Sildenafil potentiates the antitumor activity of cisplatin by induction of apoptosis and inhibition of proliferation and angiogenesis

Figures & data

Table 1 Primary antibodies, clones, and dilutions for immunostaining

Antibody	Clone	Dilution	Cellular location	Manufacturer
VEGF	Ab46154	1/100	Cytoplasmic	Abcam
Ki-67	Ab15580	1/100	Nuclear	Abcam
Caspase-3	Ab2302	1/50	Cytoplasmic	Abcam

Note: Abcam, Cambridge, UK.

Abbreviation: VEGF, vascular endothelial growth factor.

Figure 1 Effect of sildenafil and/or cisplatin on tumor volume.

Notes: Data are presented as mean ± SEM (n=8). ^aSignificant difference from control group (P<0.05); ^bSignificant difference from sildenafil group (P<0.05); ^cSignificant difference from cisplatin group (P<0.05).
Abbreviation: SEM, standard error of the mean.

Table 2 Effect of sildenafil and/or cisplatin on angiogenin and tumor necrosis factor-alpha (TNF-α)

Groups	Control	Sildenafil	Cisplatin	Cisplatin + sildenafil
Parameters
Angiogenin (pg/mg)	553.8±29.6	203.8±11a	274.3±18.2a	57.4±3a,b,c
TNF-α (pg/mg)	61.4±2.7	25.3±2a	37.4±1.4a	19.6±1a,b,c

Notes: Data are presented as mean ± standard error of mean (n=8).

a Significant difference from control group (P<0.05);

b Significant difference from sildenafil group (P<0.05);

c Significant difference from cisplatin group (P<0.05).

Table 3 Effect of sildenafil and/or cisplatin on VEGF, Ki-67, and caspase-3

Groups	Control	Sildenafil	Cisplatin	Cisplatin + sildenafil
Parameters
VEGF	262.5±8.81	235±7.32a	215±8.66a	176.25±8.01a,b,c
Ki-67	250±12.96	241.25±11.87	191.25±6.39a	138.75±6.39a,b,c
Caspase-3	11.9±1.88	45±6.81a	88.75±9.9a	136.25±12.9a,b,c

Notes: Data are presented as mean ± standard error of mean (n=8).

a Significant difference from control group (P<0.05);

b Significant difference from sildenafil group (P<0.05);

c Significant difference from cisplatin group (P<0.05).

Abbreviation: VEGF, vascular endothelial growth factor.

Figure 2 Cytoplasmic VEGF staining in tumor sections from different groups.

Notes: VEGF is strongly positive in the cytoplasm of both control (A) and sildenafil-treated tumors (B). VEGF positivity is seen in some of the cells in tumors treated with cisplatin (C), while it is markedly reduced in tumors treated with the combination therapy (D). Magnification, ×200. Cytoplasmic positivity is shown by arrows.
Abbreviation: VEGF, vascular endothelial growth factor.

Figure 3 Ki-67 nuclear expression in different groups.

Notes: Most of the nuclei showed Ki-67 nuclear immunoreactivity in control and sildenafil-treated tumors (A and B). Nuclei of cisplatin-treated tumors and tumors treated with the combination therapy showed markedly reduced nuclear Ki-67 positivity (C and D). Magnification, (A and B) ×200; (C) ×400; and (D) ×100. Nuclear positivity is shown by arrows.

Figure 4 Caspase-3 expression in different groups of tumors.

Notes: Caspase-3 is negative in a tumor from the control group (A). Mild cytoplasmic caspase-3 expression is seen focally in this tumor from a group treated with cisplatin (B). Tumors from mice treated with sildenafil and the combination therapy showed moderate cytoplasmic positivity to caspase-3 (C and D). Magnification, ×200. Cytoplasmic positivity is shown by arrows.

Figure 5 Histopathological examination of solid tumor.

Notes: A small area of necrosis within viable tissue in tumor biopsy excised from mice received no therapy (indicated by arrow) (A), in comparison, a substantial area of necrosis is seen in tumor treated with sildenafil, cisplatin, or the combination therapy (indicated by arrow) (B). Magnification, ×40.

Figure 6 Effect of sildenafil and/or cisplatin on the cell cycle.

Notes: Cell cycle distribution was determined using DNA flow cytometry analysis, the histograms (A), (B), (C), and (D) represent the DNA content of the cells in each of the cell cycle phases (Sub-G₁, G₀/G₁, S, and G₂/M) in control, sildenafil, cisplatin, and cisplatin + sildenafil groups, respectively. Different cell phases were plotted (E) as percentage of total events. The apoptotic cells were plotted separately (F) as percentage of the total events. Proliferation index (S% phase + G₂/M% phase) of all groups was plotted (G) (n=5). ^aSignificant difference from control group (P<0.05); ^bSignificant difference from sildenafil group (P<0.05); ^cSignificant difference from cisplatin group (P<0.05).

Figure 7 Cytotoxic effect of cisplatin and/or sildenafil on MCF-7 cell line.

Notes: Cells were exposed to different concentrations of sildenafil for 48 hours (A). Cells were exposed to different concentrations of cisplatin alone and different concentrations of cisplatin in combination with sildenafil (14 µg/mL) (B). Data are presented as mean ± standard deviation (n=6).

Figure 8 Scatter plots of significant correlation between tumor volume and caspase-3 (A), proliferation index (B), angiogenin (C), VEGF (D) and TNF-α (E) in sildenafil-treated group.

Notes: The solid lines represent the linear regression and correlation coefficient (r), P is the correlation significance level, and n is the total number of mice in control and sildenafil groups.
Abbreviations: TNF-α, tumor necrosis factor-alpha; VEGF, vascular endothelial growth factor.