Prostate cancer (PCa) patients receiving the androgen ablation therapy ultimately develop recurrent castration-resistant prostate cancer (CRPC) within 1–3 years. Treatment with caffeic acid phenethyl ester (CAPE) suppressed cell survival and proliferation via induction of G1 or G2/M cell cycle arrest in LNCaP 104-R1, DU-145, 22Rv1, and C4–2 CRPC cells. CAPE treatment also inhibited soft agar colony formation and retarded nude mice xenograft growth of LNCaP 104-R1 cells. We identified that CAPE treatment significantly reduced protein abundance of Skp2, Cdk2, Cdk4, Cdk7, Rb, phospho-Rb S807/811, cyclin A, cyclin D1, cyclin H, E2F1, c-Myc, SGK, phospho-p70S6kinase T421/S424, phospho-mTOR Ser2481, phospho-GSK3α Ser21, but induced p21Cip1, p27Kip1, ATF4, cyclin E, p53, TRIB3, phospho-p53 (Ser6, Ser33, Ser46, Ser392), phospho-p38 MAPK Thr180/Tyr182, Chk1, Chk2, phospho-ATM S1981, phospho-ATR S428, and phospho-p90RSK Ser380. CAPE treatment decreased Skp2 and Akt1 protein expression in LNCaP 104-R1 tumors as compared to control group. Overexpression of Skp2, or siRNA knockdown of p21Cip1, p27Kip1, or p53 blocked suppressive effect of CAPE treatment. Co-treatment of CAPE with PI3K inhibitor LY294002 or Bcl-2 inhibitor ABT737 showed synergistic suppressive effects. Our finding suggested that CAPE treatment induced cell cycle arrest and growth inhibition in CRPC cells via regulation of Skp2, p53, p21Cip1, and p27Kip1.
Oxysterols are oxidation products of cholesterol. Cholestane-3β, 5α, 6β-triol (abbreviated as triol) is one of the most abundant and active oxysterols. Here, we report that triol exhibits anti-cancer activity against human prostate cancer cells. Treatment of cells with triol dose-dependently suppressed proliferation of LNCaP CDXR-3, DU-145, and PC-3 human prostate cancer cells and reduced colony formation in soft agar. Oral administration of triol at 20 mg/kg daily for three weeks significantly retarded the growth of PC-3 xenografts in nude mice. Flow cytometric analysis revealed that triol treatment at 10–40 µM caused G1 cell cycle arrest while the TUNEL assay indicated that triol treatment at 20–40 µM induced apoptosis in all three cell lines. Micro-Western Arrays and traditional Western blotting methods indicated that triol treatment resulted in reduced expression of Akt1, phospho-Akt Ser473, phospho-Akt Thr308, PDK1, c-Myc, and Skp2 protein levels as well as accumulation of the cell cycle inhibitor p27Kip. Triol treatment also resulted in reduced Akt1 protein expression in PC-3 xenografts. Overexpression of Skp2 in PC-3 cells partially rescued the growth inhibition caused by triol. Triol treatment suppressed migration and invasion of DU-145, PC-3, and CDXR-3 cells. The expression levels of proteins associated with epithelial-mesenchymal transition as well as focal adhesion kinase were affected by triol treatment in these cells. Triol treatment caused increased expression of E-cadherin protein levels but decreased expression of N-cadherin, vimentin, Slug, FAK, phospho-FAK Ser722, and phospho-FAK Tyr861 protein levels. Confocal laser microscopy revealed redistribution of β-actin and α-tubulin at the periphery of the CDXR-3 and DU-145 cells. Our observations suggest that triol may represent a promising therapeutic agent for advanced metastatic prostate cancer.
The qRT-PCR analysis of 139 clinical samples and analysis of 150 on-line database clinical samples indicated that AKT3 mRNA expression level was elevated in primary prostate tumors. Immunohistochemical staining of 65 clinical samples revealed that AKT3 protein expression was higher in prostate tumors of stage I, II, III as compared to nearby normal tissues. Plasmid overexpression of AKT3 promoted cell proliferation of LNCaP, PC-3, DU-145, and CA-HPV-10 human prostate cancer (PCa) cells, while knockdown of AKT3 by siRNA reduced cell proliferation. Overexpression of AKT3 increased the protein expression of total AKT, phospho-AKT S473, phospho-AKT T308, B-Raf, c-Myc, Skp2, cyclin E, GSK3β, phospho-GSK3β S9, phospho-mTOR S2448, and phospho-p70S6K T421/S424, but decreased TSC1 (tuberous sclerosis 1) and TSC2 (tuberous Sclerosis Complex 2) proteins in PC-3 PCa cells. Overexpression of AKT3 also increased protein abundance of phospho-AKT S473, phospho-AKT T308, and B-Raf but decreased expression of TSC1 and TSC2 proteins in LNCaP, DU-145, and CA-HPV-10 PCa cells. Oncomine datasets analysis suggested that AKT3 mRNA level was positively correlated to BRAF. Knockdown of AKT3 in DU-145 cells with siRNA increased the sensitivity of DU-145 cells to B-Raf inhibitor treatment. Knockdown of TSC1 or TSC2 promoted the proliferation of PCa cells. Our observations implied that AKT3 may be a potential therapeutic target for PCa treatment.
Androgen ablation therapy is the primary treatment for metastatic prostate cancer. However, 80-90% of the patients who receive androgen ablation therapy ultimately develop recurrent tumors in 12-33 months after treatment with a median overall survival time of 1-2 years after relapse. LNCaP is a commonly used cell line established from a human lymph node metastatic lesion of prostatic adenocarcinoma. We previously established two relapsed androgen receptor (AR)-rich androgen-independent LNCaP sublines 104-R1 (androgen depleted for 12 months) and 104-R2 cells (androgen depleted for 24 months) from AR-positive androgen-dependent LNCaP 104-S cells. LNCaP 104-R1 and 104-R2 mimics the AR-positive hormone-refractory relapsed tumors in patients receiving androgen ablation therapy. Androgen treatment stimulates proliferation of 104-S cells, but causes growth inhibition and G1 cell cycle arrest in 104-R1 and 104-R2 cells. We investigated the protein expression profile difference between LNCaP 104-S vs. LNCaP 104-R1, 104-R2, PC-3, and DU-145 cells as well as examined the sensitivity of these prostate cancer cells to different chemotherapy drugs and small molecule inhibitors. Compared to 104-S cells, 104-R1 and 104-R2 cells express higher protein levels of AR, PSA, c-Myc, Skp2, BCL-2, P53, p-MDM2 S166, Rb, and p-Rb S807/811. The 104-R1 and 104-R2 cells express higher ratio of p-Akt S473/Akt, p-EGFR/EGFR, and p-Src/Src, but lower ratio of p-ERK/ERK than 104-S cells. PC-3 and DU-145 cells express higher c-Myc, Skp2, Akt, Akt1, and phospho-EGFR but less phospho-Akt and phospho-ERK. Overexpression of Skp2 increased resistance of LNCaP cells to chemotherapy drugs. Paclitaxel, androgen, and inhibitors for PI3K/Akt, EGFR, Src, or Bcl-2 seem to be potential choices for treatment of advanced prostate cancers. Our study provides rationale for targeting Akt, EGFR, Src, Bcl-2, and AR signaling as a treatment for AR-positive relapsed prostate tumors after hormone therapy.
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