Par-4表达水平与胰腺癌细胞发生顺铂耐药的关系

发布时间：2018-06-08 03:07

本文选题：顺铂 + Par-4　；参考：《第三军医大学》2016年博士论文

【摘要】：研究背景与目的胰腺癌(Pancreatic cancer,PC),是发生在胰腺的恶性肿瘤。是我们肝胆外科的常见肿瘤。由于早期的胰腺癌没有明显症状,通常确诊的病人都是晚期患者。大约90%的患者就诊时,已经无法进行根治性的手术治疗。统筹整个患者群体,五年生存率低于5%。大部分患者只能选择化疗作为治疗手段。传统的化学药物对于胰腺癌的控制几乎没有什么作用。过去常用药物5-氟尿嘧啶(5‘-Fluorouracil,5-FU),对胰腺癌的反应率也很少超过25%。现在,胰腺癌的第一线标准治疗仍然是单一使用吉西他滨来化疗,其剂量建议1000 mg/m2。但吉西他滨的反应率并不突出,也不能完全治愈胰腺癌,吉西他滨的影响有限,只能稍微提升生活品质,它对正常细胞毒性较小,能够减轻疼痛,稍稍延长患者生命。目前,对胰腺癌没有非常有效的抗癌药品可供选择,吉西他滨在1998年被美国食品药物管理局(Food and Drug Administration,FDA)核准为用于治疗胰腺癌的第一个化疗药品。2005年11月,美国食品药物管理局核准了靶向治疗药物埃罗替尼(Erlotinib,即特罗凯)可联合吉西他滨合使用,作为胰腺癌的一线化疗手段。联合使用在存活期及反应率比较上有统计学意义,但是临床治疗上并不能发现其带来的明显好处。顺铂(cisplatin,CDDP)是经典的化疗药物,因为胰腺癌很容易发生顺铂耐药,故没有用在胰腺癌化疗上。而胰腺癌顺铂耐药机制,目前尚不清楚。上皮间质转化(Epithelial mesenchymal transition,EMT)是细胞由原来的上皮形态,通过一些反应,向间充质形态转化,获得间充质细胞表型的过程。最近越来越多的文献报道显示,激活EMT会使大部分上皮来源的中路细胞获得间质表型。EMT过程与肿瘤细胞的远处转移,复发,肿瘤细胞侵袭转移力增加密切相关。还有证据显示,激发EMT过程与肿瘤的化疗耐药密切相关。在肿瘤细胞中,EMT的激发依赖多种信号传导途径的调控。在胰腺癌中,Zeb-1和其他EMT的调控子可能维持了胰腺癌的化疗耐药状态,这说明在胰腺癌中,EMT与化疗耐药密切相关。因此研究EMT关键分子的调控机制、研究胰腺癌细胞的耐药机理,发展有效的治疗措施措施、探索潜在的治疗靶点,逆转耐药机制,有重要意义。前列腺凋亡反应蛋白-4(Prostate apoptosis response-4,Par-4),最早被发现在前列腺癌细胞中,是一个促凋亡反应蛋白。它是PAR4基因的表达产物。Par-4蛋白具有独特的促肿瘤细胞凋亡,却不影响正常细胞的作用,这使得Par-4蛋白的安全性非常高。再者,Par-4蛋白可以从内源性和外源性两种途径诱导肿瘤细胞的凋亡,这让它的抗肿瘤能力成倍上升。Par-4在一系列的人类癌症组织中都被发现下调包括胰腺癌,它的下调被认为是肿瘤发生的关键事件之一,而且在胰腺癌的细胞中,Par-4蛋白的低表达预示着非常差的预后。Par-4蛋白仅凭自身就可诱导肿瘤细胞的凋亡反应,而且还可通过增加肿瘤细胞对凋亡介质的敏感性,如阿霉素、TNF-α(tumor necrosis factor-α),TNF相关的凋亡反应配体,来触发肿瘤细胞凋亡反应。研究还显示,结肠癌细胞对5-FU的敏感性可被Par-4增加。特别是在胰腺癌细胞中,Par-4在调节Bcl-2家族蛋白的小分子阻断剂导致的胰腺癌细胞凋亡事件中,扮演着调节敏感性的重要角色。近年来,Par-4已经被作为一个非常重要的靶向治疗的位点,许多临床试验也已经开展。但是,Par-4的下调是否与胰腺癌的顺铂耐药有关,到底怎样参与了耐药过程,还没有相关研究。我们猜测耐顺铂的胰腺癌细胞中发生了EMT,Par-4蛋白水平更低。下面来探讨CDDP耐药的胰腺癌细胞的作用机制。材料方法及结果1、以CDDP诱导建立胰腺癌耐药细胞株BXPC-3/CDDP。以西南医院肝胆实验室的BXPC-3细胞为亲本细胞,培养液中CDDP浓度从0.1μg/ml开始,浓度逐渐升至1μg/ml。建成BXPC-3/CDDP耐药细胞株。与BXPC-3细胞相比,BXPC-3/CDDP细胞更为细长,细胞较分散,呈间充质细胞样形态改变。MTT实验检测细胞耐药性。结果显示BXPC-3/CDDP细胞较其亲本细胞BXPC-3明显耐药。BXPC-3的耐顺铂细胞株BXPC-3/CDDP建立成功。以上结果说明用成功用CDDP浓度梯度法诱导BXPC-3变为BXPC-3/CDDP。2、探索耐药细胞株BXPC-3/CDDP与EMT的关联。用western blot检测BXPC-3和BXPC-3/CDDP中EMT标志物:snail,twist,E-cadherin,N-cadherin,并作出比较。细胞Transwell侵袭实验评价其与亲本细胞侵袭转移能力。结果显示,相比BXPC-3细胞,BXPC-3/CDDP细胞中snail表达水平上升,twist1表达水平上升,E-cadherin水平下降,N-cadherin水平上升。Transwell实验显示:BXPC-3/CDDP较其亲本细胞BXPC-3侵袭力上升。在BXPC-3/CDDP中,较其亲本细胞BXPC-3,发生了EMT。以上结果说明:BXPC-3/CDDP,较其亲本细胞BXPC-3,发生了EMT改变。3、探索耐药细胞株BXPC-3/CDDP中Par-4在mRNA和蛋白水平表达的情况。Western blot检测耐药细胞株BXPC-3/CDDP中Par-4蛋白表达水平,RT-PCR检测耐药细胞株BXPC-3/CDDP中Par-4 mRNA水平。以上结果显示,较BXPC-3,BXPC-3/CDDP中Par-4在蛋白和mRNA水平均降低了。4、Par-4的过表达与BXPC-3/CDDP细胞的顺铂耐药性、EMT有关。5、在BXPC-3/CDDP中采用过表达Par-4策略,用慢病毒包装转染pcDNA3.1-Par-4后,EMT相关标志物E-cadherin,N-cadherin用western blot检测。肿瘤细胞Transwell侵袭试验用来检测其转染后与未转染的侵袭力对比。MTT试验检测耐药性。结果显示,在BXPC-3/CDDP中过表达Par-4后,E-cadherin水平上升,N-cadherin水平下降。细胞侵袭转移水平下降,耐药性降低。呈MET改变。以上结果显示在BXPC-3/CDDP中从基因层面上调PAR4,会使肿瘤细胞发生MET改变,耐药能力下降。6、干扰BXPC-3中PAR-4基因后,细胞上皮表型发生改变。在BXPC-3中采用RNA干扰策略,降低Par-4表达。转染Par-4 SiRNA(small interfering RNA,小干扰RNA)后,western blot检测EMT相关标志物E-cadherin,N-cadherin。肿瘤细胞Transwell侵袭试验用来检测干扰前后侵袭力对比。用MTT试验来检测干扰前后细胞耐药性。结果显示,在BXPC-3中敲除Par-4后,E-cadherin水平下降,N-cadherin水平上升。细胞侵袭转移水平上升,耐药性增高。呈EMT改变。以上结果证明从基因层面下调BXPC-3中PAR-4基因后,肿瘤细胞发生了EMT趋势,耐药性增加,侵袭转移能力增强。7、BXPC-3中PAR-4基因下调后,细胞发生的EMT与PI3K/Akt(phosphatidylinositol 3-kinase,磷脂酰肌醇3激酶)信号通路有关。在BXPC-3中采用RNA干扰策略。转染Par-4 Si RNA后,加入PI3K/Akt阻断剂LY294002来阻断该信号通路。EMT相关标志物E-cadherin,N-cadherin用western blot来检测。肿瘤细胞Transwell侵袭试验用来检测加阻断剂前后侵袭力对比。用MTT试验来检测加阻断剂前后肿瘤细胞耐药性。结果显示,在BXPC-3中敲除Par-4,加入PI3K/Akt阻断剂LY294002后,E-cadherin水平较未加阻断剂组上升,N-cadherin水平较未加阻断剂组下降。细胞侵袭转移水平较未加阻断剂组下降,耐药性降低。EMT改变被阻断。另一试验证实,BXPC-3转染Par-4 siRNA后随时间推移测western测p Akt/total Akt比值,呈逐渐上升趋势。以上结果表明基因层面PAR4的下调通过PI3K/Akt途径激活了EMT,导致了BXPC-3细胞顺铂耐药。8、肿瘤细胞裸鼠移植瘤实验。接种肿瘤细胞后,用顺铂腹腔注射治疗,测各组肿瘤体积变化与70天生存率。结果示:顺铂对Par-4 siRNA-transfected BXPC-3组几乎没有作用,但加入PI3K/Akt阻滞剂LY294002后,该组的顺铂耐药被逆转。以上解说表明,在体外实验中,阻断PI3K/Akt途径可以逆转Par-4下调引起的EMT介导的耐药。研究结论本研究明确了耐顺铂的胰腺癌细胞BXPC-3/CDDP中,肿瘤细胞发生了EMT改变。同时,其Par-4在蛋白和mRNA水平都被发现下降。在基因层面上调耐药细胞株BXPC-3/CDDP的PAR-4水平,可以逆转顺铂耐药,通过MET(mesenchymal epithelial transition)途径。
[Abstract]:Background and objective Pancreatic cancer (PC) is a malignant tumor occurring in the pancreas. It is a common tumor in our department of hepatobiliary surgery. Since early pancreatic cancer has no obvious symptoms, the patients who are usually diagnosed are late patients. About 90% of the patients have been unable to perform radical surgical treatment. The five year survival rate is less than 5%. most patients can only choose chemotherapy as a treatment. Traditional chemical drugs have little effect on the control of pancreatic cancer. 5- fluorouracil (-Fluorouracil, 5-FU) was used in the past, and the response rate of pancreatic cancer was rarely over 25%.. The first line of standard treatment for pancreatic cancer is still A single use of gemcitabine chemotherapy, the dosage recommended 1000 mg/m2., but the response rate of gemcitabine is not prominent, and can not completely cure pancreatic cancer. The effect of gemcitabine is limited, only slightly improve the quality of life, it is less toxic to normal cells, can reduce pain, and slightly prolong the patient's life. At present, there is no very good for pancreatic cancer. The effective anticancer drug was selected. In 1998, gemcitabine was approved by the Food and Drug Administration (FDA) as the first chemotherapeutic drug for the treatment of pancreatic cancer in November. The US Food and Drug Administration approved the combination of the target therapy drug, erlotinib (Erlotinib, special Luo Kai), and gemcitabine combined with gemcitabine. Use, as a first-line chemotherapy for pancreatic cancer. Combined use is statistically significant in survival and response rates, but clinical treatment does not find its obvious benefits. Cisplatin (CDDP) is a classic chemotherapeutic agent because pancreatic cancer is very susceptible to cisplatin resistance, so it is not used in pancreatic cancer chemotherapy. The mechanism of cisplatin resistance in adenocarcinoma is not clear. Epithelial mesenchymal transition (EMT) is the process of cell transformation from the original epithelium to mesenchyme phenotype through some reactions. More and more recent reports suggest that activation of EMT will lead to most of the epithelial sources. The process of obtaining interstitial phenotypic.EMT from the path cells is closely related to distant metastasis, recurrence, and invasion and metastasis of tumor cells. There is also evidence that the activation of the EMT process is closely related to the chemotherapeutic resistance of the tumor. In the tumor cells, the excitation of EMT depends on the regulation of a variety of signal transduction pathways. In pancreatic cancer, Zeb-1 and other EMT are in the pancreatic cancer. The regulator may maintain the chemotherapeutic resistance of pancreatic cancer, which indicates that EMT is closely related to chemotherapy resistance in pancreatic cancer. Therefore, it is of great significance to study the regulatory mechanism of the key molecules of EMT, to study the mechanism of the drug resistance of pancreatic cancer cells, to develop effective treatment measures, to explore potential therapeutic targets and to reverse the mechanism of drug resistance. Apoptosis reactive protein -4 (Prostate apoptosis response-4, Par-4), the earliest found in prostate cancer cells, is a proapoptotic reaction protein. It is the expression product of the PAR4 gene,.Par-4 protein, which has a unique tumor promoting cell apoptosis, but does not affect the function of normal cells, which makes Par-4 protein safe. And Par-4, Par-4. The protein can induce apoptosis of tumor cells from two endogenous and exogenous pathways, which makes its antitumor capacity multiplied by.Par-4 in a series of human cancer tissues, which are found to be down-regulated, including pancreatic cancer. Its downregulation is considered to be one of the key events of the tumor, and the low Par-4 protein in the cells of pancreatic cancer. The expression indicates a very poor prognosis,.Par-4 protein can induce apoptosis response only by itself, and can also trigger the apoptosis response of tumor cells by increasing the sensitivity of tumor cells to the apoptosis medium, such as adriamycin, TNF- alpha (tumor necrosis factor- a) and TNF related apoptosis reaction ligands. The sensitivity of cells to 5-FU can be increased by Par-4. Especially in pancreatic cancer cells, Par-4 plays an important role in regulating the apoptosis of pancreatic cancer cells caused by the small molecular blockers of the Bcl-2 family protein. In recent years, Par-4 has been used as a very important target therapy site, many clinical trials It has also been carried out. However, whether the down-regulation of Par-4 is associated with cisplatin resistance in pancreatic cancer and how to participate in the drug resistance process, there is no related study. We speculate that EMT and Par-4 protein levels have occurred in pancreatic cancer cells resistant to cisplatin. The mechanism of action of CDDP resistant pancreatic cancer cells is discussed below. Material methods and results are 1, CD DP induced the establishment of pancreatic cancer resistant cell line BXPC-3/CDDP. with BXPC-3 cells in the liver and gallbladder Laboratory of Southwest Hospital as parental cells. The concentration of CDDP in the culture medium started from 0.1 mu g/ml, and the concentration gradually increased to 1 g/ml. to build the BXPC-3/CDDP resistant cell lines. Compared with BXPC-3 cells, the BXPC-3/CDDP cells were more slender, and the cells were more dispersed and mesenchyme cells. The results showed that BXPC-3/CDDP cells were more successful than the BXPC-3 resistant cisplatin resistant.BXPC-3 cell line BXPC-3/CDDP, which was significantly more resistant to BXPC-3. The results showed that BXPC-3 became BXPC-3/CDDP.2 with the CDDP concentration gradient method successfully, and the association between BXPC-3/CDDP and EMT was explored. Tern blot detected the EMT markers in BXPC-3 and BXPC-3/CDDP: snail, twist, E-cadherin, N-cadherin, and made a comparison. Cell Transwell invasion test evaluated the invasion and metastasis of parental cells. The dherin level rising.Transwell experiment showed that BXPC-3/CDDP was more aggressive than its parent cell BXPC-3. In BXPC-3/CDDP, the result of EMT. above the parent cell BXPC-3 showed that BXPC-3/CDDP, compared with the parent cell BXPC-3, occurred EMT change.3, and explored the expression of the protein level in the resistant fine cell BXPC-3/CDDP. The expression level of Par-4 protein in drug resistant cell line BXPC-3/CDDP was detected by.Western blot, and RT-PCR was used to detect the level of Par-4 mRNA in the drug-resistant cell line BXPC-3/CDDP. P was used to express the Par-4 strategy. After the transfection of pcDNA3.1-Par-4 with lentivirus package, the EMT related marker E-cadherin and N-cadherin were detected by Western blot. The tumor cell Transwell invasion test was used to detect the resistance of the transfected and untransfected invasive.MTT test to detect the resistance. The results showed that the Par-4 was over expressed in BXPC-3/CDDP. The level of dherin increased and the level of N-cadherin decreased. The level of cell invasion and metastasis decreased and the drug resistance decreased. The results showed that the up-regulation of PAR4 from the gene level in BXPC-3/CDDP could cause the MET change of the tumor cells and the decrease of resistance to.6. The epithelial phenotype changes after the interference of PAR-4 gene in BXPC-3. RN in BXPC-3 uses RN. A interference strategy, reducing the expression of Par-4. After transfection of Par-4 SiRNA (small interfering RNA, small interference RNA), Western blot tests EMT related E-cadherin, and the tumor cell invasion test is used to detect the invasion force before and after interference. In addition to Par-4, the level of E-cadherin decreased and the level of N-cadherin increased. The level of cell invasion and metastasis increased and the drug resistance increased. The results showed that after the gene level downregulation of the PAR-4 gene in BXPC-3, the tumor cells had the EMT trend, the resistance increased, the invasion and transfer energy increased.7, and the PAR-4 gene in BXPC-3 was down, and the cell occurred. EMT is related to the signaling pathway of PI3K/Akt (phosphatidylinositol 3-kinase, phosphatidylinositol 3 kinase). RNA interference strategy is used in BXPC-3. After Par-4 Si RNA, PI3K/Akt blocker LY294002 is used to block the signal pathway.EMT related markers. The MTT test was used to detect the resistance of tumor cells before and after the addition of blockers. The results showed that after knocking out Par-4 and adding PI3K/Akt blocker LY294002 in BXPC-3, the level of E-cadherin was higher than that of the non blocking agent group, and the level of N-cadherin was lower than that of the non blocking agent group. The level of cell invasion and metastasis was not the same. With the decrease of the blocker group and the decrease of the resistance to.EMT, the other test confirmed that the P Akt/total Akt ratio measured by BXPC-3 transfection to Par-4 siRNA showed a gradual increase. The above results showed that the downregulation of the gene level PAR4 was activated by PI3K/Akt pathway, leading to the cisplatin resistance of the BXPC-3 cells, the tumor cells. The tumor cells were inoculated in nude mice. After inoculation of tumor cells, cisplatin was intraperitoneally injected to test the changes of tumor volume and 70 natural survival rate. The results showed that cisplatin had little effect on Par-4 siRNA-transfected BXPC-3 group, but after the addition of PI3K/Akt blocker LY294002, the cisplatin resistance of the group was reversed. PI3K/Akt pathway can reverse the EMT mediated resistance induced by Par-4 downregulation. Conclusion this study identified the EMT changes in the tumor cells in the pancreatic cancer cell BXPC-3/CDDP resistant to cisplatin. At the same time, the Par-4 and mRNA levels were found to be reduced. The PAR-4 level of the drug resistant cell line BXPC-3/CDDP was up-regulated at the gene level. Resistance to cisplatin was passed through the MET (mesenchymal epithelial transition) pathway.
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.9

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