Sorafenib decreases proliferation and induces apoptosis of prostate cancer cells by inhibition of the androgen receptor and Akt signaling pathways

Abstract

Antihormonal and chemotherapy are standard treatments for nonorgan-confined prostate cancer. The effectivity of these therapies is limited and the development of alternative approaches is necessary. In the present study, we report on the use of the multikinase inhibitor sorafenib in a panel of prostate cancer cell lines and their derivatives which mimic endocrine and chemotherapy resistance. (3)H-thymidine incorporation assays revealed that sorafenib causes a dose-dependent inhibition of proliferation of all cell lines associated with downregulation of cyclin-dependent kinase 2 and cyclin D1 expression. Apoptosis was induced at 2 μM of sorafenib in androgen-sensitive cells, whereas a higher dose of the drug was needed in castration-resistant cell lines. Sorafenib stimulated apoptosis in prostate cancer cell lines through downregulation of myeloid cell leukemia-1 (MCL-1) expression and Akt phosphorylation. Although concentrations of sorafenib required for the antitumor effect in therapy-resistant sublines were higher than those needed in parental cells, the drug showed efficacy in cells which became resistant to bicalutamide and docetaxel respectively. Most interestingly, we show that sorafenib has an inhibitory effect on androgen receptor (AR) and prostate-specific antigen expression. In cells in which AR expression was downregulated by short interfering RNA, the treatment with sorafenib increased apoptosis in an additive manner. In summary, the results of the present study indicate that there is a potential to use sorafenib in prostate cancers as an adjuvant therapy option to current androgen ablation treatments, but also in progressed prostate cancers that become unresponsive to standard therapies.

Figure 1

Dose-dependent inhibition of proliferation in prostate cancer cell lines by sorafenib. LNCaP, 22Rv1, PC3, and LNCaP-IL6+ cells were exposed to increasing concentrations of sorafenib in HITES medium for 48 h. (A) Proliferation was assessed by ³H-thymidine incorporation. (B) Protein expression of CDK2 and cyclin D1 was detected by western blotting. Bands were scanned densitometrically and normalized to expression levels of GAPDH. Representative western blots from at least three independent experiments are shown. (A and B) Statistical significances are calculated against the DMSO-treated cells and values indicated are mean±s.e.m., n≥3. */# P<0.05; **/## P<0.01; ***/### P<0.001. POI, protein of interest.

Figure 2

Differential sensitivities of prostate cancer cell lines to sorafenib-mediated apoptosis through downregulation of MCL-1 and Akt pathway. LNCaP, 22Rv1, PC3, and LNCaP-IL6+ cells were exposed to increasing concentrations of sorafenib in HITES medium for 48 h. (A) Apoptosis was determined by PE/Annexin V staining and flow cytometry. LNCaP cells treated with 4 μM sorafenib underwent massive apoptosis resulting in an insufficient number of cells to perform assays. (B) Expression of MCL-1 was determined by western blotting. Bands were scanned densitometrically and normalized to expression levels of GAPDH. (C) Expression levels and phosphorylation status of Akt and GSK-3β in LNCaP and PC3 cells were determined by western blotting. GAPDH served as loading control. (A and B) Statistical significances are calculated against the DMSO-treated cells and values indicated are mean±s.e.m., n≥3. * P<0.05; ** P<0.01; *** P<0.001. Representative western blots from at least three independent experiments are shown.

Figure 3

Antiproliferative effects of sorafenib in therapy-resistant models of human prostate cancer. LNCaP-Bic and PC3-DR were exposed to increasing concentrations of sorafenib in HITES medium for 48 h. (A) Proliferation was assessed by ³H-thymidine incorporation. For comparison purposes results from Fig. 1A (LNCaP and PC3) are shown again. (B) Protein expression of CDK2 and cyclin D1 was detected by western blotting. Bands were scanned densitometrically and normalized to expression levels of GAPDH. Representative western blots from at least three independent experiments are shown. Statistical significances are calculated against the DMSO-treated cells and values indicated are mean±s.e.m., n≥3. */# P<0.05; **/## P<0.01; ***/### P<0.001. POI, protein of interest.

Figure 4

Therapy-resistant cells have decreased apoptotic sensitivity to sorafenib. LNCaP-Bic and PC3-DR were exposed to increasing concentrations of sorafenib in HITES medium for 48 h. (A) Apoptosis was determined by PE/Annexin V staining and flow cytometry. For comparison purposes results from Fig. 2A (LNCaP and PC3) are shown again. (B) Activity of the executioner caspases 3 and 7 after addition of the specific substrate. (C) Expression levels and phosphorylation status of MCL-1, Akt, and GSK-3β were determined by western blotting. Representative western blots from at least three independent experiments are shown. (A and B) Statistical significances are calculated against the DMSO-treated cells or parental cells and values indicated are mean±s.e.m., n≥3. * P<0.05; ** P<0.01; *** P<0.001. RLU, relative light units.

Figure 5

Sorafenib suppresses AR expression and decreases PSA secretion. (A) LNCaP, 22Rv1, and LNCaP-Bic were exposed to increasing concentration of sorafenib in HITES medium for 48 h. (A) Protein expression of AR was detected by western blotting. Bands were scanned densitometrically and normalized to expression levels of GAPDH. Representative western blots from at least three independent experiments are shown. (B) LNCaP and LNCaP-Bic cells were treated with sorafenib or bicalutamide in HITES medium for 48 h. Secreted PSA in the supernatants was measured and normalized to the respective cell number. (A and B) Statistical significances are calculated against the DMSO-treated cells and values indicated are mean±s.e.m., n≥3. * P<0.05; ** P<0.01; *** P<0.001.

Figure 6

Downregulation of AR by siRNA enhances apoptotic sensitivity of androgen-sensitive cells to sorafenib. LNCaP and 22Rv1 cells were transfected with 10 nM AR LBD siRNA and exposed on the next day to 2 μM sorafenib or DMSO in HITES medium for 48 h. (A) Protein expression of AR was detected by western blotting. GAPDH served as a loading control. Representative western blots from at least three independent experiments are shown. (B) Activity of the executioner caspases 3 and 7 after addition of the specific substrate. Values indicated are mean±s.e.m., n≥3. * P<0.05; ** P<0.01; *** P<0.001. RLU, relative light units.