Study On Mechanism Of Isoliquiritigen On Inhibiting Prostate Cancer And Benign Prostatic Hyperplasia By Activating Aldo-keto Reductase Which Regulated Androgen Metabolic Pathway

Objectives:In the previous study of our research group,isoliquiritigenin(ISL)inhibited the proliferation of prostate cancer cells LNCa P,22RV1 and PC-3,and promoted the apoptosis of prostate cancer cells in the traditional Chinese medicine compound PC-SPES for treating prostate cancer.The results of PC-3 xenografts in nude mice indicated that ISL can inhibit the growth of PC-3 xenografts.Further studies showed that ISL significantly down-regulated the expression of Androgen receptor(AR)and decreased the nuclear transcriptional activity of AR after acting on prostate cancer LNCa P cells;and the m RNA microarray gene chip of AKR1C2,the main metabolic enzyme of DHT,was studied and it was found that ISL can up-regulate its m RNA expression,suggesting that ISL may play an anti-prostate role by up-regulating the androgen-metabolizing enzyme AKR1C2 and affecting the AR signaling pathway.Based on the results of previous studies,study in the text aims to investigate the role of aldo-keto reductase in the regulation of androgen metabolism and the proliferation of prostate cancer cells,and to explore the role of aldo-keto reductase in the pathogenesis of prostate cancer and the mechanism of action against prostate cancer of ISL may play effecting on androgen metabolic pathway.Methods:1.The effects of different fetal bovine serum concentration on the expression of AKR1C2,AR and PSA in prostate cancer cells and the effect of ISL on the expression of AKR1C2,AR and PSA in prostate cancer cells were detected by q RT-PCR and Western blotting.2.q RT-PCR and Western blotting were used to investigate the changes of androgen receptor AR expression in prostate cancer cells VCa P,22RV1 and LNCa P by overexpressing AKR1C2,and the best time for transfection was selected.The effect of DHT-induced prostate cancer cell proliferation induced by overexpression of AKR1C2 were detected by MTT assay;flow cytometry was used to detect the effect of AKR1C2 overexpression on prostate cancer cell apoptosis.3.q RT-PCR and Western blotting experiments were used to investigate the changes of NRF2 protein expression and the changes of its target genes after ISL treatment on22RV1 and LNCa P cells.The substrate of AKR1C2 was determined by HPLC-MS technique after ISL treatment on PCa cells.Their metabolites were separately quantified.4.MTT assay were used to detect the effect of ISL on the proliferation of prostatic hyperplasia cells BPH-1.5.Western blotting was used to observe the effect of ISL on the expression of estrogen and androgen receptor and AKR1C2 protein in BPH-1 cells of benign prostatic hyperplasia cells.6.q RT-PCR was used to observe the effect of ISL on the expression of AKR1C2 m RNA in BPH-1 cells of benign prostatic hyperplasia cells.Results:1.Serum concentration had no evident effect on the expression of AKR1C2,AR and PSA in prostate cancer cells;ISL increased the expression of AKR1C2,decreased the expression of AR and decreased the expression of PSA m RNA in prostate cancer cells.2.Vcap cells transfected at 72 h,LNCa P cells transfected at 96 h and 22RV1 cells transfected at 72 h showed the best transfection effect.There was no significant change in the expression of androgen receptor AR protein and m RNA in LNCa P and22RV1 cells after transfection.The expression of AR m RNA decreased,the expression of androgen receptor AR protein and PSA m RNA in VCa P cells showed no significant changes.AKR1C2 can inhibit DHT-induced PCa cell proliferation;after overexpression of AKR1C2,early apoptosis of prostate cancer cells increases.3.The expression of NRF2 protein in LNCAP cells was increased when ISL was used at 25 ?M concentration,but the expression of NRF2 protein increased when ISL was used in 22RV1 cells.ISL increased the expression of AKR1C1,AKR1C2,AKR1C3,NQO1 and HO-1 m RNA in LNCAP cells,and the expression of Nrf2,MRP2 and Keap-1 m RNA did not change significantly.ISL increased the expression of AKR1C1,AKR1C2,HO-1 and NQO1 m RNA in 22RV1 cells.There was no significant change in Nrf2,AKR1C3,MRP2,and Keap-1 m RNA expression.Androgen T and DHT decreased when ISL was used in LNCa P cells.4.ISL inhibits BPH-1 proliferation in benign prostatic hyperplasia cells in a dose-dependent manner.5.ISL decreased the expression of estrogen receptor ER-? protein and increased the expression of AR protein in BPH-1,the expression of AKR1C2 protein increased significantly,and the expression of PSA protein decreased.6.ISL significantly increased the expression of AKR1C2 m RNA in BPH-1.Conclusions:1.Activation of aldosterone reductase AKR1C2 can inhibit the transcriptional activity of androgen receptor,promote the metabolism of intracellular androgen,inhibit the proliferation of prostate cancer cells,and induce apoptosis of prostate cancer cells.2.The mechanism of action of ISL may be to increase the expression and activity of Nrf2,increase the expression of AKR1C2,promote the metabolism of androgen in cells,inhibit the transcriptional activity of AR,and thereby inhibit the proliferation of prostate cancer cells.At the same time,ISL inhibits AR expression and inhibits proliferation of prostate cancer.3.ISL up-regulates the gene expression of phase II detoxification enzymes HO-1and NQO1 to promote the protection of the body.4.ISL inhibits BPH-1 proliferation in benign prostatic hyperplasia cells in a dose-dependent manner.5.ISL may inhibit the proliferation of BPH-1 in benign prostatic hyperplasia cells through the effects of estrogen receptor ER-?,androgen receptor AR,and aldosterone reductase AKR1C2.6.ISL decreased the expression of estrogen receptor and increased the expression of androgen receptor,which inhibited the proliferation of prostatic hyperplasia cells BPH-1.ISL also increased the expression of androgen metabolism enzyme AKR1C2 and inhibited the proliferation of BPH-1 cells.