menadione induced in HSC ROS production and apoptosis that were prevented by pyrrolidine dithiocarbamate, showing HSC to be susceptible to oxidative stress-induced apoptosis. Addition of exogenous GSH or its precursor NAC potentiated the apoptotic action of MDMA but blocked apoptosis induced by menadione. Pre-treatment of HSC with the cytochrome P450 inhibitor quinine diminished the extent of apoptosis caused by MDMA, suggesting the involvement of a metabolic derivative of MDMA on its apoptotic effect. Nuclear factor NF-kappaB was activated by MDMA in a oxidative stress independent fashion and played a protective role in the apoptotic response, since inhibition of NF-kappaB by treatment with parthenolide or by viral infection with a dominant-negative form of NIK (Ad5dnNIK) resulted in an increase of MDMA-induced cell death. In summary, MDMA-induced apoptosis of HSC is accompanied, but not caused by oxidative stress; a metabolic derivative of the drug is responsible for the apoptotic effect of MDMA, which is partially blocked by NF-kappaB activation.     

HMGB1对宫颈癌HeLa细胞凋亡的影响及其作用机制

目的 观察HMGB1对人宫颈癌HeLa细胞生长及凋亡的影响并研究其可能的作用机制.方法 构建HMGB1高表达质粒和RNA干扰质粒,将HMGB1高表达质粒与RNA干扰质粒分别转染到HeLa细胞中,采用MTT实验、PI单染流式细胞术和Annexin V-PI双标法流式细胞术检测HeLa细胞的增殖活性、细胞周期和凋亡情况.采用RT-PCR和western blot法检测HMGB1基因沉默对Caspase-3、bcl-2 和细胞色素C表达的影响.结果 HMGB1高表达载体(pEGFP-N1-HMGB1)与阴性对照质粒分别转染到HeLa细胞,RT-PCR和Western blot结果显示HMGB1重组质粒转染HeLa后可明显增加HMGB1的mRNA 及蛋白表达(P＜0.05).HMGB1干扰病毒载体(HMGB1 SiRNA)与空白病毒载体分别转染到HeLa细胞中,RT-PCR和Western blot结果显示HMGB1 SiRNA转染HeLa后可明显抑制HMGB1的mRNA及蛋白表达(P＜0.05).MTT法检测HMGB1高表达及基因沉默后HeLa的细胞生长曲线,结果显示HMGB1高表达可明显提高HeLa的细胞生长速度(P＜0.05),HMGB1沉默后HeLa的细胞生长明显受到抑制(P＜0.05).PI 染色流式细胞仪结果显示:HMGB1过表达后,细胞正常增殖周期被加速(P＜0.05);HMGB1沉默后,细胞增殖周期被抑制(P＜0.05).Annexin V-PI双标法流式细胞仪结果显示,HMGB1过表达后,HeLa细胞凋亡率明显下降(P＜0.05);HMGB1沉默后,HeLa细胞凋亡率明显上升(P＜0.05).RT-PCR方法检测不同细胞组Caspase-3和bcl-2 mRNA的表达,结果显示:HMGB1基因沉默后,Caspase-3 mRNA的表达明显增加(P＜0.05),bcl-2 mRNA的表达明显减少(P＜0.05).Western blot结果显示:HMGB1 基因沉默后,细胞色素C和Caspase-3蛋白表达水平增加,bcl-2蛋白表达水平减少(P＜0.05).结论 HMGB1可提高HeLa细胞生长速度、增殖周期,抑制HeLa细胞凋亡.HMGB1基因沉默可抑制Hela细胞增殖,促进其凋亡.影响Caspase-3—细胞色素C途径及调节bcl-2的表达可能是其主要作用机制.