Inhibition And Molecular Mechanisms Of Matrine On Human Androgen-independent Prostate Cancer DU145 And PC-3 Cell

Matrine is a naturally occurring alkaloid extracted from the Chinese herb Sophora flavescens. It has been demonstrated to exhibit antiproliferative properties, promote apoptosis and inhibit cell invasion in a number of cancer cell lines. It has also been shown to improve the efficacy of chemotherapy when it is combined with other chemotherapy drugs. Although matrine has been clinically used in recent years to treat a number of types of cancer, the therapeutic efficacy of matrine in androgen-independent prostate cancer and the exact mechanisms remain poorly understood. In the present study, we investigated the impact of matrine on the proliferation, migration, invasion, cell cycle and apoptosis of androgen-independent human prostate cancer cell lines DU 145 and PC-3, and explored the mechanisms underlying the antitumor activity of matrine on these androgen-independent prostate cancer cells by high-throughput sequencing,bioinformatics software, Real-time PCR technique and Immunoblot method testing . Our aim was to develop new strategies for the treatment of androgen-independent prostate cancer.[Objective]This study is designed to explore the therapeutic efficacy and the exact mechanisms of matrine in human androgen-independent prostate cancer cell lines DU145 and PC-3.[Methods](1) Anti-proliferative effects of matrine on DU145 and PC-3 cells were examined with MTS assay. At the same time, anti-migrative and anti-invasive effects of matrine on DU 145 and PC-3 cells were examined with transwell assay.(2) The DU 145 and PC-3 cell lines were cultured in vitro for 24 hours, then Matrine/control serum were used to intervene the cells synchronously for 48 hour and total RNA samples were extracted. HiSeq 2500 high-throughput sequencing platform developed by Illumina was used to sequence total RNA. A gene library was established and the quality analysis of read data was carried out. Integrated database of DAVID website was used to analyze gene ontology and KEGG pathway of the differential genes which were screened by 2 times method.(3)Cell cycle analysis and apoptosis analysis was performed with matrine-treated DU 145 and PC-3 cells by Flow cytometry (FCM) assay.(4) Immunoblot method testing was performed to investigate the expression levels of the inhibitor of P65, pP65, IKKα/β, pIKKa/p, IKBa and IKBa in cells treated with or without matrine.(5) Real-time PCR technique was performed to investigate the mRNA expression levels of FOXO1 A, FOXO3A, FOXO4, FOX06 and PI3K in matrine-treated DU145 and PC-3 cells.Immunoblot method testing was performed to investigate the protein expression levels of FOXO1 A, FOXO3A, FOX04, FOXO6 and PI3K in matrine-treated PC-3 and DU 145 cells.[Results](1) MTS assay showed that matrine inhibited both DU 145 and PC-3cells proliferation in a dose-dependent and time-dependent manner. Transwell assay showed that matrine inhibited both DU 145 and PC-3 cells migration and invasion in a dose-dependent and time-dependent manner.(2)By analyzing RNA with high throughput sequencing, bioinformatics software and GO analysis, it was found that differential genes before and after giving treatment played an important role in cell metabolic process, growth process, anatomical structure formation,cellular component organization, biological regulation and so on. It was found by KEGG signal pathway analysis that FOXO receptors signal pathways and PI3K _ AKT signal pathways had significant difference) 。(3) Cell cycle analysis showed that both Du 145 cells and PC-3 cells treated with 2.0 to 4.0g/l of matrine showed a significant increase in the percentage of G0/G1 phase cells and a significant decrease in the percentages of S phase cells, compared with the untreated control cells. Flow cytometry,as well as Annexin-V/PI staining,showed a significant and dose-dependent increase in the number of early,as well as late stage apoptotic cells in both DU 145 and PC-3 cells compared to untreated cells.(4) Western blot analysis showed that matrine treatment led to the degradation of P65, pP65,IKKα/β, pIKKa/p, IKBa and IKBa of both DU 145 and PC-3 cells.(5)By real-time PCR technique it was found that after being treated with Matrine , the mRNA expression levels of FOXOIA , FOX03A, FOX04 and FOX06 in DU145 and PC -3 cells all increased(P<0.01),but the PI3K mRNA expression levels decreased (P< 0.01 or P < 0.05 ). By Immunoblot method testing,it was found that after treating with Matrine, the protein expression levels of FOXOIA, FOX03A, FOX04 and FOX06 in both PC-3 and DU 145 cells increased (P < 0.01), and PI3K protein expression levels decreased (P < 0.01).[Conclusion](1)Matrine could inhibit cell proliferation, migration and invasion of both DU 145 and PC-3 cells in a dose-dependent and time-dependent manner.(2) Matrine had a broad regulating effect on the mRNA expression profiles of both PC-3 and DU 145 cells. The Gene Ontology analysis suggested that the differentially expressed genes mainly enriched in the cellular process, regulation,death and apoptosis and so on.The KEGG pathway analysis suggested that the NF-κB, FOXO and PI3K-AKT pathway was the important regulation target.(3) The antitumor effects of matrine on both DU 145 and PC-3 cells may be associated with the G0/G1 cell cycle arrest (shorten-S cell cycle) and promoting apoptosis.(4) Real-time PCR technique and Immunoblot method testing suggested that Matrine may inhibit cell proliferating, migrating as well as invading, and induce apoptosis in both PC-3 and DU 145 cells through NF-κB, FOXO and PI3K-AKT signaling pathway.(5) Matrine may be a promising reagent for treating androgen-independent prostate cancer.