Brazilian red propolis induces apoptosis-like cell death and decreases migration potential in bladder cancer cells

Abstract

Natural products continue to be an invaluable resource of anticancer drug discovery in recent years. Propolis is known for its biological activities such as antimicrobial and antitumor effects. This study assessed the effects of Brazilian red propolis (BRP) on apoptosis and migration potential in human bladder cancer cells. The effect of BRP ethanolic extract (25, 50, and 100 μg/mL) on 5637 cells was determined by MTT, LIVE/DEAD, and migration (scratch assay) assays. Apoptosis induction was investigated through flow cytometry and gene expression profile was investigated by qRT-PCR. Results showed cytotoxicity on MTT and LIVE/DEAD assays, with IC50 values of 95 μg/mL in 24 h of treatment. Cellular migration of 5637 cells was significantly inhibited through lower doses of BRP ethanolic extract (25 and 50 μg/mL). Flow cytometry analyses showed that BRP induced cytotoxicity through apoptosis-like mechanisms in 5637 cells and qRT-PCR revealed increased levels of Bax/Bcl-2 ratio, p53, AIF, and antioxidant enzymes genes. Data suggest that BRP may be a potential source of drugs to bladder cancer treatment.

Figure 1

ESI(+)-MS fingerprint of red propolis ethanolic extract.

Figure 2

Brazilian red propolis ethanolic extract increased antiproliferative and cytotoxicity effect in 5637 cells (a). Cell proliferation in 5637 and CHO-K1 was investigated by MTT assay. Data are expressed as means ± SEM from three independent experiments. Different letters (A/a, B/b, and C/c) indicate significant differences between the means. Uppercase letter indicates difference between the treatments in 5637 cells. Lowercase letter indicates difference between the treatments in CHO-K1 cells. The differences were considered significant at P < 0.05. VC = vehicle control (EtOH-H₂O). (b) Brazilian red propolis ethanolic extract increased 5637-cell death. Bladder cancer cells were treated with the BRP extract for 24 h. Analysis of cell death was estimated by LIVE/DEAD assay with 20x optical zoom. Untreated cells (A); 25 μg/mL treatment (B); 50 μg/mL treatment (C); 100 μg/mL treatment (D).

Figure 3

BRP ethanolic extract promotes apoptosis in 5637 cells. (a) Flow cytometry graphs for Annexin V-7AAD analysis of 5637 cells treated with 25, 50, and 100 μg/mL of BRP ethanolic extract for 24 h. Control group (A), 25 μg/mL group (B), 50 μg/mL group (C), and 100 μg/mL group (D). (b) Percentage of apoptotic cells after treatment with 25, 50, and 100 μg/mL of BRP ethanolic extract for 24 h. Data are expressed as means ± SEM from three independent experiments. Different letters (A, B, and C) indicate significant differences between the means and differences were considered significant at P < 0.05. (c) Selectivity of BFP in normal and tumoral cells after treatment with 100 μg/mL for 24 h. Data are expressed as means ± SEM from three independent experiments. Different letters (A, B, and C) indicate significant differences between the means and differences were considered significant at P < 0.05. (∗) Early and late apoptosis together.

Figure 4

BRP ethanolic extract increased DNA fragmentation in 5637 cells. (a) Flow cytometry graphs for TUNEL analysis of cells treated with 25, 50, 100, and 200 μg/mL of BRP ethanolic extract for 24 h. Control group (A), 25 μg/mL group (B), 50 μg/mL group (C), and 100 μg/mL group (D). (b) Percentage of cells with DNA fragmentation after treatment with 25, 50, and 100 μg/mL of BRP ethanolic extract for 24 h. Data are expressed as means ± SEM from three independent experiments. Different letters (A and B) indicate significant differences between the means and differences were considered significant at P < 0.05.

Figure 5

BRP ethanolic extract increased the Bax/Bcl-2 ratio in 5637-cell line after 24 hours of treatment. The gene expression profile was determined by qRT-PCR and data were normalized using GAPDH levels. (a) Proapoptotic Bax gene expression. (b) Antiapoptotic Bcl-2 gene expression. (c) Bax/Bcl-2 ratio. Data are expressed as means ± SEM from three independent experiments. Different letters (A, B, and C) indicate significant differences between the means and (∗) indicates difference between 100 μg/mL treated group and no treatment control group. The differences were considered significant at P < 0.05.

Figure 6

Effect of BRP ethanolic extract in apoptotic-related gene expression of 5637-cell line. (a) AIF; (b) Endo G. Data are expressed as means ± SEM from three independent experiments. Different letters (A, B, and C) indicate significant differences between the means and differences were considered significant at P < 0.05.

Figure 7

BRP ethanolic extract induced changes in the mRNA levels in 5637 cells. 5637 cells were treated with the indicated concentrations of BRP ethanol extract for 24 h. RT-PCR were performed to caspase-9, caspase-8, caspase-3, and p53 genes and data were normalized using GAPDH levels. Different letters (A, B, and C) indicate significant differences between the means and differences were considered significant at P < 0.05.

Figure 8

BRP ethanolic extract changes antioxidant enzymes gene expression in 5637 cells. The gene expression profile was determined by qRT-PCR. (a) Mn-SOD; (b) TRx; (c) GST; (d) CAT; (e) Cu/Zn-SOD; (f) GLUT. Data are expressed as means ± SEM from three independent experiments. Different letters (A, B, and C) indicate significant differences between the means and differences were considered significant at P < 0.05.

Figure 9

BRP ethanol extract decreased migration of 5637-cell line after 25 and 50 μg/mL treatments. (a) Migration of 5637 cells after 24 hours of treatment with 25 and 50 μg/mL of BRP ethanolic extract. (b) Percentage of free width in the scratch-wound healing after treatment with 25 and 50 μg/mL of BRP ethanolic extract in 0, 8, and 12 h. (c) Number of cells that cross the scratch-wound healing after treatment with 25 and 50 μg/mL of BRP ethanolic extract in 8 and 12 h. Data are expressed as means ± SEM from three independent experiments. Different letters (A and B) and asterisk (∗) indicate significant differences between the means and differences were considered significant at P < 0.05. ^# P < 0.01.