Inhibition of Colon Cancer Cell Growth by Imidazole Through Activation of Apoptotic Pathway

Abstract

BACKGROUND This study aimed to investigate the inhibitory effect of imidazole on colon cancer cell proliferation and understand the mechanism involved. MATERIAL AND METHODS MTT assay and flow cytometry using Hoechst 33258 staining were used to assess cell proliferation and morphology, respectively. Changes in protein expression was determined by western blotting assay. The reactive oxygen species (ROS) production in DLD-1 cells was analyzed by flow cytometry using DCFH-DA (2',7'-dichlorofluorescein diacetate) stain. RESULTS DLD-1 and HCT-116 cell viability was suppressed by imidazole in a concentration-based manner. At the concentration of 36 μM, imidazole reduced DLD-1 and HCT-116 cell viability to 22% and 28%, respectively. Treatment with imidazole led to chromatin material condensation, detaching of cells, and apoptotic nuclei. In imidazole treated cells, the G1/G0 phase cell proportion increased, whereas in the S and G2/M phases the cell proportion decreased. Imidazole treatment of DLD-1 cells markedly promoted activation of caspase-3, caspase-8, and caspase-9. The level of cleaved PARP1 was also upregulated in DLD-1 cells with imidazole treatment. Treatment of DLD-1 cells with imidazole suppressed Bcl-2 and promoted Bax, p53, and cytc expression. The Akt activation was suppressed by imidazole treatment in DLD-1 cells. ROS generation in DLD-1 cells was enhanced markedly by treatment with imidazole. CONCLUSIONS The present study demonstrated that imidazole inhibited colon cancer cell viability through activation of apoptosis and cell cycle arrest by increasing the generation of ROS, caspase activation, and apoptotic protein expression. Therefore, imidazole can act as a therapeutic molecule for the treatment of colon cancer.

Figure 1

Imidazole suppressed DLD-1 and HCT-116 cell viability. Changes in cell viability by different concentrations of imidazole at 48 hours were measured by MTT assay. The values presented average of triplicate measurements. * P<0.05, ** P<0.02, and *** P<0.01 versus untreated cells.

Figure 2

Effect of imidazole on DLD-1 cell morphological features. Flow cytometry was used to assess the morphological features of Hoechst 33258 stained DLD-1 cells at 48 hours of treatment with 12, 24, and 36 μM concentrations of imidazole.

Figure 3

Imidazole arrested the DLD-1 cell cycle in the G1/G0 phase. At 48 hours of treatment with 12, 24, and 36 μM concentrations of imidazole, cell cycle was analyzed by DNA content of cells using flow cytometry. Magnification 200×.

Figure 4

Imidazole caused the onset of apoptosis in DLD-1 cells. In DLD-1 cells, treatment with the indicated concentrations of imidazole was followed by Annexin-V/PI staining.

Figure 5

Imidazole upregulated caspase activation in DLD-1 cells. Western blotting of DLD-1 cells at 48 hours of treatment with 12, 24, and 36 μM concentrations of imidazole is shown.

Figure 6

Effect of imidazole on pro-apoptotic proteins in DLD-1 cells. Western blotting was employed to analyze changes in Bcl-2, Bax, p53, cyt-c, and Akt levels in DLD-1 cells by imidazole treatment.

Figure 7

Effect of imidazole on reactive oxygen species (ROS) production in DLD-1 cells. Production of ROS in DLD-1 cells following treatment with different concentrations of imidazole was measured by flow cytometry of DCFH-DA stained cells. Magnification 200×.