The Effect Of Arsenic Trioxide On Hormone-independent Prostate Cancer Pc-3 Cell Line In Vitro

Prostate cancer is the most common malignant tumor of genitourinary system malignancy in male.The incidence of prostate cancer has increased for the past few years.But the majority of patients with prostate cancer were advanced cancer when they were diagnosed and could only be treated by endocrine therapy.After few years, major of prostate cancer relapse to the status of hormone-independent prostate cancer. Despite the availability of various therapeutic approaches,none has provided a marked therapeutic effectiveness for such patients.Therefore,investigation of novel therapies for hormonal-independent prostate cancer is urgently needed.The main mechanism of endocrine therapy is that it can inhibit the growth of hormone-dependent prostate cancer cells by intercepting the combination between androgen and androgen receptor(AR).The abnormal expression of AR is one of the main reasons of the formation of hormone-independent prostate cancer.The researches report,About 1/3 of the hormone-independent prostate cancer,loss of AR expression.The mechanism of AR gene expression silencing is probably associated with the CpG island methylation in the core Promoter of AR gene.Thus, demethylation aimed at AR gene becomes a new therapeutic strategy for AR-negative prostate cancer.Arsenic trioxide(As₂O₃) has been confirmed that it has the function to retain the growth of tumor cells,and also has the function of demethylation.It shows the promising prospect for clinical application. In this experiment,cell culture is used to observe the inhibition of As₂O₃ on the carcinoma cell line PC-3 growth,the effect of As₂O₃ on AR expression,and the Possible function mechanisms are initially discussed.This will Provide experimental basis for further application to the zooscopy of this cell and possible clinical application in the future.ObjectiveObserve the inhibition of As₂O₃ with different concentration on PC-3 cells growth, the effect of As₂O₃ on AR expression,and initially discuss the mechanism of As₂O₃ effection.Materials and Methods1.Human prostate cancer cell line(AR-negative PC-3) was acquired here. Waiting for well growth state,take and adjust concentration of PC-3 cells to 1×10⁶ pieces/ml,change RPMI 1640 culture medium including As₂O₃ to different concentration:1.0μmol/l,2.0μmol/l,4.0μmol/l,8.0μmol/l,10.0μmol/l,and cultivate continually to 48 hours.At the same term,using the PC-3 cells as negative comparison which unsettled by As₂O₃.2.The morphologic changes of PC-3 cells were studied after 48h treatment with different concentration As₂O₃ in vitro by the inverted microscope.3.MTT assay was used to observe inhibitory effect after 48h treatment with different concentration As₂O₃ on Proliferation of PC-3 cells.4.Flow Cytometry was used to assess cell cycle progression after 48h treatment with different concentration As₂O₃ on PC-3 cells.5.Immunocytochemical technique was used to measure the express of AR protein after 48h treatment with different concentration As_O3 on PC-3 cells.6.Experimental datas are disposed by SPSS 13.0 statistics soft ware,deploying one-way analysis of variance,significance of difference standard(a=0.05).Results:1.The changes of cell morphology of PC-3 cell after the 48h treatment with As₂O₃.Under observation by inverted microscope:the control group,the PC-3 cells assumed the shape of shuttle or tall and slender shuttle,growing with adherence, homogeneous transparent endoehylema,and good refraction;for the 2.0μmol/l group, the PC-3 cells began to reduce adherent and appear exfoliated.Some cells lost the normal shape,and began to round.The refraction became to weaken.In the cells, there appeared the non-homogeneous granulation;for the 4.0μmol/l group,there were a massive reduction of the adherence cells,and the cells felt off increased.Most cells became round,and the cell bodies crimpled distorted.In the cells,the grana increased; for the 8.0μmol/l group,the mission cells were separate,and the cells felt off.The cell bodies crimpled.By chance the cell debris were seen.2.The inhibition to the growth multiplication of the PC-3 cells after the 48h treatment with As₂O₃ in vitro.MTT assay observed that As₂O₃ of different concentrations all could inhibit the growth of the prostate cancer cell line PC-3,and its inhibition strengthened along with the dosage increasing,assuming the dose dependence(P＜0.001).Flow Cytometry assessed that As₂O₃ could mediate PC-3 cell cycle and arrest PC-3 cells in G₂/M stage.After incubated with As₂O₃ for 48h,the arrest rate of G₂/M cells in PC-3 cells is 18.9%,27.7%,34.4%,36.9%respectively when the concentration of As₂O₃ is 0,1,2,4μmol/l.3.The effect of As₂O₃ on the AR protein expression.For the negative control group,there was no AR expression;for the medication group,there had AR positive expression,which was ranging fron yellow brown to dark brown and lied in nucleus.Comparing the medication groups with the negative control group,AR positive-expression rate has great increased.Using the positive area average gradation value,to compare any medication group with the negative control group,there was a significant difference between them on the AR average positive-expression rate(P＜0.001);to comparing any two of the AR groups,there was also a significant difference between them on the AR average positive-expression rate(P＜0.001).Conclusions:1.As₂O₃ could mediate PC-3 cells growth inhibition through dose-depend manner, and it could arrest PC-3 cell in G₂/M stage. 2.As₂O₃ could induce AR protein re-expression in human prostate cancer cell line PC-3 through dose-depend manner.