The Study On Action Mechanism Of Noscapine Reverse Cisplatin Resistance In Human Ovarian Cancer Cells

Objectives：At present, ovarian cancer is the most common malignantcancer in gynecology, characterized by some biology features, such as easyrelapse, metastasis and multidrug resistance(MDR). Chemotherapy is one ofthe most important treatments on the basis of ovarian cancer cytoreductivesurgery. In Compounds of Pt, cisplatin(DDP) is the most effective of thevarious kinds of medicines. However, multidrug resistance of the cancer leadsto the failure of the chemotherapy. Multidrug resistance during chemotherapyis that after tumor cells are resistant to a certain medicine, the cells are alsoresistant to antitumor agents which are never touched and which have differentstructures and functions. The reasons for the resistance are numerous, but theabnormal expression of apoptosis control gene is one of the main factors thatlead to multidrug resistance of ovarian cancer. Researches find that cellsignaling and tumor suppress gene can promote cell proliferation and survivalabilities, but reduce the sensibility of cisplatin. The mediation of variousfactors such as apoptosis-inducing factor including wild P53，Bax, Caspase-3and apoptosis inhibition factor including XIAP, Bcl-2and Survivin lead to thefailure of antitumor agents. Multidrug resistance factors mutant XIAP,Caspase-3, Bcl-2and Survivin have abnormal expression in multi tumortissues and multi tumor drug resistance cells. They are also involved in thedevelopment of multidrug resistance.Microtubule inhibitors noscapine (NOS) is a kind of benzylisoquinolinealkaloids, which is widely used as antitussive drug in the past few decades. Inrecent years, people have found that noscapine and its derivatives haveantitumor effect. Studies show that noscapine can inhibit microtubule dynamicinstability, block cell cycle, induce cell apoptosis and can also prevent thedevelopment of glioma, myeloid leukemia, lymphoma, melanoma, breast cancer, cervical cancer and prostate cancer. However, its mechanism is notfully understood. Perhaps multiple pathways are involved, including inhibitionof mitotic phase G2/M and JNK pathway, P53participation, the cascadereaction, mitochondrial pathway, and NF-κB signal pathway adjustment. Somestudies have also shown that when microtubule inhibitors such as paclitaxelproduce resistance effect, noscapine and its derivatives can play a certainanti-tumor effect, and have few toxic side effects and no obvious crossresistance, which can prove that noscapine may have the effect of reversal ofMDR. At present, the effect of noscapine cisplatin resistance in ovarian cancercell line SKOV3/DDP has not been reported, which needs to be furtherstudied.This experiement makes use of cisplatin to induce the resistance ofovarian cancer cells and establish SKOV3/DDP cells. The changes of XIAP,Caspase-3, Bcl-2and Survivin after the use of noscapine effect are observed invitro experiments. It aims to make a research on the reversal effect and itsmechanism of noscapine to ovarian cancer drug resistance. Vivo experimentsare to implant ovarian cancer cell line in subcutaneous layer of nude mice andestablish animal model. We observe the change of nude mice transplantationtumor volume and its organization by giving different concentrations ofnoscapine and the combined dose of noscapine and cisplatin. At the same timewe analyze the impact of the expression of noscapine to XIAP, Caspase-3,Bcl-2and Survivin. This experiment has employed various experimentalmethods to detect XIAP, Caspase-3, Bcl-2and Survivin gene, the expressionof protein in normal ovarian tissue, junction of ovarian tumor and epithelialovarian carcinoma, and to observe the correlation between occurrence ofresistance and ovarian cancer. We expect to make a research on the effect andrelative mechanism of noscapine to reverse multidrug resistance effects ofhuman ovarian cancer SKOVS/DDP cells. If it can be developed to reversemultidrug resistance, it has wide clinical application prospects. Methods：1The establishment of drug resistance in ovarian cancer SKOV3/DDPcells and its multidrug resistance protein expressionSKOV3cells in logarithmic growth phase were inoculated in lowconcentration of cisplatin (0.02mg/L) medium with continued drug exposureconcentration increasing induction method. We continuously improve the drugconcentrations according to the cell growth, until the cells can grow stably inculture medium of cisplatin concentration0.2mg/L. It lasts8months, and thecell is named SKOV3/DDP cells. MTT method was used to detect cell growthinhibition rate of SKOV3cells, SKOV3/DDP cells induced by cisplatin, andto calculate the median inhibitory concentration (IC50). We get to know theresistance index (RI).FCM method can detect apoptosis and the epression of XIAP, Caspase-3,Bcl-2and Survivin protein after the intervention of cisplatin to SKOV3/DDPcells. Realtime-PCR can detect the expression of XIAP, Survivin mRNA.Cisplatin drug intervenes the growth of SKOV3and SKOV3/DDP cells.2Cisplatin resistance and its mechanism of reversal of SKOV3/DDP cellsby noscapineMTT method is used to detect growth inhibition rate affected bynoscapine, cisplatin and the comnination of noscapine and cisplatin effect onSKOV3/DDP cells. FCM method is uses to detect the influence ofSKOV3/DDP apoptosis and cell cycle affected by noscapine, cisplatin and thecombination of noscapine and cisplatin. Application of Realtime-PCR, FCMcan help detect XIAP, Caspase-3, Bcl-2and Survivin protein and the changeof XIAP, Survivin mRNA after the combined effects on SKOV3/DDP cells bynoscapine and cisplatin.3Study on reversal of the resistance of ovarian carcinoma in nude miceby noscapineBALB/c nu/nu nude mice (3-4weeks of age, female,16~20g) wererandomly divided into10groups,6rats in each group.7groups were injectedSKOV3/DDP cells in nude mice subcutaneous tumor;3groups were injectedSKOV3cells subcutaneously in nude mice transplanted tumor. After one weektumor cells came into being, and treatment began. The experimental groups were given20and40mg/kg noscapine or3and6mg/kg cisplatin. Cisplatinand noscapine treatment group,3mg/kg cisplatin and the combination of20and40mg/kg noscapine. The control group was given normal saline.Noscapine, intraperitoneal injection,1time/3d, for7times. Intraperitonealinjection of cisplatin,1time/3d, a total of7times injections. SKOV3cellssubcutaneous implanted tumor model nude mice group. The experimentalgroups were given3and6mg/kg cisplatin. The control group was givennormal saline.During the experiment we need to measure nude mice weight and thelong, short diameter of subcutaneous tumor every other day.3day after drugwithdrawal the mice were killed painlessly by pulling the neck method, withsubcutaneous tumors weighed, tumor volume and weight inhibition ratecalculated. The tumor tissues were removed to have IHC, FCM detection.FCM detects cell apoptosis, cell cycle and the expression of XIAP, Caspase-3,Bcl-2and Survivin protein in subcutaneous implanted tumor tissue. IHCdetects the expression and orientation of tumor cells XIAP, Caspase-3, Bcl-2and Survivin protein.4The expression and its significance of multidrug resistance associatedprotein in ovarian carcinomaA total of80cases of ovarian epithelial carcinoma,20cases of borderlinetumors,15cases of normal ovarian tissue have been collected. In80cases ofepithelial ovarian cancer,41cases are serous cystadenocarcinoma,28mucinous cystadenocarcinoma, and11endometrial carcinoma of uterus.38cases of I, II period,42cases of III, IV period.39cases well-differentiated,26cases moderately differentiated,15cases low differentiated.39cases are withlymph node metastasis,41cases without lymph node metastasis. Theexpression of tissue XIAP, Caspase-3, Bcl-2and Survivin protein in121casesis detected by using FCM and IHC to analyze the relationship between XIAP,Caspase-3, Bcl-2and Survivin expression and clinicopathological features ofovarian epithelial carcinoma. Results：1Establishment of drug resistance in ovarian cancer SKOV3/DDP cellsand its multidrug resistance protein expressionAfter8months, the resistant cell SKOV3/DDP was successfullycultivated. Compared with SKOV3cells, SKOV3/DDP cells volume wasbigger and polygonal, and neuron grew like cells. Morphology was moreirregular. The cytoplasm was found to have vesicles, fuzzy boundaries, andpoor refraction. MTT found that the resistance index of cisplatin toSKOV3/DDP and SKOV3cells24h,48h and72h of RI were respectively2.43,2.95and3.78.FCM detected that expression of XIAP, Bcl-2and Survivin protein inSKOV3/DDP cells was significantly higher than that in SKOV3cells (P<0.01);Caspase-3protein expression decreased obviously (P<0.01). And theapoptosis of cisplatin to SKOV3/DDP cells decreased obviously after48h(P<0.01).Realtime-PCR detected that the expression level of XIAP mRNA inSKOV3and SKOV3/DDP cells had no significant difference (P>0.05). Theexpression of Survivin mRNA in SKOV3/DDP cells was significantly higherthan that in the sensitive cell SKOV3cells. There was significant differencebetween them (P<0.05).2Cisplatin resistance and its mechanism of reversal of SKOV3/DDP cellsby noscapineThe results of MTT showed, IC50of noscapine on SKOV3/DDP cells24h,48h and72h were respectively52.84μmol/L,28.04μmol/L and21.58μmol/L.It had an obvious inhibitory effect on the growth of SKOV3/DDP cells, anddepended on the concentration and time (P<0.05). Compared the combinedeffect of noscapine and cisplatin group with noscapine and cisplatin groupseparately, the SKOV3/DDP cells growth inhibition rate was significantlyincreased (P<0.05).The results of FCM showed, compared the combined effect of noscapineand cisplatin group with noscapine and cisplatin group separately, theSKOV3/DDP cells growth inhibition rate was significantly increased (P<0.05). The percentage of G0/G1phase cells decreased, and the percentage of G2/Mphase cells increased (P<0.05).Realtime-PCR and FCM results showed that compared the combinedeffect of noscapine and cisplatin group with noscapine and cisplatin groupseparately, XIAP, Survivin mRNA expression was significantly reduced inSKOV3/DDP cells (P<0.05). The expression of XIAP, Bcl-2and Survivinprotein was significantly reduced(P<0.05), while the expression of Caspase-3protein was significantly increased (P<0.05).3Study on reversal of the resistance of ovarian carcinoma in nude miceby noscapineThe tumor models of human ovarian carcinoma xenograft weresuccessfully constructed, with the tumor formation rate of100%. In the sameconcentration, the volume and weight of the subcutaneous implanted tumorinoculation of SKOV3cells were significantly increased (P<0.05). Comparedthe combined effect of noscapine and cisplatin group with noscapine andcisplatin group separately, volume and weight of SKOV3/DDP cellssubcutaneous implanted tumor were significantly decreased (P<0.05).FCM results showed that compared the combined effect of noscapine andcisplatin group with noscapine and cisplatin group separately, apoptosis rate ofSKOV3/DDP cells subcutaneous tumor was significantly increased (P<0.05).The percentage of G0/G1phase cells decreased, and the percentage of G2/Mphase cells increased (P<0.05). The results of FCM showed that compared thecombined effect of noscapine and cisplatin group with noscapine and cisplatingroup separately, XIAP, Bcl-2and Survivin protein expression wassignificantly decreased in SKOV3/DDP cells (P<0.05), while the expressionof Caspase-3protein was significantly increased (P<0.05). In addition, theXIAP, Caspase-3, Bcl-2and Survivin protein positive expression by IHC wasalso consistent with FCM results.4The expression and its significance of multidrug resistance associatedprotein in ovarian carcinomaIHC results show that XIAP positive products were located in the cell cytoplasm. Bcl-2positive products were located in the cell cytoplasm andmembrane. Survivin, Caspase-3was localized in the cell nucleus andcytoplasm. The positive rate of XIAP, Bcl-2and Survivin protein expressionin ovarian cancer tissue (78.8%,48.8%and55.0%) and borderline ovariantumor (60.0%,30.0%and25.0%) was significantly higher than that of normalovarian tissue (0,0and6.7%)(P<0.01). The positive rate of Caspase-3expression in ovarian carcinoma(43.8%) and borderline ovariantumor(55%)was significantly lower than that in normal ovarian tissues(93.3%)(P<0.01).FCM and IHC results showed that, the positive rates and its expressionlevels of Bcl-2, Survivin, XIAP and Caspase-3proteins in differenthistological types had no significant difference (P>0.05). In the high, medium,low differentiation groups, positive rate of protein and protein expression levelincreased gradually, and the difference between groups had statisticalsignificance (P<0.01). The positive rate of Bcl-2protein and Survivin proteinsexpression level in metastasis group was significantly higher than that in nometastasis group, and the difference was statistically significant (P<0.01). Thedifferences in XIAP and Caspase-3proteins also had no significant difference(P>0.05). The positive rate and the protein expression level of XIAP, Bcl-2and Survivin protein of clinical stage (III~IV) group was higher than that ofearly (I~II) group, and the difference was statistically significant (P<0.01).Caspase-3protein in early clinical (I~II) group was higher than that of late(III~IV) group, and the difference has statistical significance (P<0.01). Theexpression of XIAP and Caspase-3in ovarian cancer, and the expression ofSurvivin and Bcl-2proteins (P<0.01; P<0.01) had a correlation. Theexpression of XIAP and Bcl-2, and the expression of Survivin and Caspase-3were not correlated with each other (P>0.05; P>0.05).Conclusions：1With the application of cisplatin sustained contact with increasingconcentrations induced method, ovarian cancer SKOV3/DDP cells wassuccessfully established. inhibition and apoptosis rate of SKOV3/DDP cells was lower than that of SKOV3cells after Cisplatin treatment. The resultsuggested that SKOV3/DDP cells had cisplatin-resistant. Compared withSKOV3cells, there was higher expression of XIAP, Bcl-2and Survivinprotein or Survivin mRNA, and lower expression of Caspase-3inSKOV3/DDP cells, so as to reverse its drug resistance.2It was the first time to discuss noscapine reversal of ovarian cancerresistant mechanism, and there was no related reports at home and abroad.Noscapine effectively inhibited proliferation of SKOV3/DDP cells, and hadthe time and the concentration dependence. Compared the combined effect oflow dose noscapine and cisplatin group with cisplatin group, the expression ofXIAP, Bcl-2and Survivin proteins or mRNA decreased in SKOV3/DDP cells,and the expression of Caspase-3protein increased. It also increased thecytotoxicity of cisplatin on SKOV3/DDP cells, so as to reverse the drugresistance of SKOV3/DDP cells to cisplatin.3Tumor model of SKOV3and SKOV3/DDP cells in nude mice wasestablished by subcutaneous ovarian cancer cell cultivation. The resultsshowed that under the combined effects of noscapine and cisplatin, cisplatininhibitory ability on human nude mouse ovarian cancer cell subcutaneousimplanted tumor was significantly increased, which provides the experimentalbasis for noscapine to become ovarian cancer multidrug resistance reversalagent. Multidrug resistance reversal mechanism of noscapine was related todowngrade of XIAP, Bcl-2and Survivin protein and increase of Caspase-3protein expression in tumor tissue of nude mice, which suggested that theseproteins may be involved in ovarian cancer cisplatin resistant.4In the process of epithelial ovarian cancer lesions, the expression rateand intensity of XIAP, Bcl-2and Survivin in normal ovarian tissues,borderline ovarian tumor and epithelial ovarian cancer were graduallyincreased, and Caspase-3decreased gradually, which suggested that theoccurrence and development of inhibitor of apoptosis may be involved inovarian tumors. Expression of XIAP, Caspase-3, Bcl-2and Survivin proteinwas closely related to clinical staging and malignant degree of ovarian cancer, which was a reference index for diagnosis of ovarian tumors. The expressionof XIAP and Caspase-3proteins in ovarian cancer, and the expression ofSurvivin and Bcl-2proteins had a correlation. The expression of those fourproteins of nude mice transplantation tumor organization on ovariancarcinoma cisplatin-resistant was similar to that of the patient ovarian cancerorganization, which indicated that noscapine may have the same effect inreversal of drug resistance of person and nude mice ovarian cancer.