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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

8m induction of apoptotic gene and protein expression. (A) HCT116 cells were treated with 8m at 5 μmol/L for 4 h before real-time PCR was performed. Gene expression of three indicated genes as shown relative to control cells (bP<0.05 vs control). (B) HCT116 cells were treated with the specified concentration of 8m for 24 h, and whole cell extracts were analyzed by Western blot using the indicated antibodies. β-Actin protein was used as an internal control. (C) HCT116 cells were treated with 5 μmol/L 8m for 4, 8, 12, or 24 h. Whole cell extracts were analyzed by Western blot using the indicated antibodies.
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fig2: 8m induction of apoptotic gene and protein expression. (A) HCT116 cells were treated with 8m at 5 μmol/L for 4 h before real-time PCR was performed. Gene expression of three indicated genes as shown relative to control cells (bP<0.05 vs control). (B) HCT116 cells were treated with the specified concentration of 8m for 24 h, and whole cell extracts were analyzed by Western blot using the indicated antibodies. β-Actin protein was used as an internal control. (C) HCT116 cells were treated with 5 μmol/L 8m for 4, 8, 12, or 24 h. Whole cell extracts were analyzed by Western blot using the indicated antibodies.

Mentions: To ascertain the underlying mechanisms responsible for enhancement of apoptotic cell death, we examined the effects of 8m on the expression of apoptosis-related genes and proteins. To determine whether 8m acts through the extrinsic apoptotic pathway, the expression levels of two members of the tumor necrosis factor (TNF) receptor superfamily, DR4 and DR5, which are upstream mediators of the extrinsic apoptosis pathway6, were examined after 8m treatment. Real-time PCR analysis revealed increased DR4 and DR5 mRNA levels, with DR5 increasing more significantly than DR4, suggesting that DR5 may play a more important role than DR4 in mediating 8m-induced apoptosis (P<0.05, Figure 2A). We then examined the expression of DR5 at the protein level and found that DR5 protein expression was induced by 8m in a concentration- and time-dependent manner. The cleavage and truncation of Bid and caspase-8, two downstream effectors, were induced by 8m, as indicated by western blotting (Figure 2B, 2C). These results suggested that 8m induced apoptosis through the extrinsic apoptotic pathway.


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)

8m induction of apoptotic gene and protein expression. (A) HCT116 cells were treated with 8m at 5 μmol/L for 4 h before real-time PCR was performed. Gene expression of three indicated genes as shown relative to control cells (bP<0.05 vs control). (B) HCT116 cells were treated with the specified concentration of 8m for 24 h, and whole cell extracts were analyzed by Western blot using the indicated antibodies. β-Actin protein was used as an internal control. (C) HCT116 cells were treated with 5 μmol/L 8m for 4, 8, 12, or 24 h. Whole cell extracts were analyzed by Western blot using the indicated antibodies.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4561968&req=5

fig2: 8m induction of apoptotic gene and protein expression. (A) HCT116 cells were treated with 8m at 5 μmol/L for 4 h before real-time PCR was performed. Gene expression of three indicated genes as shown relative to control cells (bP<0.05 vs control). (B) HCT116 cells were treated with the specified concentration of 8m for 24 h, and whole cell extracts were analyzed by Western blot using the indicated antibodies. β-Actin protein was used as an internal control. (C) HCT116 cells were treated with 5 μmol/L 8m for 4, 8, 12, or 24 h. Whole cell extracts were analyzed by Western blot using the indicated antibodies.
Mentions: To ascertain the underlying mechanisms responsible for enhancement of apoptotic cell death, we examined the effects of 8m on the expression of apoptosis-related genes and proteins. To determine whether 8m acts through the extrinsic apoptotic pathway, the expression levels of two members of the tumor necrosis factor (TNF) receptor superfamily, DR4 and DR5, which are upstream mediators of the extrinsic apoptosis pathway6, were examined after 8m treatment. Real-time PCR analysis revealed increased DR4 and DR5 mRNA levels, with DR5 increasing more significantly than DR4, suggesting that DR5 may play a more important role than DR4 in mediating 8m-induced apoptosis (P<0.05, Figure 2A). We then examined the expression of DR5 at the protein level and found that DR5 protein expression was induced by 8m in a concentration- and time-dependent manner. The cleavage and truncation of Bid and caspase-8, two downstream effectors, were induced by 8m, as indicated by western blotting (Figure 2B, 2C). These results suggested that 8m induced apoptosis through the extrinsic apoptotic pathway.

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