Study On The Mechanism Of Inhibitory Effect In Human Prostate Cancer By Breviscapine

1.Background and Purpose:Prostate cancer is the most common male malignancy in the male reproductive system.Endocrine therapy is the standard treatment for advanced prostate cancer.Although endocrine therapy is effective at early stage,most patients are more likely to have castration resistance and the existing clinical treatment is not satisfactory.Breviscapine is from the Erigeron breviscapus extract flavonoids active ingredients,its therapeutic effect on the disease,such as stroke and cardiovascular disease.In recent years,breviscapine have been reported to have the effect on inhibiting some cancer,however,the inhibiton effect and molecular mechanisms of breviscapine on prostate cancer is unclear.The purpose of this study is to investigate the effect of breviscapine on prostate cancer and the related molecular mechanism in vivo and in vitro to provide a new idea for the treatment of advanced prostate cancer.2.Methods:Different concentrations of breviscapine were used to treat LNCap and PC3 cells.Morphological changes were observed under microscope.The viability of prostate cancer LNCap and PC3 cells was detected by MTT assay.The normal prostate cells RWPE-1 and normal liver cell L-02 were treated with different concentrations of breviscapine,and the cell viability of normal prostate cells RWPE-1 and normal liver cell L-02 were detected by MTT assay.Flow cytometry was used to evaluate the cell cycle distribution of LNCap and PC3 cells treated with breviscapine at different concentrations.Alexa Fluor 488 Annexin V/PI double staining method was used to detect the apoptosis of LNCap and PC3 cells after different concentrations of breviscapine.Cell cycle checkpoint-related proteins(p-ATM,p-ATR,p-CHK1,p-CHK2),cell cycle-related protein(PCNA,Cyclin D1,p21,p27,Rb,p-Rb)and apoptosis-related factors(active Caspase-3,activate PARP,Bax,Bcl-2)were measured on LNCap and PC3 cells by Western blot.The expression of MCM7 and γH2AX protein in LNCap and PC3 cells treated with breviscapine at different concentrations were measured.The MCM7 overexpression plasmid was transfected into LNCap cells with breviscapine treatment.The expression of MCM7 and γH2AX protein was detected by Western blot.Cell viability,the cell cycle and the percentage of apoptotic cells were detected.γH2AX siRNA transfected LNCap cells with Breviscapine treatment,cell viability changes,cell cycle changes and the proportion of apoptotic cells were measured.Positive and negative test whether breviscapine does inhibit the proliferation of prostate cancer cells by MCM7/γH2AX,induce cell stasis and apoptosis.LNCap and PC3 cells were harvested in nude mice.The nude mice were injected intraperitoneally with different doses of breviscapine.The health status of nude mice and the growth of transplanted tumor were observed.Cell cycle checkpoint-related proteins(p-ATM,p-ATR,p-CHK1,p-CHK2),cell cycle-related protein(PCNA,Cyclin D1,p21,p27,Rb,p-Rb)and apoptosis-related factors(active Caspase-3,activate PARP,Bax,Bcl-2)were detected on transplanted tumor by Western blotting.The expression of MCM7,Ki67 and TUNEL were detected by immunohistochemistry.The expressions of MCM7 and γH2AX protein were detected by immunohistochemistry.The expression of PCNA,Cyclin D1,p21 and p27 mRNA was detected by qPCR.3.Results:MTT assay showed that breviscapine had a significant inhibitory effect on the growth of LNCap and PC3 prostate cancer cells in a dose-dependent manner.Breviscapine has low toxicity to normal prostate cells and liver cells.Cell cycle G0/G1 ratio of LNCap and PC3 cells increased in a dose-dependent manner.The percentage of apoptosis in LNCap and PC3 cells increased in a dose-dependent manner.Western blot analysis showed that Cell cycle checkpoint-related proteins(p-ATM,p-ATR,p-CHK1,p-CHK2),cell cycle-related protein(PCNA,Cyclin D1,p21,p27,Rb,p-Rb)and apoptosis-related factors(active Caspase-3,activate PARP,Bax,Bcl-2)in LNCap and PC3 cells were increased with the increase of breviscapine concentration.With the increase concentration of breviscapine,MCM7 protein expression was down-regulated while γH2AX protein expression was up-regulated.Overexpression of MCM7 gene can partially reverse the expression of downstream γH2AX protein and growth inhibition,cell cycle arrest and apoptosis induction of breviscapine.Silencing of γH2AX can partially reverse the effect of breviscapine.Breviscapine treatment on nude mice transplanted tumor results showed that breviscapine treatment group significantly slowed the tumor growth in a dose-dependent manner.Immunohistochemical staining showed that the expression of MCM7 and Ki67 decreased with the increase of breviscapine dose,while the stain of TUNEL increased.With the increase of breviscapine concentration,western blot analysis showed that the expression of γH2AX was up-regulated with MCM7 down-regulated,the cell cycle checkpoint related protein(p-ATM,p-ATR,p-CHK1,p-CHK2)up-regulated,the expression of PCNA and Cyclin D1 downregulation,p21 and p27 upregulation,pRb down-regulation,and apoptosis-related factors expression changed(activated Caspase-3,activated PARP upregulation,Bax up-regulation,Bcl-2 down-regulation).4.Conclusions:Breviscapine have inhibitory effect on prostate cancer cells in a dose-dependent manner.Breviscapine induced G1 phase arrest and apoptosis of prostate cancer cell.Moreover,breviscapine was shown to induce cycle G1 arrest and apoptosis of prostate cancer cell through the regulation of MCM7/γH2AX signal pathway.