Cabazitaxel suppresses the proliferation and promotes the apoptosis and radiosensitivity of castration-resistant prostate cancer cells by inhibiting PI3K/AKT pathway

Abstract

Background: Cabazitaxel has been applied to the treatment of castration-resistant prostate cancer (CRPC), but the molecular mechanism remained to be fully understood.

Methods: After treatment with Cabazitaxel alone or in combination with ionizing radiation (IR), CRPC cell viability, proliferation and apoptosis were determined by Cell Counting Kit-8 (CCK-8) assay, colony formation, and flow cytometry, respectively. Tumor volume was measured after the establishment of animal xenograft model. Relative expressions of proteins related to apoptosis (B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X protein (Bax), and cleaved caspase 3) and phosphoinositide 3-kinase (PI3K)/AKT pathway were measured by Western blot, and the phosphorylated-PI3K/PI3K and p-AKT/AKT ratios were determined as well.

Results: Cell viability and proliferation were suppressed, and apoptosis was promoted in CRPC cells after Cabazitaxel treatment alone, accompanied with upregulated expressions of Bax and cleaved caspase 3 and downregulated Bcl-2 expression. Also, a single treatment with Cabazitaxel resulted in suppression of PI3K/AKT pathway activation, along with downregulated expressions of p-PI3K and p-AKT and a reduced ratio of p-PI3K/PI3K to p-AKT/AKT. Meanwhile, Cabazitaxel enhanced the effects of IR on suppressing survival and promoting apoptosis in CRPC cells through downregulating Bcl-2 and upregulating Bax and cleaved caspase 3. However, Cabazitaxel suppressed IR-induced PI3K/AKT pathway activation via downregulating p-PI3K and p-AKT, leading to a reduced ratio of p-PI3K/PI3K to p-AKT/AKT. Furthermore, Cabazitaxel further promoted the effects of IR on suppressing tumor growth in vivo.

Conclusion: Cabazitaxel inhibited the proliferation and promoted the apoptosis and radiosensitivity of CRPC cells, which is related to the suppression of PI3K/AKT pathway, providing a therapeutic method for CRPC in clinical practice.

Keywords: Castration resistant prostate cancer; apoptosis; cabazitaxel; phosphoinositide 3-kinase (PI3K)/AKT pathway; proliferation; radiosensitivity.

Figure 1

Cabazitaxel suppressed the viability and proliferation of CRPC cells. A. Chemical structure of Cabazitaxel. B. Relative viability of CRPC 22Rv1 and PC-3 cells after treatment with different concentrations of Cabazitaxel was determined by CCK-8 assay. C, D. Relative colony formation rate of CRPC cells after treatment with different concentrations of Cabazitaxel was measured by colony formation assay. All experiments have been performed in triplicate and data were expressed as mean ± standard deviation (SD). *P<0.05, ***P<0.001, vs. 0 nmol/L. ^^P<0.01, ^^^P<0.001, vs. Control. CRPC: Castration Resistant Prostate Cancer; CCK-8: Cell Counting Kit-8.

Figure 2

Cabazitaxel promoted CRPC cell apoptosis. A, B. Apoptosis of CRPC 22Rv1 and PC-3 cells after treatment with different concentrations of Cabazitaxel was detected by flow cytometry. C-F. Relative protein/β-actin expressions of apoptosis-related factors (Bcl-2, Bax, and cleaved caspase 3) after treatment with different concentrations of Cabazitaxel were quantified by Western blot. β-actin was used as internal control. All experiments have been performed in independent triplicates and the data were expressed as mean ± standard deviation (SD). ^P<0.05, ^^P<0.01, ^^^P<0.001, vs. Control. Bcl-2: B-cell lymphoma 2; Bax: Bcl-2-associated X protein.

Figure 3

Cabazitaxel suppressed PI3K/AKT pathway activation in CRPC cells. (A, B) Relative protein/β-actin expressions of PI3K/AKT pathway-related proteins after treatment of 22Rv1 cells with different concentrations of Cabazitaxel were measured by Western blot. β-actin was used as internal control. (C, D) Ratios of p-PI3K/PI3K (C) and p-AKT/AKT (D) were measured. (E, F) Relative protein/β-actin expressions of PI3K/AKT pathway-related proteins after treatment of PC-3 cells with different concentrations of Cabazitaxel were measured by Western blot. β-actin was used as internal control. (G, H) Ratios of p-PI3K/PI3K (G) and p-AKT/AKT (H) were quantified. All the experiments have been performed in independent triplicates and the data were expressed as mean ± standard deviation (SD). ^P<0.05, ^^P<0.01, ^^^P<0.001, vs. Control.

Figure 4

Cabazitaxel enhanced the suppressive effects of IR on CRPC cell survival. (A-D) Colony formation assay was used to determine the effects of Cabaziatxel and IR on the survival of CRPC 22Rv1 (A, B) and PC-3 (C, D) cells. All the experiments were performed in triplicates and the data were expressed as mean ± standard deviation (SD). *P<0.05, **P<0.01, ***P<0.001, vs. Control. IR: ionizing radiation; SER: sensitivity enhancement ratio.

Figure 5

Cabazitaxel enhanced the effects of IR on promoting CRPC cell apoptosis. A, B. Apoptosis of CRPC 22Rv1 and PC-3 cells after Cabazitaxel and IR treatment was detected by flow cytometry. C-F. Relative protein/β-actin expressions of apoptosis-related factors (Bcl-2, Bax, and Cleaved caspase 3) after Cabazitaxel and IR treatment were quantified by Western blot. β-actin was used as internal control. All the experiments have been performed in independent triplicates and the data were expressed as mean ± standard deviation (SD). *P<0.05, **P<0.01, ***P<0.001, vs. Control; ^P<0.05, ^^P<0.01, ^^^P<0.001, vs. IR.

Figure 6

Cabazitaxel suppressed IR-induced PI3K/AKT pathway activation in CRPC cells. (A, B) Relative protein/β-actin expressions of PI3K/AKT pathway-related proteins after treatment of 22Rv1 cells with Cabazitaxel and IR were measured by Western blot. β-actin was used as internal control. (C, D) Ratios of p-PI3K/PI3K (C) and p-AKT/AKT (D) were measured. (E, F) Relative protein/β-actin expressions of PI3K/AKT pathway-related proteins after treatment of PC-3 cells with Cabazitaxel and IR were measured by Western blot. β-actin was used as internal control. (G, H) Ratios of p-PI3K/PI3K (G) and p-AKT/AKT (H) were quantified. All the experiments have been performed in independent triplicates and the data were expressed as mean ± standard deviation (SD). *P<0.05, **P<0.01, ***P<0.001, vs. Control; ^^^P<0.001, vs. IR.

Figure 7

Cabazitaxel enhanced the inhibitory effects of IR on tumor growth. A, B. Tumor volume was measured after injection of the PC-3 cells treated with Cabazitaxel and IR into the nude mice. All the experiments have been performed in independent triplicates and the data were expressed as mean ± standard deviation (SD). ***P<0.001, vs. Control; ^^^P<0.001, vs. IR.