Chitosan/pshRNA plasmid nanoparticles targeting MDR1 gene reverse paclitaxel resistance in ovarian cancer cells

In order to investigate the effect of chitosan/pshRNA plasmid nanoparticles targeting MDR1 genes on the resistance of A2780/TS cells to paclitaxel, chitosan/pshRNA plasmid nanoparti- cles were synthesized by means of a complex coacervation technique and transfected into A2780/TS cells. The cells transfected with MDRl-targeted chitosan/pshRNA plasmid nanoparticles were experimental cells and the cells transfected with chitosan/pGPU6/GFP/Neo no-load plasmid nanoparticles served as negative control cells. Morphological features of the nanoparticles were observed under transmission electron microscope （TEM）. MDR1 mRNA expression was assessed by RT-PCR. Half-inhibitory concentration （IC50） ofpaclitaxel for A2780/TS cells was determined by MTT method. TEM showed that the nanoparticles were round-shaped, smooth in surface and the diameters varied from 80 to 120 nm. The MDR1 mRNA in the transfected cells was significantly decreased by 17.6%, 27.8% and 52.6% on the post-transfection day 2, 4 and 7 when compared with that in A2780/TS cells control （P〈0.05）. MTT assay revealed that the relative reversal efficiency was increased over time and was 29.6%, 51.2% and 61.3% respectively in the transfected cells 2, 4, 7 days after transfection and IC_50 （0.197±0.003, 0.144±0.001, 0.120±0.004） were decreased with difference being significant when compared with that in A2780/TS （0.269±0.003） cells control （P〈0.05）. It was concluded that chitosan/pshRNA plasmid nanoparticles targeting MDR1 can effectively reverse the paclitaxel resistance in A2780/TS cells in a time-dependent manner.     

卵巢癌紫杉醇耐药的蛋白质组学研究

目的：研究与卵巢癌紫杉醇耐药相关的蛋白质。方法：应用双向凝胶电泳（2-DE）和基质辅助激光解吸电离飞行时间质谱（MALDI-TOF-MS）寻找紫杉醇耐药细胞和敏感细胞的差异表达蛋白质。使用Western印迹技术对其中2个蛋白质进行验证。结果：通过对2组细胞总蛋白质双向凝胶电脉图谱进行分析,找到差异蛋白质点40个;通过质谱分析,24个蛋白质得到鉴定。这些蛋白质包括增殖细胞核抗原（PCNA）、nm23蛋白、prohibitin（PHB）分子伴侣蛋白、脂皮质素（annexin）、α-烯醇化酶（α-enolase）以及热休克蛋白（HSP）等。结论：通过蛋白质组学技术,发现了卵巢癌紫杉醇耐药细胞系和敏感细胞系之间差异表达蛋白质24个,这些差异蛋白质可能参与卵巢癌细胞紫杉醇耐药过程。     

卵巢癌顺铂耐药细胞系的建立

目的 :建立人卵巢癌体外耐药模型 ,研究卵巢癌对顺铂的耐药特征。方法 :模仿临床卵巢癌化疗模型 ,采用中等浓度、间歇作用法 ,从人卵巢癌细胞株SKOV3培养出对顺铂耐药细胞系SKOV3/DDP ;倒置显微镜下观察细胞形态 ;MTT法、单克隆形成法检测耐药细胞的耐药指数 ;流式细胞仪检测两种细胞的细胞周期分布。结果 :SKOV3/DDP细胞形态无明显改变 ;耐药指数用MTT法检测为 1 8,单克隆形成法检测为 8;SKOV3主要分布于G0 -G1期 ,SKOV3/DDP主要分布于S期及G2 -M期。结论 :中等剂量、间歇作用法可以诱导出卵巢癌细胞株SKOV3对顺铂耐药性。此项研究结果可为临床卵巢癌化疗方案的制定、复发癌二线化疗药物的选择提供理论依据     

The effect of methylseleninic acid on paclitaxel efficacy in A2780 ovarian cancer cells

Objective:The role of methylseleninic acid (MSeA), a selenium compound, has been documented in cancer chemoprevention. However, the therapeutic effect of MSeA in combination with paclitaxel, a chemotherapeutic agent used to treat ovarian cancer, is unknown. In this study, we investigated the effect of combination treatment of MSeA and paclitaxel against ovarian cancer cells. Methods:Ovarian cancer cells(A2780) were treated with different concentrations of MSeA, paclitaxel alone or in combination. The individual and combined concentrations of drugs that achieved certain cells growth/death were determined using a sulforhodamine B(SRB) assay. Drug effects on cell viability were further confirmed using floating cell count and trypan blue exclusion assay. The mean values±standard deviation were calculated and compared between treatment groups using unpaired t test. Results: The concentration of paclitaxel alone that inhibited 50% of cell growth(IC50) was 0.5μmol/L. This concentration increased to 1.2μmol/L when paclitaxel was given in sequential combination with MSeA. The number of dead cells after the combination treatment did not show a significance increase when compared with drug alone. Conclusion:Pretreatment with MSeA did not enhance the paclitaxel effect against A2780 ovarian cancer cells.     

Paclitaxel-induced apoptosis in human ovarian cancer cell line COC1

Summary To elucidate the pattern of paclitaxel-induced apoptosis in human ovarian cancer cell line COC₁ and the role of paclitaxel in chemotherapy of ovarian cancer, apoptosis was investigatedin vitro by applying cytohistochemical techniques, DNA gel electrophoresis and flow cytometry. COC₁ cells manifested typical apoptotic morphologic features after exposure to paclitaxel. The rate of apoptosis was enhanced within the test concentration range in a concentration-dependent pattern. At low paclitaxel concentration, the rate of apoptosis were low and the levels of mitotic arrest were high. Whereas at higher paclitaxel concentration, the rate of apoptosis were higher and the levels of mitotic arrest were relatively lower. It is concluded that the antitumor effect of paclitaxel was correlated with druginduced apoptosis. Apoptosis induced by paclitaxel was concentration-dependent and was not significantly correlated with the mitotic arrest.     

姜黄素逆转卵巢癌对紫杉醇耐药的实验研究

目的:通过观察姜黄素作用人卵巢癌A2780/Taxol细胞株后,逆转该细胞株对紫杉醇的耐药作用,研究卵巢癌细胞对紫杉醇耐药的原因及姜黄素逆转耐药机制。方法:人卵巢癌A2780/Taxol细胞株在体外培养,将细胞分为对照组（A组）和实验组（不同浓度姜黄素组作用细胞）。用四甲基偶氮唑盐微量酶反应比色法检测2组细胞与相同浓度紫杉醇联用后细胞的生长抑制情况;Westernblot方法检测2组细胞内P-糖蛋白（P-gp）和蛋白激酶C-α（PKC-α）的表达情况。结果:四甲基偶氮唑盐微量酶反应比色法得出联合用姜黄素各组细胞生长抑制率均高于A组（P<0.05～P<0.01）;Western blot检测联合用姜黄素各组多药耐药蛋白-1/P-gp和PKC-α表达均较A组显著降低（P<0.01）。结论:姜黄素通过阻碍卵巢细胞中多药耐药蛋白-1/P-gp和PKC-α的表达,增加紫杉醇对细胞的毒性,降低细胞的耐药性。     

Effect of Mad2 on paclitaxel-induced cell death in ovarian cancer cells

In this article, the status of spindle assembly checkpoint and the alteration of its major component, Mad2 protein level were examined in A2780 and SKOV3 ovarian cancer cell lines.Recombinant eukaryotic expression plasmid pEGFP-Mad2 was transfected into paclitaxel-resistant SKOV3 cells and Mad2 protein was knocked down by Mad2-specific siRNA in paclitaxel-sensitive A2780 cells.Then the expression level of Mad2 gene was detected by Western blotting.Flow cytometry revealed that SKOV3 cells were not fully arrested in G2/M phase in contrast to A2780 cells in the presence of paclitaxel.However, paclitaxel sensitivity assay showed that sensitivity to paclitaxel was reversed after the transfection in both cell lines in terms of number of cells arrested at G2/M phase and the expression of Bcl-2 was significantly changed.These results suggest that weakened spindle checkpoint with reduced expression of Mad2 is associated with resistance to paclitaxel in ovarian cells and Bcl-2 may be involved in this process.     

卵巢癌干细胞对5种化疗药物的耐药性及其机制

目的探讨卵巢癌干细胞（Ovarian Cancer Stem cells,OCSCs）针对化疗药物耐药的作用机制。方法通过免疫磁选方法分离和纯化OCSCs,对比普通卵巢癌细胞（EOC）,观察在紫杉醇、卡铂、顺铂、5-FU、阿霉素5种化疗药物作用下OCSCs耐药的情况。通过定量PCR,分析EOC与OCSCs中ABCG2、ABCB1、Nanog、MMP-2和MMP-9基因的表达水平。结果从HO8910细胞分离的CD44＋CD117＋细胞,对卡铂、顺铂、紫杉醇、阿霉素和5-FU等化疗药物的耐药性明显强于HO8910细胞（P〈0.05）。定量PCR检测发现CD44＋CD117＋细胞中ABCG2、ABCB1、Nanog、MMP-2和MMP-9基因的表达显著高于HO8910细胞（P〈0.05）。结论与EOC相比,OCSCs对多种化疗药物的耐药性更为明显的机制可能与其较高水平表达ABCG2,ABCB1,Nanog,MMP-2和MMP-9基因有关。     

Molecular mechanism of indirubin-3′-monoxime and Matrine in the reversal of paclitaxel resistance in NCI-H520/TAX25 cell line

Background  Multidrug resistance (MDR) is a main reason for paclitaxel (TAX) treatment failure. Indirubin-3′-monoxime (IRO) and Matrine are traditional Chinese medicines, which may reverse the resistance of tumor cells to some chemotherapy drugs, but the relationship between paclitaxel resistance and Matrine is still unclear. The aim of this study was to explore the potential molecular mechanism of IRO and Matrine in reversal of TAX resistance.

Methods  In this study, MTT assay was used to measure the non-cytotoxic dosage of IRO and Matrine on NCI-H520/TAX25 cells and determine the reversal extent of TAX resistance under non-toxic doses. In addition, RT-PCR and Western blotting were used to evaluate the mRNA expression and the protein level of survivin, Oct-4, and Sox-2 in NCI-H520/TAX25 cells using semi-quantitative methods.

Results  There was no obvious inhibition on sensitive cell strains and drug-resistant strains, when the final concentration was at lest 4 µmol/L for IRO and 100 µmol/L for Matrine. So 4 µmol/L of IRO and 100 µmol/L of Matrine were considered as the reversal dosage. When 4 µmol/L of IRO or 100 µmol/L of Matrine were used together with TAX, the sensitivity to TAX increased evidently in NCI-H520/TAX2 cells; the reversal rate of IRO and Matrine was about 1.92 (43.56/22.6 nmol/L) and 1.74 (43.56/25.0 nmol/L), respectively. The mRNA expression and the protein level of survivin, Oct-4, and Sox-2 in NCI-H520/TAX25 decreased significantly (P <0.05) after addition of IRO or Matrine in TAX treatment, compared to that of TAX treatment alone.

Conclusion  The decrease in both mRNA expression and protein level of survivin, Oct-4, and Sox-2 might be the molecular mechanism, by which IRO and Matrine mediate the reversal of TAX resistance.

     

红景天苷下调KRT17增加紫杉醇对卵巢癌细胞的化疗敏感性

[目的] 红景天苷是从景天科植物大株红景天的干燥根及根茎或干燥全草中提取的一种化合物,具有多种药理作用。研究旨在探讨红景天苷下调角蛋白家族成员17（KRT17）影响紫杉醇对卵巢癌细胞的化疗敏感性。[方法] 红景天苷、紫杉醇单独或联用处理SKOV-3细胞,将细胞随机分为4组：对照组、红景天苷单独处理组、紫杉醇单独处理组和联合处理组,Cell Counting Kit-8（CCK-8）法检测细胞毒性,5-乙炔基-2’-脱氧尿苷（EDU）检测细胞增殖,Hoechst 33258检测细胞凋亡。免疫荧光法检测自噬体形成情况,蛋白免疫印迹法检测KRT17和细胞自噬相关蛋白表达水平。[结果] 与对照组比较,红景天苷、紫杉醇单独处理组随浓度的升高,对SKOV-3细胞的毒性增强,细胞增殖数、P62蛋白表达降低,细胞凋亡率、LC3荧光斑点数、LC3-Ⅱ/LC3-I比值、Beclin1蛋白表达升高。且红景天苷与紫杉醇协同处理效果优于红景天苷、紫杉醇单独处理组,可能是红景天苷通过下调KRT17蛋白表达实现的。[结论] 红景天苷下调KRT17增加紫杉醇对卵巢癌细胞的化疗敏感性。     

RNAi抑制EGFR基因增强卵巢癌细胞对泰素敏感性的研究

目的 探讨表皮生长因子受体(EGFR)基因在卵巢癌细胞对泰素敏感性的增强作用.方法 构建针对EGFR基因序列特异性shRNA的表达载体,用脂质体转染卵巢癌SKOV3细胞及G418筛选阳性克隆.采用RT-PCR、Western blot检测EGFR基因的抑制情况,用MTT法检测卵巢癌细胞对泰素的敏感性.结果 靶向EGFR的序列特异性shRNA可明显抑制EGFR基因的表达,EGFR mRNA和蛋白的抑制率分别为77.5%和76.1%;序列特异性shRNA-EGFR可明显提高卵巢癌细胞对泰素的敏感性,差异有统计学意义(P<0.01).结论 体外实验证实抑制EGFR基因的表达可提高卵巢癌细胞的化疗敏感性.     

血清中耐药相关的差异表达蛋白质在诊断卵巢上皮性癌铂类耐药中的临床价值

目的 分析血清耐药相关的差异表达蛋白质在诊断卵巢上皮性癌铂类耐药患者中的价值,为非手术卵巢上皮性癌患者化疗药物方案的制定提供参考.方法 选择川北医学院附属医院妇产科2015年3月至2016年3月收治的经铂类药物治疗无效的卵巢癌患者40例设为观察组,选择同期收治的经铂类药物治疗获得改善的卵巢癌患者40例作为对照组,采集两组受试者静脉血,采用酶联免疫法(ELISA)检测并比较两组患者的血清耐药相关蛋白(SERPINA1、FN1、ORM1、annexinA3、destrin、IDHl))的表达情况,所有患者均行病理穿刺,获取病理组织,采用逆转录一聚合酶链反应(RT-PCR)检测方法对病理组织中的上述耐药相关蛋白进行检测.分析血清标本检测结果和病理组织检测的SERPINA1、FN1、ORM1、destrin、annexinA3、IDHl结果之间的差异性和相关性.结果 观察组患者血清中的SERPINA1、FN1、ORM1、destrin、annexinA3、IDHl水平分别为(753.6±138.6)μg/μL、(223.5±51.5)μg/μL、(72.2±40.9)μg/μL、(331.5±21.8)μg/μL、(344.2±40.6)μg/μL、(44.5±21.3)μg/μL,病理组织中上述指标分别为(38.69±6.7)％、(32.32±6.6)％、(35.48±6.1)％、(29.44±5.4)％、(26.63±4.8)％、(11.37±1.5)％,对照组患者血清中的SERPINA1、FN1、ORM1、destrin、annexinA3、IDHl水平分别为(512.6±113.4)μg/μL、(125.5±41.4)μg/μL、(64.6±41.4)μg/μL、(215.7±21.1)μg/μL、(23.42±5.3)μg/μL、(115.5±21.3)μg/μL,病理组织中上述指标分别为(27.49±5.7)％、(21.38±5.4)％、(23.41±5.2)％、(23.41±5.2)％、(21.22±4.6)％、(29.43±2.3)％.观察组患者血清中SERPINA1、FN1、ORM1、destrin、annexinA3均明显高于对照组,但血清中IDHl水平明显低于对照组,差异均有统计学意义(P<0.05);观察组患者卵巢癌病理组织中的SERPINA1、FN1、ORM1、destrin、annexinA3水平均明显高于对照组,但IDHl水平明显低于对照组,差异均有统计学意义(P<0.05);血清中检测的SERPINA1、FN1、ORM1、destrin、annexinA3、IDHl水平与病理组织中检测的结果的spearman相关性系数分别为0.734、0.683、0.702、0.682、0.593、0.618,均呈正相关(P<0.05).结论 血清中耐药相关的差异表达蛋白质检测结果与病理组织中的检测结果密切相关,对于无法行手术探查或者治疗的患者,采用血清样本检测耐药相关差异蛋白质可为化疗药物方案的制定提供重要的参考,具有较高的临床价值.     

阿霉素诱导人卵巢癌细胞耐药株的建立及多药耐药表型测定

[目的]探讨卵巢癌细胞多药耐药的发生机制,寻找克服及逆转卵巢癌多药耐药的方法。[方法]采用阿霉素较大剂量间歇诱导和浓度梯度递增培养法,建立人卵巢癌耐药细胞株（SK—OV-3／adr）。运用MTT法检测药物敏感性和细胞生长规律变化,流式细胞仪检测细胞周期分布,荧光显微镜观察耐药株细胞内药物含量变化。[结果]建立的SK—OV-3／adr对阿霉素、5-氟尿嘧啶、长春新碱、紫杉醇、足叶乙甙有明显的交叉耐药,耐药指数分别为5．6（296．7／53．4ng·mL^-1）、6．3（12008．4／1921．6ng·mL^-1）、4．6（1176．2／254．3ng·mL^-1）、12．5（4269．6／342．0ng·mL^-1）、〉10．9（〉20000／1830．1ng·mL^-1）。与卵巢癌细胞敏感株（SK—OV-3）相比,半数细胞抑制剂量IC50明显增加,差异有统计学意义（P〈0．01）。给予P-糖蛋白抑制剂（盐酸维拉帕米）时,交叉耐药性减弱。与SK—OV-3相比,SK—OV-3／adr细胞内阿霉素荧光强度明显变弱。[结论]建立了具有多药耐药表型的SK—OV-3／adr细胞株。     

益气活血解毒方加减治疗卵巢癌紫杉醇化疗后外周神经毒性临床观察

[目的]观察益气活血解毒方加减治疗卵巢癌患者经紫杉醇化疗后导致的外周神经毒性的疗效。[方法]采用治疗前后自身对照方法进行观察,符合标准的患者均予益气活血解毒方加减口服及外洗治疗。1个月后观察90例患者神经毒性的等级及周围神经的传导速度的变化。[结果]两种不同兼夹症外周神经毒性发生例数减少,治疗前后比较差异均有统计学意义(P0.01)。治疗后正中神经和腓神经的运动和感觉神经传导速度均有变化,除湿邪阻络者正中神经的感觉神经传导速度差异无统计学意义(P0.05),其余治疗前后比较差异均有统计学意义(P0.05)。[结论]益气活血解毒方加减的内服和外用有改善卵巢癌患者经紫杉醇化疗后外周神经毒性的作用。     

Gab2在卵巢癌中的表达及其与铂类药物化疗耐药的相关性研究

目的 通过检测Gab2在卵巢癌中的表达,探讨其与卵巢癌铂类化疗耐药的相关性.方法 利用免疫组织化学及蛋白质印迹法(Western blotting)对该院妇产科2011年1月至2015年6月手术切除的107例患者浆液性卵巢癌组织、12例膀胱癌根治术中切除正常卵巢组织中Gab2的表达进行检测,分析Gab2表达与临床分期,以及与以铂类药物为基础的联合化疗疗效的相关性.结果 卵巢癌组织中Gab2表达水平明显高于正常卵巢组织,且与肿瘤的国际妇产科联盟(FIGO)临床分期有关,分期越晚表达越高,差异均有统计学意义(P<0.05);61例卵巢癌患者化疗敏感,46例发生耐药,化疗耐药患者Gab2表达水平明显高于化疗敏感者,差异有统计学意义(P<0.05).结论 Gab2可能成为预测铂类药物化疗敏感性的有效指标之一.     

卵巢癌顺铂耐药细胞系建立及部分耐药基因的表达

目的建立卵巢癌顺铂(cisplatin,DDP)耐药细胞系,并比较耐药与非耐药卵巢癌细胞系之间部分耐药相关基因的表达差异。方法采用浓度梯度递增法建立人卵巢癌DDP耐药细胞系SKOV-3/DDP。验证其有关的生物学指标;并用免疫组化法及RT-PCR技术检测耐药与非耐药卵巢癌细胞系之间P-糖蛋白(P-glycoprotein,P-gp)及部分耐药相关基因的表达差异。结果耐药细胞系对DDP、氟尿嘧啶(fluorouracil)和多柔比星(doxorubicin)的耐药指数(RI)分别为13.94、10.83、4.52;细胞周期比例SKOV-3/DDP细胞系G0/G1期细胞明显少于SKOV-3细胞系(P<0.01),S期、G2/M期细胞明显多于SKOV-3细胞系(P<0.01);P-糖蛋白(P-gp)阳性细胞率SKOV-3/DDP细胞系为65%±5%;SKOV-3细胞系为6%±2%,两者差异极显著(P<0.001)。在检测的耐药相关基因中MDR1、Sur-vivin在SKOV-3/DDP细胞系呈阳性表达,在SKOV-3细胞系未表达;TopoⅡβ表达情况与此相反。结论顺铂诱导的卵巢癌细胞耐药可对其他不同作用机制的药物产生交叉耐药,多药耐药涉及多个相关基因的表达,说明耐药的发生是一个多途径的复杂过程。     

Reversal of multidrug resistance and inhibition of phosphorylation of AKT in human ovarian cancer cell line by wild-type PTEN gene

Summary The reversing effect of wild-type PTEN gene on resistance of C13K cells to cisplatin and its inhibitory effect on the phosphorylation of protein kinase B (AKT) were studied. The expression of PTEN mRNA and protein in OV2008 cells and C13K cells were semi-quantitatively detected by using RT-PCR and Western blotting. Recombinant eukaryotic expression plasmid containing human wild-type PTEN gene was transfected into C13K cells by lipofectamine2000. The expression of PTEN mRNA was monitored by RT-PCR and the expression of PTEN, Akt, p-Akt protein were analyzed by Western blotting in PTEN-transfected and non-transfected C13K cells. Proliferation and chemosensitivity of cells to DDP were measured by MTT, and cell apoptosis was detected by flow cytometry after treatment with cisplatin. The expression of PTEN mRNA and protein in OV2008 cells were significantly higher than those in C13K cells. After transfection with PTEN gene for 48 h, the expression of PTEN mRNA and protein in C13K cells were 2.04 ± 0.10, 0.94 ± 0.04 respectively and the expression of p-Akt protein (0.94 ± 0.07) was lower than those in control groups (1.68 ± 0.14, 1.66 ± 0.10) (P < 0.05). The IC₅₀ of DDP to C13K cells transfected with PTEN (7.2 ± 0.3 μmol/L) was obviously lower than those of empty-vector transfected cells and non-transfected cells (12.7 ± 0.4 μmol/l, 13.0 ± 0.3 μmol/L) (P<0.05). The apopototis ratio of wild-type PTEN-transfected, empty vector transfected and non-transfected C13K cells were (41.65 ± 0.87)%, (18.61 ± 0.70)% and (15.28 ± 0.80)% respectively, and the difference was statistically significant (P<0.05). PTEN gene plays an important role in ovarian cancer multidrug resistance. Transfection of PTEN could increase the expression of PTEN and restore drug sensitivity to cisplatin in human ovarian cancer cell line C13K with multidrug-resistance by decreasing the expression of p-Akt. This project was supported by a grant from the National Natural Sciences Foundation of China (No. 30571950) and National Key Basic Research Program Foundation (NO.2002CB513107).     

人卵巢癌紫杉醇耐药细胞株的建立和生物学特性评价

目的建立人卵巢癌紫杉醇耐药细胞株(Skov-3/PTX),并对其生物学性状进行检测和鉴定。方法采用紫杉醇浓度梯度递增法,建立人卵巢癌紫杉醇耐药株。通过细胞形态学观察、生长曲线和群体倍增时间测定、药物敏感试验、细胞内Rh-123和紫杉醇含量研究及MDR1、MRP和GST-πmRNA水平的测定,评价Skov-3/PTX生物学特性。结果成功建立了Skov-3/PTX耐药株,耐药指数为45.90,对吉非替尼、9-硝基喜树碱和阿霉素产生明显的交叉耐药性。与Skov-3细胞相比,Skov-3/PTX耐药细胞异形明显;耐药细胞倍增时间显著延长(P<0.05);耐药细胞内Rh-123和紫杉醇量显著减少(P<0.05),环孢菌素可以增加细胞内Rh-123和紫杉醇含量;耐药细胞的MDR1、MRP和GST-πmRNA水平显著增高。结论Skov-3/PTX细胞具有典型多药耐药特性,为进一步研究耐药逆转途径提供了实验基础。     

Reversal of Multidrug Resistance and Inhibition of Phosphorylation of AKT in Human Ovarian Cancer Cell Line by Wild-type PTEN Gene

The reversing effect of wild-type PTEN gene on resistance of C13K cells to cisplatin and its inhibitory effect on the phosphorylation of protein kinase B (AKT) were studied. The expression of PTEN mRNA and protein in OV2008 cells and C13K cells were semi-quantitatively detected by using RT-PCR and Western blotting. Recombinant eukaryotic expression plasmid containing human wild-type PTEN gene was transfected into C13K cells by lipofectamine2000. The expression of PTEN mRNA was monitored by RT-PCR and the expression of PTEN, Akt, p-Akt protein were ana- lyzed by Western blotting in PTEN-transfected and non-transfected C13K cells. Proliferation and chemosensitivity of cells to DDP were measured by MTT, and cell apoptosis was detected by flow cytometry after treatment with cisplatin. The expression of PTEN mRNA and protein in OV2008 cells were significantly higher than those in C13K cells. After transfection with PTEN gene for 48 h, the expression of PTEN mRNA and protein in C13K cells were 2.04±0.10, 0.94±0.04 respectively and the expression of p-Akt protein ( 0.94±0.07) was lower than those in control groups (1.68±0.14, 1.66±0.10) (P< 0.05). The IC50 of DDP to C13K cells transfected with PTEN (7.2±0.3 μmol/L) was obviously lower than those of empty-vector transfected cells and non-transfected cells (12.7±0.4 μmol/l, 13.0±0.3 μmol/L) (P<0.05). The apopototis ratio of wild-type PTEN-transfected, empty vector transfected and non-transfected C13K cells were (41.65±0.87)%, (18.61±0.70)% and (15.28 ±0.80)% respectively, and the difference was statistically significant (P<0.05). PTEN gene plays an important role in ovarian cancer multidrug resistance. Transfection of PTEN could increase the ex- pression of PTEN and restore drug sensitivity to cisplatin in human ovarian cancer cell line C13K with multidrug-resistance by decreasing the expression of p-Akt.