凋亡抑制蛋白XIAP和促凋亡因子Smac在胰腺癌化疗抵抗中的调控作用

Objective： To investigate the relation of X-linked inhibitor of apoptosis （XIAP） and second mitochondria-derived activator of caspase （Smac） signaling pathway to chemoresistance in human pancreatic cancer Panc-1 and BXPC-3 cells. Methods： Apoptosis and the changes of XIAP expression in permeabilized cells induced by cisplatin and 5-fluorouracil （FU） were measured by flow cytometry. The cytosolic expression of XIAP, Smac and caspase-3 was detected by Western blot. A recombinant plasmid vector pEGFP-N1/Smac was constructed and transfected into of Pancol cells. The effect of cytosolic overexpression of Smac on apoptosis of Panc-1 cells was evaluated by flow cytometry. Results： Panc-1 was more resistant to cisplatin or 5-FU induced apoptosis than BXPC-3. Western blot revealed that chemoresistant Panc-1 highly expressed XIAP, and increased cytosolic expression of Smac might be responsible for the marked down-regulation of XIAP in chemo-sensitive BXPC-3 cells after exposure to cisplatin or 5-FU. Furthermore, cytosolic overexpression of Smac could significantly down-regulate the levels of XIAP and promote the activity of caspase-3, as well as sensitize Panc-1 cells to anticancer drug-induced apoptosis. Conclusion： Anticancer drug-induced apoptosis requires mitochondrial release of Smac and downregulation of XIAP, which may be an important determinant of chemo-sensitivity in pancreatic cancer cells. Up-regulation of cytosolic expression of Smac may act as an effective modifying signal to overcome apoptosis resistance to chemotherapy in pancreatic cancer cells.     

凋亡抑制蛋白XIAP和促凋亡因子Smac在胰腺癌化疗抵抗中的调控作用

目的探讨凋亡抑制蛋白XIAP和促凋亡因子Smac在胰腺癌细胞化疗抵抗中的作用及其分子机制。方法应用流式细胞术检测顺铂、5-FU介导的Panc-1、BXPC-3的凋亡率及胞浆染色分析细胞XIAP表达变化,Western-blot分析XIAP、Smac、Caspase-3表达水平；构建pEGFP-N1／Smac真核表达载体并转染胰腺癌Panc-1细胞,流式细胞术检测转染胞浆表达型 Smac基因对Panc-1细胞凋亡敏感性的作用。结果与BXPC-3细胞相比,Panc-1对顺铂或5-FU介导的凋亡具有较强抵抗性,Western blot分析显示Panc-1细胞高表达XIAP,在化疗药物作用下化疗敏感细胞BXPC-3胞浆内XIAP水平下降明显多于Panc-1细胞,而且凋亡的BXPC-3细胞释放入胞浆内的成熟 Smac蛋白水平明显高于Panc-1细胞。转染胞浆表达型Smac基因至化疗抵抗Panc-1细胞,可明显下调其XIAP表达水平,促进效应 Caspase-3分子活化,显著提高顺铂、5-FU诱导的细胞凋亡率。结论在化疗药物诱导的凋亡中,线粒体释放Smac下调XIAP是胰腺癌化疗敏感性的重要决定因素,而上调Smac活性蛋白的胞浆表达作为一种有效调节信号,通过拮抗XIAP的凋亡抑制作用协同化疗药物促进胰腺癌细胞凋亡。     

XIAP regulates Akt activity and caspase-3-dependent cleavage during cisplatin-induced apoptosis in human ovarian epithelial cancer cells

Chemoresistance is a major hurdle for successful cancer therapy. Although multiple mechanisms have been implicated to be involved in cisplatin resistance, recent evidence has suggested that X-linked inhibitor of apoptosis protein (XIAP) may be a key determinant in chemosensitivity in ovarian cancer. Cell fate is determined by a balance between cell survival and apoptotic signaling. Whereas phosphatidylinositol 3-kinase (PI 3-K) and XIAP are believed to be important cell survival factors in human ovarian surface epithelial cancer cells, if and how they interact to confer resistance to chemotherapy is not known. In the present study, we have investigated the role of XIAP in the regulation of the PI 3-K/Akt survival pathway in chemosensitive (A2780-s, OV2008, and OVCAR-3) and resistant (A2780-cp) ovarian cancer cell lines and the nature of this interaction in cell death/survival signaling. Cisplatin decreased XIAP protein levels and induced Akt cleavage and apoptosis in chemosensitive, but not in resistant, ovarian cancer cells. Cisplatin also induced cleavage of caspase-9 and caspase-3, a process blocked by XIAP overexpression. Pretreatment of ovarian cancer cells and their whole cell lysate with tetrapeptide inhibitors of caspases in vitro significantly decreased Akt cleavage induced by cisplatin and exogenous active caspase-3. Adenoviral sense XIAP cDNA expression increased XIAP protein levels and increased Akt phosphorylation, indicative of activation of Akt and, likely, of PI 3-K. This was associated with a decrease in cisplatin-induced apoptosis. In a cell line (OVCAR-3) where basal phosphorylated Akt levels were high, XIAP overexpression failed to increase further the level of this phosphoprotein. XIAP down-regulation induced Akt cleavage and apoptosis, and treatment of whole cell lysate with human recombinant active caspase-3 resulted in a similar pattern of Akt cleavage. In the presence of the PI 3-K inhibitor (LY294002), XIAP overexpression failed to block cisplatin-induced apoptosis and to induce Akt phosphorylation, suggesting that the site of action of XIAP is upstream of Akt in this cell survival pathway. Taken together, the results indicate that XIAP prevents apoptosis through a PI 3-K-dependent inhibition of the caspase cascade. These results demonstrate a novel mechanism by which XIAP regulates apoptosis and the possible involvement of the PI 3-K/Akt survival pathway in XIAP-mediated chemoresistance of ovarian cancer cells.     

Restoration of XAF1 expression induces apoptosis and inhibits tumor growth in gastric cancer

XAF1 (XIAP‐associated factor 1) is a novel XIAP binding protein that can antagonize XIAP and sensitize cells to other cell death triggers. Our previous results have shown that aberrant hypermethylation of the CpG sites in XAF1 promoter is strongly associated with lower expression of XAF1 in gastric cancers. In our study, we investigated the effect of restoration of XAF1 expression on growth of gastric cancers. We found that the restoration of XAF1 expression suppressed anchorage‐dependent and ‐independent growth and increased sensitivity to TRAIL and drug‐induced apoptosis. Stable cell clones expressing XAF1 exhibited delayed tumor initiation in nude mice. Restoration of XAF1 expression mediated by adenovirus vector greatly increased apoptosis in gastric cancer cell lines in a time‐ and dose‐dependent manner and sensitized cancer cells to TRAIL and drugs‐induced apoptosis. Adeno‐XAF1 transduction induced cell cycle G2/M arrest and upregulated the expression of p21 and downregulated the expression of cyclin B1 and cdc2. Notably, adeno‐XAF1 treatment significantly inhibited tumor growth, strongly enhanced the antitumor activity of TRAIL in a gastric cancer xenograft model in vivo, and significantly prolonged the survival time of animals bearing tumor xenografts. Complete eradication of established tumors was achieved on combined treatment with adeno‐XAF1 and TRAIL. Our results document that the restoration of XAF1 inhibits gastric tumorigenesis and tumor growth and that XAF1 is a promising candidate for cancer gene therapy. © 2009 UICC     

X-linked inhibitor of apoptosis (XIAP) and XIAP-associated factor-1 expressions and their relationship to apoptosis in human hepatocellular carcinoma and non-cancerous liver tissues

The X-linked inhibitor of apoptosis (XIAP) belongs to the inhibitor of apoptosis (IAP) family, and the action of XIAP is inhibited by XIAP-associated factor-1 (XAF1). In the present study, XIAP and XAF1 protein expressions and their relationship to apoptosis were investigated in hepatocellular carcinoma (HCC). We examined immunohistochemical expressions of XIAP and XAF1, and the number of apoptotic HCC cells in surgically resected tissues of 24 HCCs, consisting of 7 well-, 10 moderately and 7 poorly differentiated HCCs. As a result, XIAP and XAF1 expressions were identified in the cytoplasm of non-neoplastic and neoplastic hepatocytes. In the 24 HCCs, XIAP expression was not different according to the histological grade of HCC. In contrast, XAF1 expression was significantly lower in poorly differentiated than that in well- or moderately differentiated HCCs (P=0.001), or XIAP expression in poorly differentiated HCC (P<0.001). Apoptotic HCC cell number was significantly lower in poorly differentiated than that in well- or moderately differentiated HCCs (P<0.01). A significant relationship was observed between XAF1 expression and apoptotic cell number in HCC tissues. In conclusion, the present findings suggest that significantly low XAF1 expression, but not XIAP expression, in poorly differentiated HCC may relate to resistance to apoptosis.     

XIAP及其相关因子1在急性淋巴细胞白血病中表达意义

目的：探讨X染色体连锁凋亡抑制蛋白（XIAP）及其相关因子（XAF1）在急性淋巴细胞白血病（ALL）中的表达及其临床意义,并评估其在临床治疗及预后中的价值。方法：采用病例对照研究,应用实时荧光定量聚合酶链反应（RQ-PCR）检测85例ALL患者骨髓标本中XIAP及XAF1的mRNA表达水平。结果：XIAP mRNA表达水平初诊ALL组高于CR组和对照组（P〈0.05）,而低于复发组（P〈0.05）,CR组表达水平高于对照组（P〈0.05）;而XAF1在ALL时呈低表达或不表达,CR组表达高于ALL其它组（P〈0.05）,与对照组差异无统计学意义（P〉0.05）。XIAP及XAF1二基因表达水平在T系ALL与B系ALL,成人与儿童,男女性别之间表达水平差异无统计学意义（P〉0.05）。XIAP/XAF1比值在ALL患者中初诊组和复发组明显高于对照组和缓解组（P〈0.05）,缓解组高于对照组（P〈0.05）。结论：ALL患者XIAP基因高表达,而XAF1呈现低表达或不表达,提示XIAP可能通过抑制白血病细胞凋亡参与了ALL的发生发展,并与预后不良及治疗反应相关。ALL中XIAP与XAF1表达水平的不平衡,可能是ALL预后不良及复发的一项重要因素之一。抑制XIAP及上调XAF1基因来治疗ALL,将为ALL的基因治疗提供新思路。     

XIAP及其相关因子1在急性淋巴细胞白血病中表达意义

目的:探讨X染色体连锁凋亡抑制蛋白(XIAP)及其相关因子(XAF1)在急性淋巴细胞白血病(ALL)中的表达及其临床意义,并评估其在临床治疗及预后中的价值。方法:采用病例对照研究,应用实时荧光定量聚合酶链反应(RQ-PCR)检测85例ALL患者骨髓标本中XIAP及XAF1的mRNA表达水平。结果:XIAP mRNA表达水平初诊ALL组高于CR组和对照组(P<0.05),而低于复发组(P<0.05),CR组表达水平高于对照组(P<0.05);而XAF1在ALL时呈低表达或不表达,CR组表达高于ALL其它组(P<0.05),与对照组差异无统计学意义(P>0.05)。XIAP及XAF1二基因表达水平在T系ALL与B系ALL,成人与儿童,男女性别之间表达水平差异无统计学意义(P>0.05)。XIAP/XAF1比值在ALL患者中初诊组和复发组明显高于对照组和缓解组(P<0.05),缓解组高于对照组(P<0.05)。结论:ALL患者XIAP基因高表达,而XAF1呈现低表达或不表达,提示XIAP可能通过抑制白血病细胞凋亡参与了ALL的发生发展,并与预后不良及治疗反应相关。ALL中XIAP与XAF1表达水平的不平衡,可能是ALL预后不良及复发的一项重要因素之一。抑制XIAP及上调XAF1基因来治疗ALL,将为ALL的基因治疗提供新思路。     

三氧化二砷联合顺铂对人类非小细胞肺癌细胞生长及凋亡抑制基因XIAP表达的影响

背景与目的 在中国肺癌居各种肿瘤之首位,寻求肺癌有效的治疗方法已成为人们关注的热点.本研究探讨三氧化二砷(As2O3)注射液与顺铂(DDP)联合应用对人类非小细胞肺癌细胞的生长、细胞凋亡及其X染色体连锁的凋亡抑制蛋白(XIAP)的影响.方法 采用四甲基偶氮唑蓝(MTT)比色法观察As2O3注射液和/或DDP对人类非小细胞肺癌细胞H460生长的影响;应用流式细胞术观察As2O3注射液和/或DDP处理前后细胞凋亡率;应用半定量RT-PCR检测As2O3注射液和/或DDP处理前后H460细胞中XIAP mRNA表达变化.结果 与单药作用相比,As2O3与DDP联合应用可显著抑制H460的增殖,增加细胞的凋亡率,并对细胞XIAP mRNA表达有增强抑制的作用.结论 与单药相比较,As2O3注射液联合DDP能够明显提高非小细胞肺癌对化疗的敏感性,其机制可能与抑制非小细胞肺癌的凋亡抑制蛋白XIAP的表达有关.     

Tumor suppressor XIAP‐Associated factor 1 (XAF1) cooperates with tumor necrosis factor‐related apoptosis‐inducing ligand to suppress colon cancer growth and trigger tumor regression

BACKGROUND:

XIAP‐associated factor 1 (XAF1) antagonizes the anticaspase activity of XIAP (X‐linked inhibitor of apoptosis) and functions as a tumor suppressor in colon cancer. The tumor necrosis factor‐related apoptosis‐inducing ligand (TRAIL) is known as a potential anticancer agent. In this study, the synergistic effect of XAF1 and TRAIL on colon cancer growth was investigated.

METHODS:

Adeno‐XAF1 virus was generated and purified. Cell apoptosis was detected by flow‐cytometry and terminal deoxynucleotidyl transferase‐mediated dUTP nick‐end labeling assay. Protein expression of the different genes was determined by Western blot analysis. Tumorigenesis and tumor growth were assessed in subcutaneous nude mouse xenograft experiments.

RESULTS:

Stable overexpression of XAF1‐sensitized colon cancer cells to TRAIL‐induced apoptosis significantly increased the activity of caspase 3, 7, 8, and 9; released cytochrome c; and down‐regulated XIAP, survivin, and c‐IAP‐2. The restoration of XAF1 expression mediated by adenovirus (adeno‐XAF1) directly induced apoptosis, and synergized TRAIL‐induced apoptosis in colon cancer cells. Ex vivo transduction of adeno‐XAF1 suppressed colon cancer formation in vivo. Furthermore, adeno‐XAF1 treatment of mice significantly inhibited tumor growth, strongly enhanced TRAIL‐induced apoptosis and antitumor activity in colon cancer xenograft models in vivo, and markedly prolonged the survival. Notably, the combined treatment with adeno‐XAF1 and TRAIL completely eradicated the established tumors without detectable toxicity in normal tissue.

CONCLUSIONS:

The combined restoration of XAF1 expression and TRAIL treatment may be a potent strategy for colon cancer therapy. Cancer 2010. © 2010 American Cancer Society.     

Effect of Diallyl Trisulfide on Human Ovarian Cancer SKOV-3/DDP Cell Apoptosis

Aim: To investigate the effects of diallyl trisulfide (DT) on apoptosis of cisplatin (DDP)-resistant humanepithelial ovarian cancer SKOV-3 cells (SKOV-3/DDP), and the role of p53 upregulated modulator of apoptosis(PUMA). Methods: SKOV-3/DDP cells were randomly divided into control, DT, DPP and DPP+DT groups,which were treated with DT or combined DT and DDP. All cells were incubated for 48 h. and apoptosis rateswere assessed by flow cytometry. mRNA and protein expression of PUMA, Bax and Bcl-2 was determined byRT-PCR and Western blot assays, respectively. Results: Compared with control group, the apoptosis rates ofSKOV-3/DDP cells in DT groups were obviously increased, with dose-dependence (P < 0.05), the mRNA andprotein expressions of PUMA, Bax also being up-regulated (P < 0.05), while those of Bcl-2 were down-regulated(P < 0.05). Compared with DT groups, the apoptosis rate in the DDP+DT group was significantly increased (P< 0.05). After knockdown of PUMA with specific siRNA, the apoptosis rate of SKOV-3/DDP cells was obviouslydecreased (P < 0.05). Conclusion: DT can promote the apoptosis of SKOV-3/DDP cells with PUMA playing acritical role.     

MLH1 expression sensitises ovarian cancer cells to cell death mediated by XIAP inhibition

Background:

The X-linked inhibitor of apoptosis protein (XIAP), an endogenous apoptosis suppressor, can determine the level of caspase accumulation and the resultant response to apoptosis-inducing agents such as cisplatin in epithelial ovarian cancer (EOC). In addition, the mismatch repair protein, hMLH1, has been linked to DNA damage-induced apoptosis by cisplatin by both p53-dependent and -independent mechanisms.

Methods:

In this study, hMLH1 expression was correlated with clinical response to platinum drugs and survival in advanced stage (III–IV) EOC patients. We then investigated whether MLH1 loss was a determinant in anti-apoptosis response to cisplatin mediated by XIAP in isogenic and established EOC cell lines with differential p53 status.

Results:

The percentage of cells undergoing cisplatin-induced cell killing was higher in MLH1-proficient cells than in MLH1-defective cells. In addition, the presence of wild-type hMLH1 or hMLH1 re-expression significantly increased sensitivity to 6-thioguanine, a MMR-dependent agent. Cell-death response to 6-thioguanine and cisplatin was associated with significant proteolysis of MLH1, with XIAP destabilisation and increased caspase-3 activity. The siRNA-mediated inhibition of XIAP increased MLH1 proteolysis and cell death in MLH1-proficient cells but not in MLH1-defective cells.

Conclusion:

These data suggest that XIAP inhibitors may prove to be an effective means of sensitising EOC to MLH1-dependent apoptosis.     

Tumor suppressor XAF1 induces apoptosis,inhibits angiogenesis and inhibits tumor growth in hepatocellular carcinoma

X-linked inhibitor of apoptosis (XIAP)-associated factor 1 (XAF1), a XIAP-binding protein, is a tumor suppressor gene. XAF1 was silent or expressed lowly in most human malignant tumors. However, the role of XAF1 in hepatocellular carcinoma (HCC) remains unknown. In this study, we investigated the effect of XAF1 on tumor growth and angiogenesis in hepatocellular cancer cells. Our results showed that XAF1 expression was lower in HCC cell lines SMMC-7721, Hep G2 and BEL-7404 and liver cancer tissues than that in paired non-cancer liver tissues. Adenovirus-mediated XAF1 expression (Ad5/F35-XAF1) significantly inhibited cell proliferation and induced apoptosis in HCC cells in dose- and time- dependent manners. Infection of Ad5/F35-XAF1 induced cleavage of caspase -3, -8, -9 and PARP in HCC cells. Furthermore, Ad5/F35-XAF1 treatment significantly suppressed tumor growth in a xenograft model of liver cancer cells. Western Blot and immunohistochemistry staining showed that Ad5/F35-XAF1 treatment suppressed expression of vascular endothelial growth factor (VEGF), which is associated with tumor angiogenesis, in cancer cells and xenograft tumor tissues. Moreover, Ad5/F35-XAF1 treatment prolonged the survival of tumor-bearing mice. Our results demonstrate that XAF1 inhibits tumor growth by inducing apoptosis and inhibiting tumor angiogenesis. XAF1 may be a promising target for liver cancer treatment.     

XAF1 as a prognostic biomarker and therapeutic target in pancreatic cancer

XAF1 (X chromosome‐linked inhibitor of apoptosis [XIAP]‐associated factor 1) is a novel XIAP modulator that negatively regulates the anti‐apoptotic effects of XIAP and sensitizes cells to other cell death triggers. It has been reported to be downregulated in a variety of human cancer cell lines. However, the role of XAF1 in pancreatic carcinogenesis remains unclear. In the present study, we investigated the prognostic values of XAF1 expression and its regulation in cancer cell growth and apoptosis both in vitro and in vivo. From the immunohistochemistry staining of tissue microarray, 40 of 89 (44.9%) pancreatic specimens showed low levels of XAF1 expression. Statistical analysis suggested the downregulation of XAF1 was significantly correlated with tumor staging (P = 0.047) and those patients with low XAF1 levels had shorter survival times (P = 0.0162). Multivariate analysis indicated that XAF1 expression was an independent prognostic indicator of the survival of patients with pancreatic cancer (P = 0.007). Furthermore, we found that restoration of XAF1 expression mediated by Ad5/F35 virus suppressed cell proliferation and induced cell cycle arrest and apoptosis, accompanied by the activation of caspases 3, 8, and 9 and poly(ADP‐ribose) polymerase as well as increased level of cytochrome c and Bid cleavage. Notably, XAF1 restoration robustly decreased survivin expression rather than XIAP. In addition, in vivo s.c. xenografts from Ad5/F35‐XAF1 treatment, which showed less cellular proliferation and enhanced apoptosis, were significantly smaller than those from control groups. Our findings document that XAF1 is a valuable prognostic marker in pancreatic cancer and could be a potential candidate for cancer gene therapy. (Cancer Sci 2009)     

siRNA Silencing EZH2 Reverses Cisplatin-resistance of Human Non-small Cell Lung and Gastric Cancer Cells

Clinical resistance to chemotherapeutic agents is one of the major hindrances in the treatment of humancancers. EHZ2 is involved in drug resistance and is overexpressed in drug-resistant cancer cell lines. In this study,we investigated the effects of EHZ2 on cisplatin -resistance in A549/DDP and AGS/DDP cells. EHZ2 mRNA andprotein were found to be significantly overexpressed in A549/DDP and AGS/DDP cells, compared to parentalcells. EHZ2 siRNA successfully silenced EHZ2 mRNA and protein expression. Proliferation was inhibited anddrug resistance to cisplatin was improved. Flow cytometry showed that silencing of EHZ2 arrested A549/DDPand AGS/DDP cells in the G0/G1 phase, increasing apoptosis, rh-123 fluorescence intensity and caspase-3/8activities. Silencing of EHZ2 also significantly reduced the mRNA and protein expression levels of cyclin D1 andMDR1,while up-regulating p15, p21, p27 and miR-218 in A549/DPP cells. Furthermore, silencing of EHZ2 alsosignificantly increased the expression level of tumor suppressor factor miR-218. We also found down-regulatingEHZ2 expression increased methylation in A549/DDP and AGS/DDP cells. This study demonstrates that drugresistance can be effectively reversed in human cisplatin-resistant lung and gastric cancer cells through deliveryof siRNAs targeting EHZ2.     

Survivin反义寡核苷酸对卵巢癌耐药细胞COC1/DDP抑制的实验研究

背景与目的: Survivin是近年发现的一种细胞凋亡抑制基因, 它与卵巢癌细胞的生长及耐药性密切相关,本研究探讨经脂质体介导的 survivin反义寡核苷酸 (Lip-ASODN)对人卵巢癌耐药细胞 COC1/DDP生长、凋亡及细胞周期的影响.方法:将脂质体介导的 survivin-ASODN转染 COC1/DDP细胞;细胞动力学检测、 MTT法观察细胞生长情况; RT-PCR检测 survivin mRNA的表达;通过 Western杂交检测 caspase-3蛋白及活性;流式细胞仪分析细胞凋亡率及细胞周期变化.结果:与空脂质体及 SODN组相比,经 survivin-ASODN作用后的 COC1/DDP细胞的生长受到明显抑制, 72 h的细胞生长抑制率可达( 68.3± 6.2)%( P< 0.05). Survivin mRNA的表达明显下降,而 caspase-3的活性增加,并呈时间依赖性. ASODN组细胞周期发生了明显变化,细胞被阻滞于 G0/G1期,占 79.21%, G2/M及 S期分别为 4.92%、 15.87%,均明显下降;细胞凋亡率为 33.18%,明显高于 SODN组及空脂质体组( P< 0.05).结论: Survivin ASODN能抑制人卵巢癌耐药细胞 COC1/DDP生长,降低 survivin mRNA的表达并诱导 COC1/DDP细胞凋亡.     

RNA干扰survivin基因对卵巢癌细胞生长凋亡及药物敏感性的影响

目的 探讨下调survivin基因对卵巢癌顺铂(DDP)耐药细胞株SKOV3/DDP细胞生长、凋亡及药物敏感性的影响.方法 构建survivin基因的小干扰RNA(siRNA)表达载体pSilencer-survivin,用脂质体方法转染SKOV3/DDP细胞株,另设未转染组和转染pSilencer-control组作为对照.显微镜下观察转染前后细胞的变化,逆转录聚合酶链反应(RT-PCR)和Western blot分别检测survivinmRNA及蛋白的表达,二苯基溴化四氮唑蓝(MTT)法检测细胞生长情况,流式细胞仪检测细胞凋亡及周期的变化.各组细胞加入DDP,检测其对DDP药物敏感性的影响.结果 survivin siRNA可明显下调SKOV3/DDP细胞中survivin mRNA及蛋白表达水平.SKOV3/DDP细胞生长受到明显抑制,生长曲线低平.转染48 h后,转染pSilencer-survivin组细胞凋亡率为19.1%,明显高于末转染组(2.6%)和转染pSilencer-control组(3.5%),G1/G0期细胞所占的比例增高,而G2/M期细胞所占的比例降低.转染pSilencer-survivin组细胞的IC50明显降低,为1.16 μg/mi.结论 survivin siRNA可下调SKOV3/DDP细胞中survivin的表达,使细胞生长减慢,凋亡增加,对DDP的药物敏感性增强.     

细胞色素c 介导的半胱天冬氨酸蛋白酶-3活性改变与人卵巢癌顺铂耐药的关系

目的：探讨人卵巢癌顺铂耐药细胞株A2780／DDP、COC1／DDP中抗凋亡基因bcl-XL、细胞色素c的表达和半胱天冬氨酰蛋白酶－3（caspase-3)活性对人卵巢癌顺铂耐药的影响。方法：采用逆转录聚合酶链反应（RT－PCR）和Western blot检测人卵巢癌顺铂敏感细胞株A2780、COC1和顺铂耐药株A2780／DDP、COC1／DDP中bcl-XL的表达,以及顺铂作用后细胞色素c的含量和caspase-3活性的变化,并应用流式细胞仪测定顺铂作用后A2780、COC1、A2780／DDP、COC1／DDP细胞的凋亡率。结果：bcl-XL在A2780/DDP、COC1／DDP细胞中的表达明显高于A2780、COC1细胞;顺铂作用后,细胞色素c在A2780／DDP、COC1／DDP细胞中的表达明显减少,caspase-3活性和凋亡率也较A2780和COC1细胞明显降低（P＜0．05）。结论：人卵巢部细胞对顺铂产生耐药可能与细胞内bcl-XL过度表达、细胞色素c释放受抑制和caspase-3活性下降有关。     

Sevoflurane inhibits proliferation, induces apoptosis, and blocks cell cycle progression of lung carcinoma cells

Purpose: Sevoflurane, an inhalational anesthetic, is used extensively during lung cancer surgery. However, the effect of sevoflurane on growth of lung carcinoma cells remains unclear. The purpose of this study is to investigate effects on proliferation, apoptosis, and cell cycling in the A549 human lung adenocarcinoma cell line. Methods: A549 cells were treated with 1.7%, 3.4%, and 5.1 % sevoflurane for 2, 4, and 6 hours. Cell proliferation was evaluated by the MTT assay and colony formation assay. Apoptosis and cell cycle was analyzed by flow cytometry. Expression of X-linked inhibitor of apoptosis protein (XIAP), survivin, Bcl-2, Bax, caspase-3, cyclin A, cyclin B1, and cdc2 was measured by Western blotting. Results: Sgnificant inhibition of cell proliferation and induction of apoptosis were found in A549 cells after sevoflurane treatment. Simultaneously, expression of XIAP and survivin was surpressed, while that of caspase-3 increased significantly, but Bcl-2 and Bax were not altered. Sevoflurane caused cell cycle arrest at the G2/M phase. At the same time, data revealed that cyclin A, cyclin B1, and cdc2 expression was down-regulated after sevoflurane treatment. Conclusion: This study demonstrated that sevoflurane inhibited proliferation, and induced apoptosis in human lung adenocarcinoma A549 cells, associated with down-regulated expression of XIAP and suvivin, and activating caspase-3.     

奈达铂体外诱导人卵巢癌顺铂耐药细胞凋亡的机制

目的探讨研究奈达铂（NDP）体外对人卵巢癌顺铂原发耐药细胞株SKOV3和继发耐药株SKOV3/DDP的抑制作用及其机制。方法采用MTT法检测体外不同时间不同浓度的NDP对SKOV3及SKOV3/DDP细胞的杀伤作用,计算半数抑制浓度(IC50);流式细胞术(FCM)测定给药前后细胞凋亡率及周期分布变化;半定量PCR分析凋亡基因Bcl-2,Bax,caspase-3,caspase-9及反应肿瘤细胞侵袭力的基因MMP-2的表达变化。结果NDP能明显抑制SKOV3及SKOV3/DDP生长,呈时间-剂量依赖性;流式结果显示随时间、浓度增加,SKOV3和SKOV3/DDP细胞凋亡明显增加,S期细胞的比例增加,差异均有统计学意义（P<0.05）。与对照组相比,Bcl-2、MMP-2表达减少,Bax,caspase-3,caspase-9表达增加。结论NDP可抑制SKOV3和SKOV3/DDP细胞增殖,其机制可能与诱导细胞凋亡,细胞S期阻滞,下调Bcl-2及上调Bax,caspase-3,caspase-9的表达有关;同时可下调MMP-2的表达,有利于降低卵巢癌细胞的侵袭力。