Research On Inhibitor Of 17 ?-hydroxysteroid Dehydrogenase Type 3 As A Novel Target In Prostate Cancer

Objective:Prostate cancer is one of the common malignancies in men worldwide.Due to many factors such as the aging of the population,the increase of animal fat intake and the decrease of physical activity,the incidence and mortality of prostate cancer increase year by year.Long-term use of androgen deprivation therapy centered on the androgen receptor(AR)leads to the development of castration-resistant prostate cancer(CRPC).17?-hydroxysteroid dehydrogenase III(17?-HSD3)is a new target for prostate cancer treatment independent of androgen receptor.In this study,17?-HSD3 was targeted for the curcuminoid derivatives synthesized in the lab.Screening for inhibitors to find new small molecule drugs for the treatment of prostate cancer.Method:(1)A radioimmunoassay was used to screen 17?-HSD3-sensitive curcumin derivatives by measuring the content of testosterone and progesterone in the supernatant of LC540(17?-HSD3)cells after administration;(2)Real time qPCR was used to detect the expression of 17?-HSD3 gene in LC540(17?-HSD3)cells after treatment with different concentrations of compounds;(3)MTT colorimetric assay was used to detect the effect of compound on the proliferation of LNCap,PC3 and LC540(17?-HSD3)cells;(4)Western Blot was used to detect the expression of 17?-HSD3,StAR,CYP11A1,CYP17 and 3?-HSD in the testosterone biosynthetic pathway of LC540(17?-HSD3)cell line;(5)Testis microsomes were extracted and subjected to enzyme kinetics reaction to detect the effect of compound H10 on 17?-HSD3 protease activity;(6)SD rats were given for seven consecutive days,and blood samples were taken after 7 days to detect the hormone content;(7)LNcap tumor-bearing nude mice models were constructed and randomly divided into 5 groups:normal saline group(A);solvent group(B);H10 low(10 mg/kg,C),medium(30 mg/kg,D),high(50 mg/kg,E)Three dose groups were administered intraperitoneally(ip).The tumor volume and body weight of each group of nude mice during the administration period were recorded,and serum levels of nude mice were measured before sacrifice;(8)The acute toxicity test of mice was used to preliminarily examine the safety of the target compound H10.After the animals were sacrificed,the organs were taken for histomorphological observation.Result:(1)Three potential curcumin derivatives were selected using the LC540(17?-HSD3)cell line:H1,H10,and H11.H10 significantly reduced testosterone levels in the supernatant of LC540(17?-HSD3)cells,but had no effect on progesterone levels;while H1 and H11 also reduced testosterone and progesterone levels,while other compounds had no effect on testosterone and progesterone levels.;(2)H10 treatment of the LC540(17?-HSD3)cell line down-regulated the expression of the 17?-HSD3 gene in a dose-dependent manner.H1 concentrations of 0.25,0.5,and 1 ?M significantly down-regulated the expression of 17?-HSD3 protein(P<0.05),but had no significant effect on the expression of StAR,CYP11A1,CYP17 and 3?-HSD on the testosterone synthesis pathway;(3)After 0,10,20 and 40 ?mol/L H10 treatment of testicular microsomes,the testosterone concentrations in the system were 1.19±0.07 ng/ml,0.90±0.10 ng/ml,0.56±0.06 ng/ml,and 0.49±0.09 ng/ml,respectively;(4)After 7 days(10,30,and 50 mg/kg/d)of continuous administration of SD rats,the level of testosterone decreased significantly in a dose-dependent manner,while the level of progesterone remained unchanged;(5)The tumor volume growth of nude mice in the H10 group was significantly slower than that in the saline group and the solvent group(P<0.05).The tumor volumes in the saline group,solvent group,and H10-administered group(low,medium,and high)were 510.80±99.08 mm3,494.49±81.91 mm3,424.45±95.24 mm3,243.67±58.49 mm3,and 269.44±78.98 mm3,respectively;The serum testosterone levels in nude mice were 1.53±0.56 ng/ml,1.41 ±0.65 ng/ml,0.41 ±0.30 ng/ml,0.22±0.07 ng/ml,and 0.19±0.16 ng/ml(means±SD,n=6),respectively;(6)The mice were intraperitoneally injected with H10(the highest dose for solubility reasons was 100 mg/kg),no adverse effects on KM mice in the experimental dose range,histological sections showed normal heart,liver,spleen,lung,kidney and testicular tissue.Conclusion:In this study,17?-HSD3 was used as a target,and its selective inhibitor H10 was screened from a series of curcumin derivatives,and the inhibitory effect of H10 on the expression of 17?-HSD3 gene and protein was further verified by in vitro and in vivo experiments.In the nude mouse model of LNCap cell line prostate cancer,H10 can effectively reduce testosterone levels in nude mice and inhibit tumor growth.The result shows that the curcumin derivative H10 is a potential small molecule inhibitor targeting 17?-HSD3 and can be used as a therapeutic drug for prostate cancer for further study.