Abstract
Combination therapies that display cancer-killing activities through either coexistent targeting of several cellular factors or more efficient suppression of a specific pathway are generally used in cancer treatment. Sildenafil, a specific phosphodiesterase type 5 (PDE5) inhibitor, has been suggested to display both cardioprotective and neuroprotective activities that provide a rationale for the combination with vincristine on the treatment against castration-resistant prostate cancer (CRPC). In the present work, vincristine arrested cells in the metaphase stage of mitosis. Vincristine-induced mitotic arrest was identified by Cdk1 activation (i.e., increased Cdk1Thr161 phosphorylation and decreased Cdk1Tyr15 phosphorylation), cyclin B1 upregulation, and increased phosphorylation of multiple mitotic proteins and stathmin. Sildenafil synergistically potentiated vincristine-induced mitotic arrest and a dramatic increase of mitotic index. Furthermore, sildenafil potentiated vincristine-induced mitochondrial damage, including Mcl-1 downregulation, Bcl-2 phosphorylation and downregulation, Bak upregulation and loss of mitochondrial membrane potential, and sensitized caspase-dependent apoptotic cell death. Sildenafil-mediated synergistic effects were mimicked by other PDE5 inhibitors including vardenafil and tadalafil, and also by PDE5A knockdown in cells, suggesting PDE5-involved mechanism. Notably, sildenafil amplified vincristine-induced phosphorylation and cleavage of BUBR1, a protein kinase in spindle assembly checkpoint (SAC) function and chromosome segregation. Sildenafil also significantly decreased kinetochore tension during SAC activation. Moreover, sildenafil synergized with vincristine on suppressing tumor growth in an in vivo model. In conclusion, the data suggest that sildenafil, in a PDE5-dependent manner, potentiates vincristine-induced mitotic arrest signaling, and sensitizes mitochondria damage–involved apoptosis in CRPC. Both in vitro and in vivo data suggest the combination potential of PDE5 inhibitors and vincristine on CRPC treatment.
Highlights
Prostate cancer is the second most commonly occurring cancer in men worldwide
The data were substantiated using flow cytometric quantitation of DNA content showing that sildenafil synergistically increased vincristine-induced apoptotic sub-G1 cell population in PC-3 cells (Figure 1B)
The synergism between vincristine and sildenafil was assessed through constructing isobolograms and calculating combination index (CI) values using Chou–Talalay method [20]
Summary
Prostate cancer is the second most commonly occurring cancer in men worldwide. Prostate cancer that keeps growing regardless of androgen-deprivation therapy in the situation of very low serum testosterone levels is considered castration-resistant prostate cancer (CRPC). The combination of Vinca alkaloids with several anticancer drugs in CRPC treatment has been demonstrated to display favorable activity and a low toxicity profile in several clinical studies [5,6,7]. These combination therapies fulfill the purpose of mechanism-based killing cancer and reduction of toxic effect through decreased doses of individual drugs and suggest that Vinca alkaloids are options in combination with other therapeutic drugs in CRPC treatment. Combination of sildenafil with standard chemotherapy agents (vincristine/etoposide/cisplatin) significantly enhances anticancer effect against medulloblastoma [18] These studies suggest the feasibility and therapeutic anticancer potential between the combination of sildenafil with vincristine. To the best of our knowledge, this is the first study dealing with the underlying mechanism related to perturbation of spindle checkpoint protein and microtubule–kinetochore interactions in sildenafil-sensitized anticancer effect
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