| 【Objective】 Chemotherapy resistance remains a complex problem for clinical treatment of advanced prostate cancer,and the mechanisms of drug resistance have not been fully illuminated.Recently,studies have shown that the tumor microenvironment plays an important role in disease progression and chemotherapy resistance for cancer treatment.To explore the resistance to docetaxel and its underlying mechanism in the chemotherapy of advanced prostate cancer,this study investigated the role of exosomes as intercellular communication mediators,which derived from cancer-associated fibroblasts in the tumor microenvironment of prostate cancer.【 Methods 】 In this study,firstly,PC3 cell was incubated with a gradient concentration(0-80 n M)of docetaxel for 72 hours.The effect of docetaxel on proliferative activity of the PC3 cell was detected by CCK8 assay,and then,a dose-effect curve was performed.The half maximal inhibitory concentration(IC50)of PC3 cell to docetaxel was calculated,which determined the docetaxel concentration for further experiments.Then,prostate cancer-associated fibroblasts(CAF)were isolated and purified from prostate cancer tissues surgically excised immediately.From medium which were used to cultivate CAF,CAF derived exosomes were extracted,and identification experiments and uptake assays for exosomes were performed.There were 4 treatment groups for PC3 cell: CON group(conventional medium treatment group),CAF-medium group(CAF conditioned medium treatment group),CAF-exo group(conventional medium treatment group including 50ug/ml CAF-exo),Exo-F-medium group(Exosomes-free CAF conditioned medium treatment group).After incubated for24 hours with the above medium,respectively,the PC3 cells were treated with 10 n M docetaxel for 72 hours.Then,the proliferative activity of these PC3 cells was detected by CCK8 assay to evaluate the effect of CAF-exo on the resistance to docetaxel in PC3 cell.Western blotting and q RT-PCR were used to investigate the expressions of USP8 and CX43 of the PC3 cell in each group.Furthermore,PC3cell(LV-USP8-PC3)which was infected with lentivirus highly expressing USP8 for up-regulating the expressions of USP8 and CX43(USP8/CX43 axis)was incubated with docetaxel to evaluate the effect of the USP8/CX43 axis on chemo-resistance in PC3 cell,and then the IC50 of the LV-USP8-PC3 cell to docetaxel was calculated by CCK8 assay.Finally,the abilities of proliferation,migration,and invasion of the LV-USP8-PC3 cell were detected in vitro.【 Results 】 The IC50 of PC3 cell to docetaxel was 8.5 ± 0.36 n M.CAF was successfully isolated and purified from prostate cancer tissues surgically excised.Compared with that of PC3 cell,higher drug resistance of CAF to docetaxel was found.It was confirmed that CAF-exo can be taken up by PC3 cell.The results of experiments showed that the proliferative activity of PC3 cell in CAF-medium group was higher than that in CON group(P <0.01),the proliferative activity of the PC3 cell in Exo-F-medium group was lower than that in CAF-medium group(P <0.01),while the proliferative activity of the PC3 cell in CAF-exo group was significantly higher than that in Exo-F-medium group(P <0.001),and was no significantly different from that in the CAF-medium group(P>0.05).The IC50 of PC3 cell to docetaxel in CAF-exo group(CAF-exo-PC3 cells)was 10.9 ± 0.59 n M.Western blotting results showed that the protein expressions of USP8 and CX43 of PC3 cell in CAF-medium group and CAF-exo group were lower than that in CON group(P <0.01),and the protein expressions of USP8 and CX43 of PC3 cell in Exo-F-medium group was higher than that in CAF-medium group and CAF-exo group(P <0.01).q RT-PCR results showed that the m RNA expressions of USP8 of PC3 cell in CAF-medium group and CAF-exo group were lower than that in CON group(P <0.001),and the m RNA expression of USP8 of PC3 cell in Exo-F-medium group was higher than that in CAF-medium group and CAF-exo group(P <0.001).There was no significant difference in CX43 m RNA expression of PC3 cell in each group(P > 0.05).After screened,the PC3 cell highly expressing USP8 and CX43 proteins called LV-USP8-PC3 was successfully established.q RT-PCR and Western blotting results showed that the expression of USP8 m RNA of LV-USP8-PC3 cell was 10.0 times of that of the control group(P<0.001),the expression of USP8 protein was 3.4 times of that of the control group(P <0.001),and the expression of CX43 m RNA of LV-USP8-PC3 cell was not significantly different from that of the control group(P>0.05),nevertheless,the expression of CX43 protein was 1.4 times of that in the control group(P <0.05).The results of experiments showed that the proliferative activity of the PC3 cell in LV-NC+CAF-exo group was higher than that in LV-NC+ CON group(P <0.001),whereas,the proliferative activity of the PC3 cell in LV-USP8+CAF-exo group was lower than that in LV-NC+CAF-exo group(P <0.01),and was no significantly different from that in LV-NC+CON group(P>0.05).These results indicated that up-regulation of the USP8/CX43 axis could rescue the effect of CAF-exo on docetaxel resistance in PC3 cells.The IC50 of LV-USP8-PC3 cell to docetaxel was7.0 ± 0.39 n M.The results of vitro assays showed that compared with LV-NC-PC3 cell,the proliferation,migration,and invasion abilities of LV-USP8-PC3 cell overexpressing USP8 and CX43 proteins decreased.【Conclusions】(1)CAF derived exosomes enhance resistance of PC3 cell to docetaxel;(2)CAF-exo enhances resistance to docetaxel via down-regulation of the USP8/CX43 axis in PC3 cell. |
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