Sildenafil triggers tumor lethality through altered expression of HSP90 and degradation of PKD2

Abstract

The repurposing of existing drugs has emerged as an attractive additional strategy to the development of novel compounds in the fight against cancerous diseases. Inhibition of phosphodiesterase 5 (PDE5) has been claimed as a potential approach to target various cancer subtypes in recent years. However, data on the treatment of tumors with PDE5 inhibitors as well as the underlying mechanisms are as yet very scarce. Here, we report that treatment of tumor cells with low concentrations of Sildenafil was associated with decreased cancer cell proliferation and augmented apoptosis in vitro and resulted in impaired tumor growth in vivo. Notably, incubation of cancer cells with Sildenafil was associated with altered expression of HSP90 chaperone followed by degradation of protein kinase D2, a client protein previously reported to be involved in tumor growth. Furthermore, the involvement of low doses of PU-H71, an HSP90 inhibitor currently under clinical evaluation, in combination with low concentrations of Sildenafil, synergistically and negatively impacted on the viability of cancer cells in vivo. Taken together, our study suggests that repurposing of already approved drugs, alone or in combination with oncology-dedicated compounds, may represent a novel cancer therapeutic strategy.

Figure 1.

Treatment with Sildenafil is associated with impaired proliferation of cancer cells in a dose-dependent manner. (A–F) 2 × 10⁴ MIA PaCa-2 and Panc1 pancreatic, MDA-MB-231 breast, HCT-116 and SW480 colon and A549 lung cancer cells were plated in 12 well dishes and subjected to various concentration of Sildenafil as indicated. Cell number was quantified after 72 h. One of three independent experiments performed in triplicate is shown.

Figure 2.

Sildenafil decreases proliferation of cancer cells in time-dependent manner. (A–F) 2 × 10⁴ MIA PaCa-2 and Panc1 pancreatic, MDA-MB-231 breast, HCT-116 and SW480 colon and A549 lung cancer cells were plated in 12 well dishes and subjected to 25 µM Sildenafil. Cells were allowed to grow for the next 6 days when the number was quantified. One of three independent experiments performed in triplicate is shown.

Figure 3.

Sildenafil treatment triggers cancer cell death. (A) MIA PaCa-2 pancreatic and (B) HCT-116 colon cancer cells were treated with 25 µM Sildenafil (Sil) for 3 days. Cells were subjected to Annexin V/Propidium Iodide (PI) staining and subsequent flow cytometry. The fluorescence-activated cell sorting analysis was conducted in triplicate. Data are shown as mean ± SEM. One of the three experiments is presented. (C) Protein lysates of MIA PaCa-2 and HCT-116 cells were subjected to western blot analysis with PARP antibody. β-Actin was used as loading control.

Figure 4.

Sildenafil treatment suppresses tumor growth in ovo. (A) 1.5 × 10⁶ HCT-116 colon or MIA PaCa-2 pancreatic cancer cells were implanted 8 days after fertilization. Tumors were treated 24 and 48 h with 25 µM Sildenafil (Sil). Tumor photographs 96 h after implantation are presented. Scale bar, 1 mm. (B) Quantification of tumor area is presented. Error bars represent mean ± SEM of four to seven tumors. (C) IHC of HCT-116 and MIA PaCa-2 cells growing on CAM using specific antibodies directed against Ki67 are presented. (D) Quantification of Ki67 positive cells is shown. Error bars represent mean ± SEM of at least six microscopic fields, each containing at least 200 cells.

Figure 5.

Sildenafil treatment is associated with altered HSP90 expression and PKD2 degradation. (A) Lysates of cancer cell lines incubated with 25 µM Sildenafil for 3 days were subjected to western blot analysis with HSP90β antibody. β-Actin was used as loading control. Densitometry analysis of three to four blots is presented. (B) Cleared lysates of tumor cell lines subjected to 25 µM Sildenafil for 3 days were used for western blot analysis with PKD2 antibody. β-Actin was used as loading control. (C) PKD2 overexpression was achieved via lentiviral-mediated transduction. Western blot analysis with PKD2-specific antibody after ending the selection is presented. (D–G) Cancer cells stably transduced with empty vector (Con) or PKD2 (PKD2 o.e.) were seeded in 12 well dishes. Cell proliferation was monitored for the next 3 days in the presence or absence of Sildenafil (Sil) when cell number was quantified. One representative of three experiments conducted in triplicate is shown.

Figure 6.

Sildenafil potentiates PU-H71 effects toward impaired tumor growth. (A) Lysates of cancer cell lines incubated with different amounts of PU-H71 for 24 h days were subjected to western blot analysis with PARP-specific antibody. β-Actin was used as loading control. (B) 1 × 10⁶ SW480 colon cancer cells were implanted inside a silicon ring on chicken CAM 8 days after fertilization. Tumors were treated either with 50 nM PU-H71, 15 µM Sildenafil or combination of both compounds at 24 and 48 h after implantation. Tumors were allowed to growth for additional 24 h. Scale bar, 1 mm. (C) Quantification of tumor area is presented. Error bars represent mean ± SEM of five to seven tumors. (D) IHC of SW480 cancer cells growing on CAM using specific antibodies directed against Ki67 is presented.