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New benzimidazole acridine derivative induces human colon cancer cell apoptosis in vitro via the ROS-JNK signaling pathway.

Chen K, Chu BZ, Liu F, Li B, Gao CM, Li LL, Sun QS, Shen ZF, Jiang YY - Acta Pharmacol. Sin. (2015)

Bottom Line: Knockout of JNK1, but not p38, attenuated 8m-induced apoptosis.In addition, 8m induced ROS production and mitochondrial membrane depolarization in HCT116 cells.Pretreatment with the antioxidants N-acetyl cysteine or glutathione attenuated 8m-induced apoptosis and JNK activation in HCT116 cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Tsinghua University, Beijing 100084, China.

ABSTRACT

Aim: To investigate the mechanisms underlying anticancer action of the benzimidazole acridine derivative N-{(1H-benzo[d]imidazol-2-yl)methyl}-2-butylacridin-9-amine(8m) against human colon cancer cells in vitro.

Methods: Human colon cancer cell lines SW480 and HCT116 were incubated in the presence of 8m, and then the cell proliferation and apoptosis were measured. The expression of apoptotic/signaling genes and proteins was detected using RT-PCR and Western blotting. ROS generation and mitochondrial membrane depolarization were visualized with fluorescence microscopy.

Results: 8m dose-dependently suppressed the proliferation of SW480 and HCT116 cells with IC50 values of 6.77 and 3.33 μmol/L, respectively. 8m induced apoptosis of HCT116 cells, accompanied by down-regulation of Bcl-2, up-regulation of death receptor-5 (DR5), truncation of Bid, cleavage of PARP, and activation of caspases (including caspase-8 and caspase-9 as well as the downstream caspases-3 and caspase-7). Moreover, 8m selectively activated JNK and p38 without affecting ERK in HCT116 cells. Knockout of JNK1, but not p38, attenuated 8m-induced apoptosis. In addition, 8m induced ROS production and mitochondrial membrane depolarization in HCT116 cells. Pretreatment with the antioxidants N-acetyl cysteine or glutathione attenuated 8m-induced apoptosis and JNK activation in HCT116 cells.

Conclusion: The new benzimidazole acridine derivative, 8m exerts anticancer activity against human colon cancer cells in vitro by inducing both intrinsic and extrinsic apoptosis pathways via the ROS-JNK1 pathway.

No MeSH data available.


Related in: MedlinePlus

ROS mediation of 8m-induced cell death. HCT116 cells were treated with the indicated concentrations of 8m for 24 h. (A) ROS was detected using a fluorescent microscope and the H2-DCFDA fluorescent probe. The arrows point to cells producing ROS. Scale bar, 20 μm. (B) Mitochondrial transmembrane potential dissipation was measured using the JC-1 stain. Depolarization of mitochondrial transmembrane potential is specifically indicated by a decrease in the red-to-green fluorescence intensity ratio. Scale bar, 20 μm. (C) After a 12-h incubation in standard culture conditions, HCT116 cells were pretreated with GSH (0, 1, 10, or 20 mmol/L) for 1 h and then treated with 5 μmol/L 8m for 12 h. Whole-cell extracts were prepared and analyzed by Western blotting using the specified antibodies. (D) After a 12-h incubation under standard culture conditions, HCT116 cells were pretreated with NAC (0, 1, 10, or 20 mmol/L) for 1 h and then were treated with 5 μmol/L 8m for 12 h. Whole-cell extracts were prepared and analyzed by Western blotting using the specified antibodies. (E) Cells were treated with 0, 2.5, or 5 μmol/L 8m and/or co-treated with 5 mmol/L NAC for the colony formation assay. Culture medium was changed every 3 to 4 d, and colony formation was inactivated after 14 d. (F) Schematic representation of signaling pathways through which 8m induces the apoptosis of HCT116 cells.
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fig5: ROS mediation of 8m-induced cell death. HCT116 cells were treated with the indicated concentrations of 8m for 24 h. (A) ROS was detected using a fluorescent microscope and the H2-DCFDA fluorescent probe. The arrows point to cells producing ROS. Scale bar, 20 μm. (B) Mitochondrial transmembrane potential dissipation was measured using the JC-1 stain. Depolarization of mitochondrial transmembrane potential is specifically indicated by a decrease in the red-to-green fluorescence intensity ratio. Scale bar, 20 μm. (C) After a 12-h incubation in standard culture conditions, HCT116 cells were pretreated with GSH (0, 1, 10, or 20 mmol/L) for 1 h and then treated with 5 μmol/L 8m for 12 h. Whole-cell extracts were prepared and analyzed by Western blotting using the specified antibodies. (D) After a 12-h incubation under standard culture conditions, HCT116 cells were pretreated with NAC (0, 1, 10, or 20 mmol/L) for 1 h and then were treated with 5 μmol/L 8m for 12 h. Whole-cell extracts were prepared and analyzed by Western blotting using the specified antibodies. (E) Cells were treated with 0, 2.5, or 5 μmol/L 8m and/or co-treated with 5 mmol/L NAC for the colony formation assay. Culture medium was changed every 3 to 4 d, and colony formation was inactivated after 14 d. (F) Schematic representation of signaling pathways through which 8m induces the apoptosis of HCT116 cells.

Mentions: Because oxidative stress and mitochondrial membrane depolarization can each induce cancer cell apoptosis, we examined these two indicators after 8m treatment. Using a fluorescent microscope, we determined the intracellular ROS level by measuring the oxidation of non-fluorescent H2-DCFDA to its highly fluorescent derivative 2′,7′-dichlorofluorescein (DCF). As shown in Figure 5A, 8m stimulated ROS formation in a concentration-dependent manner. JC-1 is a membrane-permeable dye whose maximal fluorescence emission changes from ∼590 nm to ∼530 nm when the mitochondrial membrane depolarizes. By measuring the shift in the fluorescence emission peak using fluorescence microscopy, we found that 8m treatment induced mitochondrial depolarization in a concentration-dependent manner (Figure 5B). Because both the DR and the mitochondrial apoptotic pathways mediated by JNK could be ROS-dependent13,39, two antioxidative ROS scavengers, GSH and NAC, were used to ascertain the relationship between 8m-induced cell death and ROS. Both scavengers reduced phosphor-JNK and the levels of other 8m-induced apoptotic proteins as well as the degree of cleavage of specific apoptotic mediators. The cleavage of caspase-7 and PARP were completely abrogated at a GSH concentration of 20 mmol/L (Figure 5C) as well as at NAC concentrations of 10 and 20 mmol/L (Figure 5D). By using the colony formation assay, we found that the numbers of colonies formed from cells co-cultured with 5 mmol/L NAC were higher and that the sizes of the colonies were also considerably larger than those in the 8m-alone culture group (Figure 5E).


New benzimidazole acridine derivative induces human colon cancer cell apoptosis in vitro via the ROS-JNK signaling pathway.

Chen K, Chu BZ, Liu F, Li B, Gao CM, Li LL, Sun QS, Shen ZF, Jiang YY - Acta Pharmacol. Sin. (2015)

ROS mediation of 8m-induced cell death. HCT116 cells were treated with the indicated concentrations of 8m for 24 h. (A) ROS was detected using a fluorescent microscope and the H2-DCFDA fluorescent probe. The arrows point to cells producing ROS. Scale bar, 20 μm. (B) Mitochondrial transmembrane potential dissipation was measured using the JC-1 stain. Depolarization of mitochondrial transmembrane potential is specifically indicated by a decrease in the red-to-green fluorescence intensity ratio. Scale bar, 20 μm. (C) After a 12-h incubation in standard culture conditions, HCT116 cells were pretreated with GSH (0, 1, 10, or 20 mmol/L) for 1 h and then treated with 5 μmol/L 8m for 12 h. Whole-cell extracts were prepared and analyzed by Western blotting using the specified antibodies. (D) After a 12-h incubation under standard culture conditions, HCT116 cells were pretreated with NAC (0, 1, 10, or 20 mmol/L) for 1 h and then were treated with 5 μmol/L 8m for 12 h. Whole-cell extracts were prepared and analyzed by Western blotting using the specified antibodies. (E) Cells were treated with 0, 2.5, or 5 μmol/L 8m and/or co-treated with 5 mmol/L NAC for the colony formation assay. Culture medium was changed every 3 to 4 d, and colony formation was inactivated after 14 d. (F) Schematic representation of signaling pathways through which 8m induces the apoptosis of HCT116 cells.
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fig5: ROS mediation of 8m-induced cell death. HCT116 cells were treated with the indicated concentrations of 8m for 24 h. (A) ROS was detected using a fluorescent microscope and the H2-DCFDA fluorescent probe. The arrows point to cells producing ROS. Scale bar, 20 μm. (B) Mitochondrial transmembrane potential dissipation was measured using the JC-1 stain. Depolarization of mitochondrial transmembrane potential is specifically indicated by a decrease in the red-to-green fluorescence intensity ratio. Scale bar, 20 μm. (C) After a 12-h incubation in standard culture conditions, HCT116 cells were pretreated with GSH (0, 1, 10, or 20 mmol/L) for 1 h and then treated with 5 μmol/L 8m for 12 h. Whole-cell extracts were prepared and analyzed by Western blotting using the specified antibodies. (D) After a 12-h incubation under standard culture conditions, HCT116 cells were pretreated with NAC (0, 1, 10, or 20 mmol/L) for 1 h and then were treated with 5 μmol/L 8m for 12 h. Whole-cell extracts were prepared and analyzed by Western blotting using the specified antibodies. (E) Cells were treated with 0, 2.5, or 5 μmol/L 8m and/or co-treated with 5 mmol/L NAC for the colony formation assay. Culture medium was changed every 3 to 4 d, and colony formation was inactivated after 14 d. (F) Schematic representation of signaling pathways through which 8m induces the apoptosis of HCT116 cells.
Mentions: Because oxidative stress and mitochondrial membrane depolarization can each induce cancer cell apoptosis, we examined these two indicators after 8m treatment. Using a fluorescent microscope, we determined the intracellular ROS level by measuring the oxidation of non-fluorescent H2-DCFDA to its highly fluorescent derivative 2′,7′-dichlorofluorescein (DCF). As shown in Figure 5A, 8m stimulated ROS formation in a concentration-dependent manner. JC-1 is a membrane-permeable dye whose maximal fluorescence emission changes from ∼590 nm to ∼530 nm when the mitochondrial membrane depolarizes. By measuring the shift in the fluorescence emission peak using fluorescence microscopy, we found that 8m treatment induced mitochondrial depolarization in a concentration-dependent manner (Figure 5B). Because both the DR and the mitochondrial apoptotic pathways mediated by JNK could be ROS-dependent13,39, two antioxidative ROS scavengers, GSH and NAC, were used to ascertain the relationship between 8m-induced cell death and ROS. Both scavengers reduced phosphor-JNK and the levels of other 8m-induced apoptotic proteins as well as the degree of cleavage of specific apoptotic mediators. The cleavage of caspase-7 and PARP were completely abrogated at a GSH concentration of 20 mmol/L (Figure 5C) as well as at NAC concentrations of 10 and 20 mmol/L (Figure 5D). By using the colony formation assay, we found that the numbers of colonies formed from cells co-cultured with 5 mmol/L NAC were higher and that the sizes of the colonies were also considerably larger than those in the 8m-alone culture group (Figure 5E).

Bottom Line: Knockout of JNK1, but not p38, attenuated 8m-induced apoptosis.In addition, 8m induced ROS production and mitochondrial membrane depolarization in HCT116 cells.Pretreatment with the antioxidants N-acetyl cysteine or glutathione attenuated 8m-induced apoptosis and JNK activation in HCT116 cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Tsinghua University, Beijing 100084, China.

ABSTRACT

Aim: To investigate the mechanisms underlying anticancer action of the benzimidazole acridine derivative N-{(1H-benzo[d]imidazol-2-yl)methyl}-2-butylacridin-9-amine(8m) against human colon cancer cells in vitro.

Methods: Human colon cancer cell lines SW480 and HCT116 were incubated in the presence of 8m, and then the cell proliferation and apoptosis were measured. The expression of apoptotic/signaling genes and proteins was detected using RT-PCR and Western blotting. ROS generation and mitochondrial membrane depolarization were visualized with fluorescence microscopy.

Results: 8m dose-dependently suppressed the proliferation of SW480 and HCT116 cells with IC50 values of 6.77 and 3.33 μmol/L, respectively. 8m induced apoptosis of HCT116 cells, accompanied by down-regulation of Bcl-2, up-regulation of death receptor-5 (DR5), truncation of Bid, cleavage of PARP, and activation of caspases (including caspase-8 and caspase-9 as well as the downstream caspases-3 and caspase-7). Moreover, 8m selectively activated JNK and p38 without affecting ERK in HCT116 cells. Knockout of JNK1, but not p38, attenuated 8m-induced apoptosis. In addition, 8m induced ROS production and mitochondrial membrane depolarization in HCT116 cells. Pretreatment with the antioxidants N-acetyl cysteine or glutathione attenuated 8m-induced apoptosis and JNK activation in HCT116 cells.

Conclusion: The new benzimidazole acridine derivative, 8m exerts anticancer activity against human colon cancer cells in vitro by inducing both intrinsic and extrinsic apoptosis pathways via the ROS-JNK1 pathway.

No MeSH data available.


Related in: MedlinePlus