Enalapril overcomes chemoresistance and potentiates antitumor efficacy of 5-FU in colorectal cancer by suppressing proliferation, angiogenesis, and NF-κB/STAT3-regulated proteins

Abstract

5-Fluorouracil (5-FU) is one of the most effective drugs for the treatment of colorectal cancer (CRC). However, there is an urgent need in reducing its systemic side effects and chemoresistance to make 5-FU-based chemotherapy more effective and less toxic in the treatment of CRC. Here, enalapril, a clinically widely used antihypertensive and anti-heart failure drug, has been verified as a chemosensitizer that extremely improves the sensitivity of CRC cells to 5-FU. Enalapril greatly augmented the cytotoxicity of 5-FU on the cell growth in both established and primary CRC cells. The combination of enalapril and 5-FU synergistically suppressed the cell migration and invasion in both 5-FU-sensitive and -resistant CRC cells in vitro, and inhibited angiogenesis, tumor growth, and metastasis of 5-FU-resistant CRC cells in vivo without increased systemic toxicity at concentrations that were ineffective as individual agents. Furthermore, combined treatment cooperatively inhibited NF-κB/STAT3 signaling pathway and subsequently reduced the expression levels of NF-κB/STAT3-regulated proteins (c-Myc, Cyclin D1, MMP-9, MMP-2, VEGF, Bcl-2, and XIAP) in vitro and in vivo. This study provides the first evidence that enalapril greatly sensitized CRC cells to 5-FU at clinically achievable concentrations without additional toxicity and the synergistic effect may be mainly by cooperatively suppressing proliferation, angiogenesis, and NF-κB/STAT3-regulated proteins.

Fig. 1. Enalapril overcomes 5-FU resistance and potentiates the proliferation-inhibitory effects of 5-FU in CRC cells.

a HCT116 and SW620 cells were treated with increasing concentrations of enalapril (EP) or 5-FU (FU) for 72 h, and the cell viability was measured by MTT assay. b HCT116 and SW620 cells were treated with EP (100 μM), FU (10 μM), or the two agents combined (E+F) for 72 h, and the cell viability was measured by MTT. c The representative morphology of SW620 cells after 72 h treatment was shown by microscopy at ×200 magnification. d HCT116 and SW620 cells were treated as described in b for 48 h and the cell apoapsis was analyzed by flow cytometry. e The primary CRC cell lines P1, P2, P3, and P4 were treated as described above, and the cell viability was measured by MTT. f The primary CRC cells were treated as described above and, after 48 h, the cell apoapsis was measured by flow cytometry. The data are presented as means ± SD from three separate experiments (n = 8 per group). Statistical analysis performed using two-tailed t-test (**P < 0.01, *P < 0.05).

Fig. 2. Combination of enalapril and 5-FU synergistically inhibits tumor growth, cell proliferation, and angiogenesis in vivo.

a SW620 cells were injected subcutaneously into the left flanks of nude mice. Twenty-four mice were allocated randomly to the control group (100 μl, p.o., daily), EP group (0.6 mg/kg, p.o., daily), FU group (30 mg/kg, i.p., 2 times/week), and E+F. The xenografts were resected on day 24 and the tumor volumes were measured and analyzed. Representative images of mice bearing tumor xenografts for each group were shown on the right (n = 6 per group). b Blood from mice was collected at the end of the experiment and the levels of plasma creatinine (Cr) and the levels of aspartate aminotransferase (AST) were analyzed. c Immunohistochemical analysis of CD31, VEGF-α, Ki67, and Caspase-3 in each treatment xenograft tumor tissues. d The number of positive cells was quantified in ten visual fields at ×400 magnification and the means ± SD are shown in the diagrams (n = 10 per group). e Western blot analysis of the expression of CD31, VEGF-α, Ki67, and Caspase-3 in SW620 cells treated with EP, FU, or E+F. β-Actin served as a control. The data are presented as means ± SD from three separate experiments (c–e). Statistical analysis performed using two-tailed t-test (**P < 0.01, *P < 0.05).

Fig. 3. Enalapril augments the effects of 5-FU against tumor cells migration, invasion, and metastasis in CRC.

a The migration capacities of SW620 and HCT116 cells were determined using the Transwell chambers after 24 h of treatment with 100 μM EP, 10 μM FU, or E+F (n = 5 per group). Representative photographs of the migrated cells were presented. The number of migrated cells was quantified in ten visual fields at ×200 magnification and the means ± SD are shown in the diagrams. The number of migratory cells in the control is set to 100%. b The invasion capacities of SW620 and HCT116 cells were detected using the Transwell chambers coated with Matrigel after 24 h of treatment as described above (n = 5 per group). The number of migrated cells was quantified in ten visual fields at ×200 magnification. c SW620 cells were injected into the splenic vein of mice and the liver metastasis was assessed after treatment with EP, FU, or E+F (n = 5) for 7 weeks. Representative photograph of the metastatic nodules in the liver was shown and d the number of liver metastatic nodules was counted and plotted. e SW620 cells were orthotopically injected to observe the metastatic spread in vivo after treatment as described above (n = 8 per group). Mice were killed after 8 weeks. f Representative photographs of the metastasis were shown. The data are presented as means ± SD from three separate experiments (a, b). Statistical analysis (a, b, and c) performed using two-tailed t-test and Mann–Whitney test (d, e) (**P < 0.01, *P < 0.05).

Fig. 4. Enalapril enhances the effects of 5-FU in suppressing EMT signaling in CRC.

a Western blot analysis of vimentin, E-cadherin, and Snail in CRC cells treated with EP, FU, or E+F. b Immunohistochemical analysis of Vimentin, E-cadherin, and Snail in tumor tissue samples treated with EP, FU, or E+F (n = 5 per group). c The number of positive cells was quantified in ten visual fields at ×400 magnification and the means ± SD are shown in the diagrams. d SW620 cells were scratched with a pipette tip and then treated with EP, FU, or E+F (n = 6 per group). The images were taken 0 and 24 h after wound formation and the percent of wound closure was analyzed. The data are presented as means ± SD from three separate experiments. Statistical analysis performed using two-tailed t-test (**P < 0.01, *P < 0.05).

Fig. 5. Enalapril enhances the efficacy of 5-FU in suppressing the expression of NF-κB/STAT3 and NF-κB/STAT3-regulated proteins in CRC.

a SW620 and HCT116 cell lysates were analyzed for p-STAT3, STAT3, p-P65, and P65 by western blotting. b Immunohistochemical analysis of p-STAT3 and p-P65 in tumor tissue samples treated with EP, FU, or E+F (n = 5 per group). The number of positive cells was quantified in ten visual fields at ×400 magnification and the means ± SD are shown in the diagrams. c Representative pictures and immunohistochemical analysis for the expression of STAT3-regulated proteins Cyclin D1, c-Myc, MPP-9, MMP-2, Bcl-2, and XIAP in tumor tissues (n = 5 per group). d The number of positive cells was quantified in ten visual fields at ×400 magnification and the means ± SD are shown in the diagrams. e Western blot analysis for the expression of NF-κB/STAT3-regulated proteins Cyclin D1, c-Myc, MPP-9, MMP-2, Bcl-2, and XIAP in SW620 cells. The data are presented as means ± SD from three separate experiments. Statistical analysis performed using two-tailed t-test (**P < 0.01, *P < 0.05).