Soluble CD40 ligands sensitize the epithelial ovarian cancer cells to cisplatin treatment

ObjectiveCD154 (CD40L) is a protein that is primarily expressed on activated T cells and is a member of the TNF superfamily of molecules. It binds to CD40 on antigen-presenting cells (APC), which leads to many effects depending on the target cell type. Being an activator of immune cells, CD40L has also been shown to directly induce apoptosis in tumor cells by multiple mechanisms. To understand the role of sCD40L in regulating the proliferation of epithelial ovarian cancer cells treated or untreated with cisplatin.MethodsEpithelial ovarian cancer cells: SKOV3 and its cisplatin-resisitant strain SKOV3/DDP cells were used to test the effect of sCD40L and cisplatin. The proliferation of SKOV3 and SKOV3/DDP cells were measured by MTT. Cell cycle was assessed by flow cytometry. The mRNA expressions of targeted genes were detected by qRT-PCR. The protein expressions were detected by Western blotting.ResultssCD40L showed a significant dose-dependence inhibitory effect on the proliferation of ovarian cancer cell lines. sCD40L in combination with cisplatin could sensitized SKOV3/DDP cells to cisplatin treatment and reversed the drug resistance of SKOV3/DDP cells. The reversal ratios of 1 μg/ml sCD40L combined with cisplatin in SKOV3 and SKOV3/DDP cells were 2.11, 2.71, while the reversal ratios of 2 μg/ml sCD40L combined with cisplatin in SKOV3 and SKOV3/DDP cells were 3.78, 5.20, respectively. sCD40L or sCD40L combined cisplatin increased tumor cells in G0/G1 phase. sCD40L in combination with cisplatin decreased the expression levels of GST-π, LRP, Survivin, p53 and Bcl-2 in both epithelial ovarian cancer cell lines. The protein expression level of GST-π, LRP and P53 protein was also decreased upon sCD40L in combination with cisplatin although the expression level of Bcl-2 and survivin protein had no significant difference.ConclusionsCD40L inhibits the proliferation of SKOV3 and SKOV3/DDP cells. The combined application of sCD40L and cisplatin can strength the inhibitory effect of cisplatin, and to a certain extent, reversing the resistance to cisplatin in SKOV3/DDP cells. sCD40L could lead a cell block in G0/G1 phase and make the cell growth restrained. sCD40L could induce SKOV3 and SKOV3/DDP cells apoptosis and reverse drug resistance through cutting GST-π mRNA, LRP mRNA, survivin mRNA, p53 mRNA and Bcl-2 mRNA and decreasing the expression of GST-π, LRP and P53 protein in SKOV3 and SKOV3/DDP cells, which provides in-vivo experiment basis to the application of sCD40L as a drug improving ovarian cancer cells sensitivity to cisplatin.     

拉帕替尼对人卵巢癌SKOV3裸鼠移植瘤的影响

【目的】探讨新型靶向治疗药物拉帕替尼对人卵巢癌SKOV3裸鼠移植瘤生长的影响。【方法】将人SKVO3细胞株接种于裸小鼠胸壁皮下建立卵巢癌裸小鼠移植瘤模型。随机将其分为空白对照组（A组）和实验组（B组）,实验组根据拉帕替尼药物浓度的不同分为两个亚组：100 mg/kg （B1）和200 mg/kg （B2）,检测各组拉帕替尼用药前后荷瘤体积及裸鼠体质量的变化。【结果】拉帕替尼可显著抑制SKOV3裸小鼠移植瘤的生长,呈剂量依赖性,B2组抑瘤率显著高于B1组,其差异有统计学意义（P＜0．05）,但小鼠体质量用药前后比较无显著性差异（P＞0．05）。【结论】拉帕替尼可显著缩小人卵巢癌细胞株SKVO3裸鼠移植瘤的体积,为卵巢癌新型靶向治疗提供了理论基础。     

沉默缺氧诱导因子-1α对卵巢癌化疗敏感性的影响

目的观察沉默缺氧诱导因子HIF-1α对卵巢癌细胞化疗敏感性的相关影响并探讨其机理。方法构建HIF-1α基因短发夹RNA真核表达质粒,并转染卵巢癌细胞SKOV3（人卵巢浆液性囊腺癌细胞株）。实验可分为空白组,非特异对照组（转染pNonspecific-siRNA,即SKOV3NS）,目的siRNA组（转染pHIF-siRNA,即SKOV3siRNA）3组,用RT-PCR及Western Blot法分别检测各组HIF-1α基因的mRNA和蛋白的表达水平。细胞经过20μmol/L顺铂作用后,用MTT法检测细胞生长抑制率,流式细胞术（FCS）检测细胞凋亡率。结果 SKOV3siRNA转染组HIF-1α基因在mRNA水平和蛋白水平表达均明显降低,SKOV3siRNA组肿瘤细胞的生长抑制率和细胞凋亡率均明显增高（P〈0.05）。结论 RNA干扰技术沉默HIF-1α能够有效地抑制卵巢癌SKOV3细胞HIF-1α基因表达,增强其对化疗药物顺铂的敏感性。     

超声微泡介导自杀基因对卵巢癌细胞的抗瘤效应

目的探讨超声微泡介导自杀基因对卵巢癌SKOV3细胞抗瘤效应的机制。方法采用最适超声转染参数,以超声介导微泡转染pORF-HSV1TK质粒,将SKOV3细胞分成6组:阴性对照组(C组)、超声辐照组(U组)、脂质体组(L组,阳性对照组)、脂质体+超声辐照组(L+U组)、脂质体+微泡+超声辐照组(L+M+U组)和微泡+超声辐照组(M+U组)。以RT-PCR检测HSV1TK的表达,以MTT测定各组细胞增殖抑制率。光镜下观察各组转染细胞数量、形态变化,以流式细胞术(FCM)检测各组细胞凋亡率及细胞周期。结果L+M+U组HSV1TK基因表达最强,细胞增殖抑制率明显高于其他组(P均<0.05),且光镜下细胞数量减少最多,形态明显异常;其细胞凋亡率为(49.13±0.82)%,且大多数细胞周期被阻断于G1期(P<0.05)。结论超声介导微泡能增强HSV1TK/GCV系统抑制SKOV3增殖和诱导SKOV3凋亡的作用,且将SKOV3细胞周期阻断在G1期,从而发挥抗瘤效应。     

18F-FDG与人卵巢癌细胞株SKOV3结合实验方法学研究

目的：建立18F-FDG与人卵巢癌细胞株SKOV3结合实验的方法学。方法：在不同条件下测定18F-FDG与人卵巢癌细胞株SKOV3结合率。①细胞数分别为5×104个/瓶、1×105个/瓶、5×105个/瓶、1×106个/瓶、5×106个/瓶;②反应时间分别为20min、40min、60min、80min、100min和120min;③18F-FDG放射性活度分别为1.85KBq、3.7KBq、7.4KBq、14.8KBq和29.6KBq;④葡萄糖的浓度分别为0mmol/L、2.78mmol/L、5.55mmol/L和11.1mmol/L。用γ测量仪测量细胞的CPM（B）及上清液CPM（F）。计算18F-FDG的细胞结合率=[B/（B+F）]×100%。结果：①18F-FDG与人卵巢癌细胞株SKOV3结合率（y）与细胞数（x1）、反应时间（x2）、葡萄糖的浓度（x3）存在线性回归关系,回归方程：y=（7.395+5.18E-0.06x1+0.094x2-2.040x3）×100%,F=106.9,P<0.05。偏回归系数P均<0.05。校正决定系数R2c=0.728,P<0.05。细胞结合率与细胞数、反应时间、葡萄糖的浓度Pearson相关系数分别为0.581、0.179、-0.599,P均<0.05。②18F-FDG与人卵巢癌细胞株SKOV3结合实验基本条件：细胞数量为1×106个/瓶,18F-FDG放射性活度3.7KBq,反应时间100min,葡萄糖浓度0mmol/L,细胞结合率为（25.45±1.66）%。结论：建立了18F-FDG与人卵巢癌细胞株SKOV3结合实验的方法学,为18F-FDG结合实验早期评价放疗和化疗对人卵巢癌细胞株SKOV3的抑制作用奠定了基础。     

Antiproliferative activity of sulforaphane in Akt-overexpressing ovarian cancer cells

Epidemiologic studies show a correlation between increased consumption of fruits and vegetables with reduced risk of ovarian cancer. One major bioactive compound found in cruciferous vegetables, particularly broccoli, is sulforaphane, derived from the breakdown of glucoraphanin. We observed potent antiproliferative effects of sulforaphane on human ovarian cancer cell line SKOV3 (IC(50) 40 micromol/L) and mouse ovarian cancer cell lines C3 and T3 (IC(50) 25 micromol/L each) by cell viability assays. The loss of viability is reflected by a down-regulation of cell cycle transition regulators cyclin D1, cyclin-dependent kinase 4 (cdk4), and cdk6. The upstream mediators of sulforaphane effects on the cell cycle in ovarian cancer are still unknown. However, because the Akt signal transduction pathway is overactivated in ovarian cancer, we investigated the effects of sulforaphane on this prosurvival pathway. Both total Akt protein and active phosphorylated levels of Akt (Ser(473)) and phosphoinositide 3-kinase were significantly decreased in sulforaphane-treated SKOV3, C3, and T3 cells with a concomitant inhibition of Akt kinase activity by sulforaphane in SKOV3 and C3 cells. This inhibitory effect of sulforaphane leads to a potent induction of apoptosis in all three cell lines, along with the cleavage of poly(ADP)ribose polymerase. Our study is the first to report the antiproliferative effects of sulforaphane in ovarian cancer and identifying the Akt pathway as a target of sulforaphane, with implications for the inhibition of carcinogenesis by diet-based chemoprevention.     

An injectable depot system for sustained intraperitoneal chemotherapy of ovarian cancer results in favorable drug distribution at the whole body, peritoneal and intratumoral levels

The current study characterizes the impact of docetaxel (DTX) distribution on efficacy following sustained intraperitoneal (IP) chemotherapy in murine models of ovarian cancer. A polymer-lipid biodegradable depot (PoLigel) was used to deliver DTX in a sustained manner over 21-days following IP administration. Distribution and efficacy studies were carried out in SCID mice bearing SKOV3 IP solid tumors or C57BL/6 mice with ID8 IP ascites fluid. In addition, a subcutaneous (SC) SKOV3 model was used to determine whether systemic drug levels that result from IP administration of the PoLigel influence antitumor efficacy. Immunostained IP and SC SKOV3 tumor sections were used to study cell death, intratumoral drug distribution and tumor penetration. Sustained concentrations of DTX were observed in plasma, tissue, tumor and ascites over the entire study period. Drug accumulation was several fold greater in tumors and ascites when compared to plasma levels. Sustained chemotherapy resulted in significant reduction in tumor burden and ascites volume. IP tumors showed greater cell death compared to the SC tumors as seen by higher TUNEL and caspase-3 expression. At the intratumoral level, DTX distributed more towards the core of IP tumors compared to the SC tumors. Tumor penetration of drug from nearest blood vessel was 1.5 fold greater in the IP tumors than the SC tumors. Overall, favorable drug distribution at the whole-body, peritoneal and intratumoral levels in combination with local and systemic sustained drug exposure contribute to the high efficacy observed. These results encourage the clinical use of IP sustained chemotherapy for ovarian cancer.     

5-氮-2’脱氧胞苷对人卵巢癌细胞RUNX3基因启动子区甲基化状态的影响

目的研究抑癌基因RUNX3启动子区在人卵巢癌细胞系（3AO、SKOV3）及正常卵巢细胞系（NOEC）中的甲基化状态,以及5-氮-2’脱氧胞苷（5-aza-2＇-deoxycytidine）对人卵巢癌细胞3AO及SKOV3增殖与凋亡及RUNX3mRNA表达的影响,探讨RUNX3基因甲基化与卵巢癌发生及发展的关系。方法用甲基化特异性PCR（MSP）法检测经特异性甲基转移酶抑制剂5-氮-2’脱氧胞苷处理前后卵巢癌细胞3AO、SKOV3以及NOEC中RUNX3基因启动子区的甲基化状态;并用0．5、5、50μmol／L 5-氮-2’脱氧胞苷处理人卵巢癌细胞株3AO及SKOV3共3d,继续常规培养5d后,采用四唑盐（MTT）比色观察细胞经药物处理前后的生长活性,以半定量RT-PCR检测细胞经药物处理前后抑癌基因RUNX3mRNA的表达,应用流式细胞仪进行细胞凋亡率的检测。结果正常卵巢细胞中未见RUNX3启动子甲基化,卵巢癌细胞3AO及SKOV3均发现RUNX3启动子甲基化现象;卵巢癌细胞3AO、SKOV3均可见RUNX3mRNA表达,但与正常卵巢细胞比较表达较弱,经药物处理癌细胞后其RUNX3mRNA的表达较前明显增强,且其表达强度与5-氮-2’脱氧胞苷的浓度存在剂量依赖关系;与加药前比较,5-氮-2’脱氧胞苷在0．5、5、50μmol／L浓度时均能明显抑制肿瘤细胞生长,随5-氮-2’脱氧胞苷浓度增加,细胞生长速率下降,加药前细胞凋亡率SKOV3为（2．71±0．81）％;3AO为（3．23±0．68）％,经5-氮-2’脱氧胞苷0．5、5、50μmol／L处理细胞后,细胞凋亡率SKOV3为（2．71±0．81）％、（15．43±1．33）％、（25．55±1．61）％;3AO为（10．66±2．09）％、（16．51±1．42）％、（25．46±1．55）％。与加药前比较,差异均有统计学意义（P〈0．01）,且凋亡率与5-氮-2’脱氧胞苷浓度呈剂量依赖关系（F=138．7;142．3,均P〈0．05）。结论特异性甲基转移酶抑制剂5-氮-2’脱氧胞苷可逆转、恢复并增强人卵巢癌细胞系3AO及SKOV3中RUNX3基因表达,抑制癌细胞生长及诱导部分癌细胞凋亡。     

熊果酸对人卵巢癌 SKOV3细胞增殖与凋亡的影响

【目的】探讨熊果酸(Ursolic acid,UA)对人卵巢癌 SKOV3细胞增殖与凋亡的影响。【方法】常规培养人卵巢癌 SKOV3细胞,应用 UA 浓度为5、10、20、40和80μmol/L 分别干预12 h、24 h 和48 h,采用 MTT 法检测细胞增殖;采用流式细胞仪检测细胞凋亡和细胞周期变化,应用 Western blot 技术检测细胞周期相关蛋白 P21、CyclinD1的表达水平。【结果】与空白对照组(0μmol/L UA)比较,40和80μmol/L UA 分别作用 SKOV3细胞12 h、24 h 和48 h 后 OD 值均显著降低(P <0.05);10、20和40μmol/L UA 作用24 h 后 SKOV3细胞早期凋亡率、晚期凋亡率和 G0/G1期比率较对照组升高,20和40μmol/L UA 作用24 h 后 SKOV3细胞周期蛋白 Cyclin D1明显降低而 P21水平显著升高(P <0.05)。【结论】体外实验中,UA 可抑制人卵巢癌 SKOV3细胞增殖和促其凋亡,其机制可能与细胞周期蛋白 Cyclin D1表达降低和 P21表达升高有关。     

MiR-139-5p通过靶向NFAT抑制卵巢癌SKOV3细胞的生长、集落形成、侵袭和迁移

目的:探究miR-139-5p对卵巢癌SKOV3细胞的生长、集落形成、侵袭能力以及迁移能力的影响及其机制。方法:采用qRT-PCR检测卵巢癌患者癌组织和卵巢癌细胞SKOV3中miR-139-5p表达情况。MiR-139-5p mimic转染SKOV3细胞,WST比色实验、集落形成实验和Transwell实验分别检测细胞的活性、集落形成、侵袭和迁移能力。采用TargetScan在线软件筛选miR-139-5p的潜在靶基因NFAT,并进一步验证。结果:MiR-139-5p在卵巢癌组织和SKOV3细胞中表达异常降低。MiR-139-5p过表达显著抑制SKOV3细胞的生长、集落形成、迁移及侵袭能力。NFAT是miR-139-5p的靶基因。过表达NFAT能逆转miR-139-5p过表达对SKOV3细胞集落形成、侵袭能力以及迁移能力与侵袭的抑制作用。结论:MiR-139-5p通过抑制靶基因NFAT来抑制卵巢癌SKOV3细胞的生长、集落形成、侵袭和迁移能力。     

Smoothened antagonists reverse taxane resistance in ovarian cancer

The hedgehog pathway has been implicated in the formation and maintenance of a variety of malignancies, including ovarian cancer; however, it is unknown whether hedgehog signaling is involved in ovarian cancer chemoresistance. The goal of this study was to determine the effects of antagonizing the hedgehog receptor, Smoothened (Smo), on chemotherapy response in ovarian cancer. Expression of hedgehog pathway members was assessed in three pairs of parental and chemotherapy-resistant ovarian cancer cell lines (A2780ip2/A2780cp20, SKOV3ip1/SKOV3TRip2, HeyA8/HeyA8MDR) using quantitative PCR and Western blot analysis. Cell lines were exposed to increasing concentrations of two different Smo antagonists (cyclopamine, LDE225) alone and in combination with carboplatin or paclitaxel. Selective knockdown of Smo, Gli1, or Gli2 was achieved using siRNA constructs. Cell viability was assessed by MTT assay. A2780cp20 and SKOV3TRip2 orthotopic xenografts were treated with vehicle, LDE225, paclitaxel, or combination therapy. Chemoresistant cell lines showed higher expression (>2-fold, P < 0.05) of hedgehog signaling components compared with their respective parental lines. Smo antagonists sensitized chemotherapy-resistant cell lines to paclitaxel, but not to carboplatin. LDE225 treatment also increased sensitivity of ALDH-positive cells to paclitaxel. A2780cp20 and SKOV3TRip2 xenografts treated with combined LDE225 and paclitaxel had significantly less tumor burden than those treated with vehicle or either agent alone. Increased taxane sensitivity seems to be mediated by a decrease in P-glycoprotein (MDR1) expression. Selective knockdown of Smo, Gli1, or Gli2 all increased taxane sensitivity. Smo antagonists reverse taxane resistance in chemoresistant ovarian cancer models, suggesting combined anti-hedgehog and chemotherapies could provide a useful therapeutic strategy for ovarian cancer.     

1,1-Bis(3'-indolyl)-1-(p-substituted phenyl)methanes inhibit ovarian cancer cell growth through peroxisome proliferator-activated receptor-dependent and independent pathways

1,1-Bis(3'-indolyl)-1-(p-t-butylphenyl)methane (DIM-C-pPhtBu) is a peroxisome proliferator-activated receptor gamma (PPARgamma) agonist, and treatment of SKOV3 ovarian cancer cells with this compound (5 micromol/L) inhibits cell proliferation, whereas up to 15 micromol/L rosiglitazone had no effect on cell growth. DIM-C-pPhtBu also inhibits G0-G1 to S phase cell cycle progression and this is linked, in part, to PPARgamma-dependent induction of the cyclin-dependent kinase inhibitor p21. DIM-C-pPhtBu induces PPARgamma-independent down-regulation of cyclin D1 and we therefore further investigated activation of receptor-independent pathways. DIM-C-pPhtBu also induced apoptosis in SKOV3 cells and this was related to induction of glucose-related protein 78, which is typically up-regulated as part of the unfolded protein response during endoplasmic reticulum (ER) stress. Activation of ER stress was also observed in other ovarian cancer cell lines treated with DIM-C-pPhtBu. In addition, DIM-C-pPhtBu induced CCAAT/enhancer binding protein homologous protein through both ER stress and c-jun NH2-terminal kinase-dependent pathways, and CCAAT/enhancer binding protein homologous protein activated death receptor 5 and the extrinsic pathway of apoptosis. These results show that DIM-C-pPhtBu inhibits growth and induces apoptosis in ovarian cancer cells through both PPARgamma-dependent and PPARgamma-independent pathways, and this complex mechanism of action will be advantageous for future clinical development of these compounds for treatment of ovarian cancer.     

三氧化二砷诱导卵巢癌细胞系SKOV3细胞凋亡的作用和机制

目的观察三氧化二砷(ATO)诱导人卵巢癌细胞系SKOV3细胞的凋亡作用和凋亡通路分子caspase 3、PARP,凋亡相关蛋白p-AKT、AKT蛋白表达的变化。方法分别用不同剂量的三氧化二砷作用人卵巢癌细胞系SKOV3细胞,应用MTT比色法检测培养48 h的细胞存活率,应用流式细胞仪测定培养48 h细胞凋亡的变化,免疫印迹法检测凋亡蛋白caspase 3、PARP和凋亡相关蛋白p-AKT、AKT的表达情况。结果 MTT示三氧化二砷能明显抑制SKOV3细胞增殖;流式细胞术显示三氧化二砷诱导SKOV3细胞凋亡,且具有明显的剂量依赖性;Western blotting检测发现药物能显著下调caspase 3、PARP、p-AKT的表达水平。结论三氧化二砷能显著抑制人卵巢癌细胞系SKOV3细胞增殖,诱导其凋亡,激活凋亡通路分子caspase 3、PARP,下调p-AKT蛋白表达水平,这可能是三氧化二砷诱导人卵巢癌细胞SKOV3细胞凋亡的作用机制。     

顺铂诱导人卵巢癌SKOV3和SKOV3/DDP细胞内质网应激差异的研究

目的研究顺铂诱导人卵巢癌SKOV3和SKOV3/DDP细胞内质网应激的差异。方法顺铂分别作用SKOV3组、SKOV3/DDP组细胞0 h、12 h、24 h。MTT检测两种细胞对顺铂敏感性差异;Western blot方法检测并比较内质网应激相关蛋白表达;RT-PCR方法检测顺铂对各时间点细胞的XBP-1表达。结果顺铂诱导两种细胞内质网应激存在显著差异。顺铂引起SKOV3细胞中内质网应激相关蛋白明显活化;而对SKOV3/DDP细胞中内质网应激相关蛋白没有明显影响;顺铂诱导SKOV3/DDP中XBP-1明显表达上调。结论顺铂诱导的内质网应激相关基因的表达差异可能是卵巢癌细胞耐药机制之一。     

Intracellular ferritin heavy chain plays the key role in artesunate-induced ferroptosis in ovarian serous carcinoma cells

Artesunate, an antimalarial drug, induces ferroptosis, but the mechanism is still unclear. In the present study, we investigated how Artesunate induces ferroptosis in ovarian serous carcinoma. Experiments were performed using the ovarian serous carcinoma cell lines CaOV3 and SKOV3ip1, and the sensitivity of CaOV3 to Artesunate was higher than that of SKOV3ip1. Ferroptosis inhibitors inhibited Artesunate-induced intracellular lipid peroxi­dation and cell death. However, unlike class 1 ferroptosis inducer erastin, Artesunate had no effect on intracellular glutathione-SH levels. We found that Artesunate-induced changes in lysosomal Fe‍²⁺ were parallel to the induction of ferroptosis. Therefore, ferritin, which oxidizes and binds intracellular Fe‍²⁺, may have an inhibitory effect on ferroptosis. Knockdown of nuclear coactivator 4, a key molecule of ferritinophagy (ferritin-specific autophagy), suppressed Artesunate-induced cell death. Knockdown of ferritin heavy chain by siRNA greatly enhanced the sensitivity to Artesunate, and overexpression of ferritin heavy chain greatly reduced the sensitivity of ovarian cancer cell lines to Artesunate. These results can explain the differential sensitivity of CaOV3 and SKOV3ip1 to Artesunate. In conclusion, enhancement of ferritinophagy is an important step involved in the mechanism of Artesunate-induced ferroptosis, and ferritin heavy chain levels may contribute to the regulation of sensitivity in Artesunate-induced ferroptosis in ovarian serous carcinoma cells.     

Smac和XIAP与卵巢癌细胞耐药及凋亡关系的研究

目的探讨Smac与卵巢癌耐药及凋亡机制。方法采用RT-PCR、Western blot方法分别从基因和蛋白水平检测卵巢癌细胞系SKOV3及其耐药细胞系SKOV3/DDP中Smac与XIAP的表达水平的差异。构建Smac真核表达载体pc DNA3.1(+)/EGFP+Smac。采用脂质体转染法将pc DNA3.1(+)/EGFP+Smac真核表达载体导入卵巢癌顺铂耐药细胞系SKOV3/DDP。运用Western blot法检测转染前后细胞内Smac与XIAP表达的变化,MTT法检测Smac表达增加后细胞生长抑制率,观察Smac基因对卵巢癌细胞凋亡的影响。结果 SKOV3、SKOV3/DDP细胞系中均表达Smac/DIABLO与XIAP,且SKOV3中Smac的相对含量高于SKOV3/DDP。SKOV3中XIAP的相对含量低于SKOV3/DDP。Western blot检测转染后耐药细胞Smac蛋白表达增加,XIAP表达无明显改变。MTT结果转染48 h与72 h后细胞生长抑制率出现明显增加,差异有统计学意义(P<0.01)。结论 Smac表达的上调对卵巢癌细胞XIAP表达无明显影响,但对肿瘤细胞生长有明显的抑制作用。提示Smac确实促进卵巢癌细胞凋亡,但其凋亡机制并不是促进XIAP降解,而是与XIAP相互结合抑制其与caspase结合,激活caspase进而起到促凋亡作用。     

Erythropoietin inhibits apoptosis induced by photodynamic therapy in ovarian cancer cells

Recombinant human erythropoietin is widely used to treat anemia associated with cancer and with the myelosuppressive effects of chemotherapy, particularly platinum-based regimens. Erythropoietin is the principal regulator of erythroid cell proliferation, differentiation, and apoptosis. Recently, the antiapoptotic and proliferative effects of erythropoietin on nonhematopoietic cells were also established. We now show the effect of erythropoietin treatment on the response of A2780 and SKOV3 ovarian carcinoma cell lines to photodynamic therapy (PDT) using hypericin. SKOV3 exhibited an increased resistance to hypericin when cells were treated with erythropoietin. This resistance was reversed by treatment of SKOV3 cells with the specific Janus kinase 2 kinase inhibitor AG490 or the tyrosine kinase inhibitor genistein. These results support a role for the specific erythropoietin-induced Janus kinase 2/STAT signal transduction pathway in PDT resistance. Evidence of erythropoietin signaling was obtained by the demonstration of Akt phosphorylation in both A2780 and SKOV3 cells. Erythropoietin-treated SKOV3 cells exhibited decreased apoptosis induced by hypericin, an effect that was blocked by the phosphoinositide 3-kinase/Akt inhibitor wortmannin. These results may have important implications for ovarian cancer patients undergoing PDT and receiving erythropoietin.     

Gli1表达抑制对卵巢癌SKOV3细胞增殖的影响

目的 抑制Glioma-associated oncogene homoglog（GLI）基因在卵巢癌SKOV3细胞中的表达,探讨GLI1表达抑制对SKOV3细胞增殖的影响.方法 构建并筛选GLI1基因的RNAi表达质粒,转染SKOV3细胞,CCK-8法比较转染前后卵巢癌细胞增殖变化,流式细胞仪检测紫杉醇诱导转染前后细胞凋亡比例,流式细胞仪检测转染前后SKOV3的细胞周期情况.Westem blot检测cyclinD、Bcl-2及Capase3蛋白表达的影响.结果 转染48 h后,在SKOV3细胞中Control组、N-Control组、GLI1 shRNA-1组、GLI1 shRNA-2组、GLI1shRNA-3组、GLI1 shRNA-4组的GLI1 mRNA水平分别为1.00±0.00、1.03 ±0.02、0.73±0.07、0.98 ±0.08、0.53±0.08、0.68±0.04,与Control组相比,GLI1 shRNA-1、GLI1 shRNA-3、GLI1 shRNA-4组GLI1 mRNA表达量均下降（P＜0.05）,转染GLI1 shRNA-3的SKOV3细胞中GLI1蛋白表达水平低于GLI1 shRNA-1、GLI1shRNA-2、GLI1 shRNA-4组（P＜0.05）,具有显著的干扰效果.转染GLI1 shRNA-3的SKOV3细胞增殖受到明显抑制（P＜0.05）,在紫杉醇的诱导下细胞凋亡比例明显增加,细胞周期主要阻滞在G1/S期.Western blot检测发现Bcl-2蛋白表达下降（P＜0.05）,Capase3蛋白表达升高（P＜0.05）,细胞周期蛋白CyclinD1蛋白表达下降（P＜0.05）.结论 GLI1 shRNh对SKOV3细胞增殖抑制作用明显,促进细胞凋亡,GLI1信号可通过抑制cyclinD1活化抑制细胞恶性增殖.     

肿瘤抑素抗肿瘤相关肽抑制卵巢癌的实验研究

目的研究肿瘤抑素（Tumstatin）185-203位氨基酸（19肽）对人卵巢癌细胞（SKOV3）的抑制作用,探讨其抑瘤作用机制。方法使用免疫组化、TUNEL方法检测肿瘤抑素19肽对SKOV3细胞增殖和凋亡的影响,以及caspase 3蛋白的表达情况。结果 19肽组抑瘤率可达36.47%,肿瘤细胞有明显的凋亡,caspase3蛋白的表达显著增加,与对照组比较差异有统计学意义（P〈0.05）。结论肿瘤抑素19肽可以明显地抑制裸鼠卵巢癌细胞的生长,其抑制卵巢癌的作用机制可能与其激活caspase通路有关。