Drastic Synergy of Lovastatin and Antrodia camphorata Extract Combination against PC3 Androgen-Refractory Prostate Cancer Cells, Accompanied by AXL and Stemness Molecules Inhibition

Abstract

Prostate cancer (PC) is the second most frequently diagnosed cancer and the fifth leading cause of cancer-related death in males worldwide. Early-stage PC patients can benefit from surgical, radiation, and hormonal therapies; however, once the tumor transitions to an androgen-refractory state, the efficacy of treatments diminishes considerably. Recently, the exploration of natural products, particularly dietary phytochemicals, has intensified in response to addressing this prevailing medical challenge. In this study, we uncovered a synergistic effect from combinatorial treatment with lovastatin (an active component in red yeast rice) and Antrodia camphorata (AC, a folk mushroom) extract against PC3 human androgen-refractory PC cells. This combinatorial modality resulted in cell cycle arrest at the G0/G1 phase and induced apoptosis, accompanied by a marked reduction in molecules responsible for cellular proliferation (p-Rb/Rb, Cyclin A, Cyclin D1, and CDK1), aggressiveness (AXL, p-AKT, and survivin), and stemness (SIRT1, Notch1, and c-Myc). In contrast, treatment with either AC or lovastatin alone only exerted limited impacts on the cell cycle, apoptosis, and the aforementioned signaling molecules. Notably, significant reductions in canonical PC stemness markers (CD44 and CD133) were observed in lovastatin/AC-treated PC3 cells. Furthermore, lovastatin and AC have been individually examined for their anti-PC properties. Our findings elucidate a pioneering discovery in the synergistic combinatorial efficacy of AC and clinically viable concentrations of lovastatin on PC3 PC cells, offering novel insights into improving the therapeutic effects of dietary natural products for future strategic design of therapeutics against androgen-refractory prostate cancer.

Figures

Figure 1

The androgen-refractory prostate cancer PC3…

Figure 1

The androgen-refractory prostate cancer PC3 cells were treated with lovastatin and AC extract,…

Figure 1 The androgen-refractory prostate cancer PC3 cells were treated with lovastatin and AC extract, individually or in combination, for 72 h. PC3 cells were treated with (A) vehicle as the control, (B) 2 μM lovastatin, (C) 40 μg/mL AC extract, and (D) 2 μM lovastatin plus 40 μg/mL AC extract for 72 h. Bar = 100 μm.

Figure 2

Combination effects of lovastatin and…

Figure 2

Combination effects of lovastatin and AC extract on cell viability. ( A )…

Figure 2 Combination effects of lovastatin and AC extract on cell viability. (A) PC3, (B) DU145 androgen-refractory prostate cancer cells, and (C) HS68 primary human foreskin fibroblast cells were treated as indicated (lovastatin and AC extract, individually or in combination) for 72 h, and then the cell viability was determined by SRB assay as described in Materials and methods.

Figure 3

Combination effects of lovastatin and…

Figure 3

Combination effects of lovastatin and AC extract on the distribution of cell-cycle phase…

Figure 3 Combination effects of lovastatin and AC extract on the distribution of cell-cycle phase and induction of apoptotic sub-G1 fraction in PC3 cells. Cells were treated as indicated for 24, 48, and 72 h. The percentages of the G0/G1 and sub-G1 fractions are shown in the respective flow cytometric histograms.

Figure 4

Combination effects of lovastatin and…

Figure 4

Combination effects of lovastatin and AC extract on cell cycle-regulating molecules in PC3…

Figure 4 Combination effects of lovastatin and AC extract on cell cycle-regulating molecules in PC3 cells. Cells were treated as indicated for 72 h. Whole-cell lysates were analyzed by Western blot for the proteins of interest. The numbers above the bands indicate the relative densitometric ratios to the bands of loading control (GAPDH). Protein size (kDa): RB (110), Cyclin A (54), CDK2 (33), Cyclin D1 (37), CDK1 (34), CDK4 (34), and GAPDH (35).

Figure 5

Combination effects of lovastatin and…

Figure 5

Combination effects of lovastatin and AC extract on aggressiveness-related molecules in PC3 cells.…

Figure 5 Combination effects of lovastatin and AC extract on aggressiveness-related molecules in PC3 cells. Cells were treated as indicated for 24, 48, and 72 h. Whole-cell lysates were analyzed by Western blot for the proteins of interest. The numbers above the bands indicate the relative densitometric ratios to the bands of loading control (GAPDH). Protein size (kDa): AXL (140), AKT (60), Survivin (16), and GAPDH (35).

Figure 6

Combination effects of lovastatin and…

Figure 6

Combination effects of lovastatin and AC extract on the ( A ) stemness…

Figure 6 Combination effects of lovastatin and AC extract on the (A) stemness molecules and (B) stemness markers of PC3 cells. Cells were treated as indicated for 72 h. Whole-cell lysates were analyzed by Western blot for the proteins of interest. The numbers above the bands indicate the relative densitometric ratios to the bands of the loading control (GAPDH). Protein size (kDa): SIRT1 (120), Notch1 (125), c-Myc (67), and GAPDH (35).