The Experimental Studies Of Inhibition Of Dexamethasone On Paclitaxel-induced Apoptosis In Human Ovarian Cancer Cell Line SKOV-3

In recent years,the chemotherapy of paclitaxel combined cisplatin has been widely used in the treatment of ovarian cancer,particularly for advanced that could not be operated,drug-refractory and metastatic ovarian cancer.Dexamethasone was routinely used as a premedication in the clinical applicantion of paclitaxel to prevent hypersensitivity and nausea.However,recently studies discovered that dexamethasone could selectively inhibit paclitaxel-induced apoptosis in a number of human solid tumor cells.It can downregulate the immune response and contribute to tumour metastasis.Pretreatment with dexamethasone interfered with the therapeutic efficacy of Paclitaxel.How is the influence of dexamethasone on paclitaxel-induced apoptosis in ovarian cancer cells? If dexamethasone can inhibit paclitaxel-induced apoptosis in ovarian cancer cells and reduce the therapeutic efficacy of paclitaxel.Therefore,we performed in vitro experiments with human ovarian cancer SKOV-3 cell line to evaluate the effects and mechanisms of dexamethasone on human ovarian cancer cell apoptosis induced by paclitaxel and to explore the influence of dexamethasone on antitumor therapeutic efficacy of paclitaxel in vivo through development of animal models bearing ovarian xenograft tumor.This finding may have a potential implication on how to use correctly dexamethasone in the clinical practice and to develop new drug route of paclitaxel.PartⅠProtective effect of dexamethasone on apoptosis of human ovarian cancer cell line SKOV-3 induced by paclitaxelObjective To observe the effects of dexamethasone on apoptosis of human ovarian cancer cell line SKOV-3 induced by paclitaxel.Methods Human ovarian cancer cell line SKOV-3 was pretreated with dexamethasone at different concentrations for 24 hours,then was treated with Paclitaxel at different concentrations for 24 hours.MTT assay was used to examine the cell proliferation and the growth inhibition of SKOV-3 cell after treatments with dexamethasone and paclitaxel.The cell apoptotic rate of human ovarian cancer cell line SKOV-3 induced by paclitaxel in the presence or absence of dexamethasone was detected by TUNEL assay.The change of cell cycle and percentage of apoptosis of human ovarian cancer cell line SKOV-3 induced by paclitaxel in the presence or absence of dexamethasone was measured by PI-staining,and then analyzed by FCM.Results Paclitaxel can inhibit the cell proliferation of human ovarian cancer cell line SKOV-3 and was relative with the concentration.It was revealed that 0.001 uM～luM dexamethasone can protected differently the inhibition of human ovarian cancer cell line SKOV-3 proliferation induced by Paclitaxel.TUNEL and DAB staining assay showed that there was rarely apoptotic cells in control group,but there were many apoptotic cells with DAB-stained buffy nuclers,karyopycnosis or chromatin aggregation in human ovarian cancer cell line SKOV-3 induced by paclitaxel in the presence or absence of dexamethasone.The apoptotic rate was 3.5±0.25%and 4.0±0.26%in control group and Dex group with no difference between each other.The apoptotic rate was 11.7±1.72% and 30.8±1.15%in paclitaxel in the presence or absence of dexamethasone group,with significant difference between each other(p＜0.01).The apoptosis percentage of human ovarian cancer cell line SKOV-3 induced by paclitaxel and pretreated with dexamethasone measured by PI-staining was 5.55±0.53%,which was less than the apoptosis percentage 13.90±1.62%treated only with Paclitaxel(p＜0.01).The percentage of cell arrested by PTX in the G2/M phase was no significant difference in paclitaxel in the presence or absence of dexamethasone group(p＞0.05).Conclusion Dexamethasone can protected differently the inhibition of human ovarian cancer cell line SKOV-3 proliferation induced by paclitaxel and was relative with the concentration of dexamethasone.Dexamethasone can inhibit apoptosis of human ovarian cancer cell line SKOV-3 induced by paclitaxel without affecting the ability of paclitaxel to induce mitotic division arrest in the G2/M phase.PartⅡPossible mechanisms of dexamethasone inhibit apoptosis of human ovarian cancer cell line SKOV-3 induced by PaclitaxelObjective To investigate the possible mechanisms of dexamethasone inhibiting apoptosis of human ovarian cancer cell line SKOV-3 induced by paclitaxel.The experiment will settle ground work for the further study in vivo.Methods The expression levels of Survivin,Bcl-2,Bcl-X_L mRNA were determined by semi- quantiative RT-PCR after cells were treated by different methods.Western blot analysis was used to detect the expression levels of Bcl-X_L anti-apoptosis protain and activity of Caspase-3.Results The expression of Bcl-X_L,Survivin mRNA treated with dexamethasoneprior to paclitaxel were higher than treated only with paclitaxel(p＜0.01).The expression of Bcl-2 mRNA treated with dexamethasone prior to paclitaxelwas nearly same compared with treated only with paclitaxel(p＞0.05).The expression of Bcl-X_L protain in dexamethasone group is higher than control group(p＜0.01).The expression of Bcl-X_L protain treated with dexamethasone prior to paclitaxel was higher than treated only with paclitaxel(p＜0.01).The expression of Cleaved caspase-3 protain treated treated with dexamethasone prior to paclitaxel was lower than treated only with Paclitaxel(p＜0.01).Conclusion Pretreatment with dexamethasone can inhibit the paclitaxel -induced apoptosis of human ovarian cancer cell line SKOV-3.The possibbe mechanism is down- regulation of caspase-3 activity by Bcl-X_L,Survivin anti- apoptosis pathways.PartⅢDexamethasone intereferes with therapeutic efficacy of paclitaxel against human ovarian cancer cell line SKOV-3 xenograft tumorsObjective We performed in vivo experiments through development of human ovarian cancer cell line SKOV-3 xenograft tumor models.We evaluate the potential effect of dexamethasone on the antitumor activity of paclitaxel in ovarian cancer cell line SKOV-3 xenograft tumors and analyze the mechanism furtherly.Methods We firstly developed the model of human ovarian cancer cell line SKOV-3 xenograft tumors,then mice were randomly divided into 4 groups(10 mice per group) and treated with various regimens on day 0(1) Control;(2) Dexamethasone alone at 1 mg/kg,ip;(3) Combination of dexamethasone and paclitaxel,in which dexamethasone was administered 12 h before paclitaxel treatment;(4) Paclitaxel alone at 20 mg/kg,iv.The same treatment regimens were repeated every 3 days for a total of 6 cycles.We observed the growth of human ovarian cancer cell line SKOV-3 xenograft tumors and drew the curve of xenograft tumors.After the mice were killed,the tumors were removed and their weights were measured.The inhibition rate of xenograft tumors growth was calculate.Immunohistochemistry assay the expression of Bcl-X_L,Cleaved caspase-3 protain and proliferation marker Ki-67 of human ovarian cancer cell line SKOV-3 xenograft tumors after 6 cycles of treatments.Under the microscope and transmission electric micro- scope,we observed the tissue morphous and ultramicrostructure.Results Nude mice bearing SKOV-3 xenograft tumors were treated with paclitaxel with pretreatment of dexamethasone,the xenograft tumors volumes were bigger than those in mice treated with paclitaxel alone(p＜0.01).The weight of xenograft tumors at the end of experiments for treated with paclitaxel with or without dexamethasone were 0.79±0.09g and 0.50±0.08g,respectively.The weight of xenograft tumors treated with paclitaxel with dexamethasone was more than treated with paclitaxel without dexamethasone.The inhibitory rate of paclitaxel alone was 65.28%.However,when the mice was pretreatment with dexamethasone,the IR of paclitaxel on SKOV-3 xenograft tumors dropped to 45.14%,which was 18-25%less than those in mice treated with paclitaxel alone.SKOV-3 xenograft tumors stained with HE,most xenograft tumor cells after the treatment of paclitaxel alone were found to become much larger,and the cellularity was reduced significantly when compared with that in any other groups.In addition,the SKOV-3 xenograft tumor cells exhibited typical apoptotic characteristics.In the group treated with combination of dexamethasone and paclitaxel,there were also many xenograft tumor cells exhibiting vacuolization and apoptotis features,but less than that in the group treated with paclitaxel alone.It was showed by electronic microscope that karyopycnosis or chromatin aggregation was more in the group treated with paclitaxel alone than in the group administered with dexamethasone prior to paclitaxel.The immunochemical results suggested that the expression of proliferation marker Ki-67 and Bcl-X_L protain in the group administered with dexamethasone prior to paclitaxel was significantly higher when compared with that in the group treated with paclitaxel alone(p＜0.0125).The expression of Cleaved caspase-3 protain in the group administered with dexamethasone prior to paclitaxel was significantly lower when compared with that in the group treated with paclitaxel alone(p＜0.0125).Conclusion Pretreatment with dexamethasone significantly interfered with the therapeutic activity of paclitaxe in human ovarian cancer cell line SKOV-3 xenograft tumors in vivo.Dexamethasone can inhibit paclitaxel-induced apoptosis in human ovarian cancer cell line SKOV-3 xenograft tumors in vivo.The possibbe mechanism is down-regulation of caspase-3 activity by up-regulation of Bcl-X_L anti- apoptosis pathways.