Study On Berberine Inhibiting The Development Of Castration-resistant Prostate Cancer By Targeting AKR1C3and Its Mechanisms

The activation of androgen receptor signaling pathway is a significant drivingforce of prostate cancer progression, consequently, androgen deprived therapy (ADT)is the mainstay strategy to decrease circulating androgens or (and) block androgenreceptor (AR) in the patients with the advanced or metastatic androgen-dependentprostate cancer. However, after the remission in a median progression-free survival of12to18months,80%-90%of patients undergoing ADT inevitably progressed tocastration-resistant prostate cancer (CRPC), wherein the median overall survival isless than2years. Therefore, it’s theoretical and practical significance to investigatethe mechanisms of CRPC and to develop reliable and effective drugs for preventionand treatment of CRPC.The latest research shows that prostate cancer cells acquired ability ofintratumoral steroidogenesis from cholesterol or adrenaline androgens, whichstimulate prostate cancer progression to CRPC. Therefore, screening and developinginhibitors targeting key enzymes in intratumoral steroidogenesis pathway become anew strategy for the prevention and treatment of CRPC.Aldo-keto reductase family1member C3(AKR1C3) is a member of thealdo/keto reductase superfamily. AKR1C3catalyzes the conversion of low activehormone precursors such as androstenedione and androsterone to highly activetestosterone and dihydrotestosterone in the last two steps of steroid synthesis inhuman prostate cancer cell. Several studies have reported low or undetectable levelsof AKR1C3in normal prostate epithelia, whereas elevated AKR1C3levels have beenfound in the localized, advanced or recurrent prostate cancer (PCa) and universallyexpressed in CRPC. Moreover, LNCaP prostate cancer cell line overexpressingAKR1C3(LNCaP-AKR1C3) made significantly higher amounts of testosterone (T)but not dihydrotestosterone (DHT). Some studies assessed the expression of severalsteroidogenic enzymes and stem cell markers in clinical prostate cancer samples andcell cultures during androgen depletion. They found that AKR1C3expression was negatively regulated by androgens in the experimental models and was increased inCRPC samples, which implies that up-regulation of AKR1C3is an adaptation to theirT/DHT deprived environment and may be related to the transformation ofhormone-dependent prostate cancer to CRPC and stem-like cancer cells. Therefore,AKR1C3is regarded as a vital therapeutic target to reduce intratumoral androgensynthesis and screening and developing inhibitor targeting AKR1C3becomes a newstrategy for the prevention and treatment of CRPC.Berberine (BBR), an isoquinoline alkaloid, has anti-inflammation, regulatingimmune function and antineoplastic properties. Recent studies have found that BBRhas a potent anticancer activity for prostate cancer, but its role and mechanism ofprevention and treatment of CRPC has rarely reported. In our previous study, wefound berberine reduced the tumor volume and extended the latent period betweenandrogen-dependent prostate cancer and CRPC in nude mouse model bearing asubcutaneous LNCaP xenograft. However, the precise mechanisms of BBR exerts inthis aspect is still not clear.In this study, we raised a few questions as following1. Are there differences of the AKR1C3expression in benign and malignantprostate tissue? Is there correlation between the expression of AKR1C3and serumprostatic sepcific antigen (PSA) or Gleason score (GS) of prostate cancer? What’reroles of AKR1C3played in the progression of CRPC?2. What is the expression level of AKR1C3in different human prostate cancercell lines? Whether BBR could inhibit the synthesis of T or the proliferation ofprostate cancer cells with AKR1C3over-expression？Whether AKR1C3is target ofBBR? If the answer is yes, what’s the molecular mechanism of BBR involved inAKR1C3?To answer these questions, we carried out our experiments in three aspects.1. AKR1C3expression and its co-relation with the progression of prostate cancer(1) In this study, immunohistochemical staining for AKR1C3in human prostateneedle biopsy tissue specimens, including benign prostate hyperplasia (BPH),prostatic intraepithelial neoplasia (PIN) and PCa, was carried out. The data alsoshowed that the distribution of AKR1C3expression was different between BPH andPCa. For BPH and PIN specimens, positive expression of AKR1C3was observed inthe stromal cells, for malignant prostate cancer specimens stronger positive staining of AKR1C3was detected in prostate cancer epithelial cytoplasm. AKR1C3expressiongradually increased with increasing GS (rs=0.396, P=0.025) This result implicatedthat the levels of AKR1C3are closely associated with the progression of PCa.(2) In classical Partin tables, GS, PSA and the age are key parameters forevaluating the progression of prostate cancer. The correlations of mean AKR1C3expression with GS, mean PSA and age were analyzed. The data showed thatAKR1C3expression gradually increased with increasing GS, as indicated by themean optical density (MOD), exhibiting a positive correlation (rs=0.396, P=0.025).Serum PSA level is negatively correlated with AKR1C3expression (rs=-0.377, P=0.036) in prostate cancer biopsies, but the AKR1C3expression was not correlatedwith serum PSA levels (rs=-0.016, P=0.979) in BPH. The data also showed that theAKR1C3expression was positively correlated with the age (rs=0.76, P=0.01) in BPH.The MOD for positive AKR1C3expression in prostate tumor tissues was significantlyhigher than that of the BPH specimens. All the above indicates that AKR1C3is abetter marker to reflect the clinicopathological stage and evaluation of PCaprogression in those patients with low serum level of PSA.(3) To further confirm the relationship between the progression of PCa and theexpression of AKR1C3, the LNCaP mouse model with or without castration wasreplicated. The data shows that the expression levels of AKR1C3in LNCaP tumors at3weeks after castration were significantly increased compared to those of the LNCaPsham tumors. These results indicate that androgen ablation likely stimulates AKR1C3gene activation and might be attributed to prostate cancer progression.2. BBR inhibited the proliferation of castration-resistant prostate cancer bytargeting AKR1C3and its mechanisms(1) The screening of AKR1C3expression human prostate cancer cell. Cellularproteins were prepared from LNCaP cells, CWR22RV1cells, C4-2B cells, PC3cellsand PC3M cells Western blot analysis confirmed the expression of AKR1C3relativelystrong in PC3M cells and CWR22RV1cells among these cell lines. We utilizedCWR22RV1cell line to carry out the following experiment for its biologiccharacteristics of castration-resistance.(2) The proliferation of cancer cells in response to12.5μM,25μM and50μMBBR,30μM indomethacin (INN) and2μM finasteride were studied by MTT assay at48hours. The data show BBR exhibited dose-dependent inhibition on the proliferation of CWR22RV1cells at48h and the inhibition was relieved by theaddition of0.1μM androstenedione. Which implied the AKR1C3might be theinhibition target for BBR.(3) Cellular testosterone systhesis experiment was carried out with25μM BBR at48h on CWR22vR1cells. Finasteride is a inhibitor of5α-reductase and was used toblocked metabolism of0.1μM△4-Adione to5α-andostane-3,17-dione and block Tto the formation of DHT. The data shows that BBR significantly reduced theformation of total testosterone. The effect of BBR was better than the classicalAKR1C3inhibitor INN.(4) The influence of BBR on the AKR1C3in the level of mRNA and protein wasdetected by the RT-PCR and western blot analysis. The data shows that after treatedCWR22vR1cells with25μM BBR for48h, AKR1C3was not changed at the level ofmRNA or protein.(5) Enzyme activity was measured as either reduction of androstendione oroxidation of S-tetralol.The data shows that both BBR and INN displayed potentinhibition on the activity of recombinant human AKR1C3, but their IC50values forAKR1C3oxydation reaction and reduction reaction were different. When usedandrostendione as the substrate, the IC50value is for BBR and INN was3.60μM and11.42μM separately. When used S-tetralol as the substrate, the IC50value is for BBRand INN was1.69μM and7.07μM separately, which means at the same concentration,the inhibitors are prone to inhibit the oxydase activity of AKR1C3.3. The molecular mechanism of BBR on AKR1C3(1) The software AutoDock Tools was used to explore the inhibition mechanismof BBR on AKR1C3.The date shows that BBR could enter the steroid/inhibitorbinding cavity of AKR1C3and formed Pi-Pi interaction in the amino-acid residuePhe306and Phe311.(2) The binding energy of BBR is higher than testosterone,5α-androstane-3,17-dione and androstenediol, lower than dehydoepiandrosterone,5α-androstane-3α,17β-diol and5α-dihydrotestosterone. The binding energy ofandrostenedione with AKR1C3was the lowest among biological substrates and itimpliedAKR1C3had better selectivity on the androstenedione.Based on the above findings, we come to a conclusion:1. AKR1C3overexpressing, as an adaptive response for the progression of PCa and may serve as a promising biomarker for evaluating prostate cancer progression.2. BBR reduced the synthesis of androgen in CWR22RV1cells in vitro andinhibited the cell growth by targeting AKR1C3.3. The computer molecular simulation implied that BBR could enter the activecenter of AKR1C3and interact with the amino-acid residue Phe306and Phe311.