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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

Effects of JNK1 on 8m-induced apoptosis. (A) Effects of JNK1 and JNK2 knockdown for 24 and 48 h. (B) After transfection with control, JNK1, or JNK2 oligonucleotides for 24 h, HCT116 cells were treated with 5 μmol/L 8m for 12 h. Whole cell extracts were analyzed by Western blotting using the indicated antibodies. (C) Effects of p38 knockdown at 24 and 48 h. (D) HCT116 cells were treated with or without 5 μmol/L 8m at 12 h after transfection with control or p38 oligonucleotides. Whole cell extracts were analyzed by Western blotting with the indicated antibodies at 24 h. (E) After transfection with control or JNK1 siRNA for 24 h, cells were treated with 5 μmol/L 8m for another 24 h, and apoptosis was detected by flow cytometry.
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fig4: Effects of JNK1 on 8m-induced apoptosis. (A) Effects of JNK1 and JNK2 knockdown for 24 and 48 h. (B) After transfection with control, JNK1, or JNK2 oligonucleotides for 24 h, HCT116 cells were treated with 5 μmol/L 8m for 12 h. Whole cell extracts were analyzed by Western blotting using the indicated antibodies. (C) Effects of p38 knockdown at 24 and 48 h. (D) HCT116 cells were treated with or without 5 μmol/L 8m at 12 h after transfection with control or p38 oligonucleotides. Whole cell extracts were analyzed by Western blotting with the indicated antibodies at 24 h. (E) After transfection with control or JNK1 siRNA for 24 h, cells were treated with 5 μmol/L 8m for another 24 h, and apoptosis was detected by flow cytometry.

Mentions: To further investigate the role of MAPK activation in 8m-induced apoptosis, we treated HCT116 cells with 8m after inhibiting the expression of JNK1, JNK2, and p38 via the addition of the indicated oligonucleotides. The effects of JNK1, JNK2, and p38 siRNA were still observable 48 h post-treatment, outlasting the duration of 8m treatment in the subsequent detection assays (Figure 4A, 4C). Compared with control siRNA, Western blotting analyses showed that JNK1 siRNA completely abolished not only the 8m-induced increase in phosphorylated-JNK (phosphor-JNK) but also partially blocked the 8m-induced up-regulation of DR5, down-regulation of Bcl-2 as well as cleavage of Bid, caspase-8, caspase-7, caspase-3, and PARP. However, other than the decrease in the intensity of the Bcl-2 band, transfection with JNK2 siRNA appeared to elicit opposite results (Figure 4B). We then examined the effects of p38 siRNA, which, unexpectedly, reduced the level of phosphorylated p38; however, p38 siRNA did not affect the levels of other apoptosis-related proteins (Figure 4D). These results suggested that JNK1 played an important role in mediating both the extrinsic and intrinsic apoptotic pathways induced by 8m. This result was further supported by our flow cytometry data, which demonstrated that JNK1 siRNA, compared with the control siRNA, rescued 8m-induced apoptosis (Figure 4E).


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)

Effects of JNK1 on 8m-induced apoptosis. (A) Effects of JNK1 and JNK2 knockdown for 24 and 48 h. (B) After transfection with control, JNK1, or JNK2 oligonucleotides for 24 h, HCT116 cells were treated with 5 μmol/L 8m for 12 h. Whole cell extracts were analyzed by Western blotting using the indicated antibodies. (C) Effects of p38 knockdown at 24 and 48 h. (D) HCT116 cells were treated with or without 5 μmol/L 8m at 12 h after transfection with control or p38 oligonucleotides. Whole cell extracts were analyzed by Western blotting with the indicated antibodies at 24 h. (E) After transfection with control or JNK1 siRNA for 24 h, cells were treated with 5 μmol/L 8m for another 24 h, and apoptosis was detected by flow cytometry.
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getmorefigures.php?uid=PMC4561968&req=5

fig4: Effects of JNK1 on 8m-induced apoptosis. (A) Effects of JNK1 and JNK2 knockdown for 24 and 48 h. (B) After transfection with control, JNK1, or JNK2 oligonucleotides for 24 h, HCT116 cells were treated with 5 μmol/L 8m for 12 h. Whole cell extracts were analyzed by Western blotting using the indicated antibodies. (C) Effects of p38 knockdown at 24 and 48 h. (D) HCT116 cells were treated with or without 5 μmol/L 8m at 12 h after transfection with control or p38 oligonucleotides. Whole cell extracts were analyzed by Western blotting with the indicated antibodies at 24 h. (E) After transfection with control or JNK1 siRNA for 24 h, cells were treated with 5 μmol/L 8m for another 24 h, and apoptosis was detected by flow cytometry.
Mentions: To further investigate the role of MAPK activation in 8m-induced apoptosis, we treated HCT116 cells with 8m after inhibiting the expression of JNK1, JNK2, and p38 via the addition of the indicated oligonucleotides. The effects of JNK1, JNK2, and p38 siRNA were still observable 48 h post-treatment, outlasting the duration of 8m treatment in the subsequent detection assays (Figure 4A, 4C). Compared with control siRNA, Western blotting analyses showed that JNK1 siRNA completely abolished not only the 8m-induced increase in phosphorylated-JNK (phosphor-JNK) but also partially blocked the 8m-induced up-regulation of DR5, down-regulation of Bcl-2 as well as cleavage of Bid, caspase-8, caspase-7, caspase-3, and PARP. However, other than the decrease in the intensity of the Bcl-2 band, transfection with JNK2 siRNA appeared to elicit opposite results (Figure 4B). We then examined the effects of p38 siRNA, which, unexpectedly, reduced the level of phosphorylated p38; however, p38 siRNA did not affect the levels of other apoptosis-related proteins (Figure 4D). These results suggested that JNK1 played an important role in mediating both the extrinsic and intrinsic apoptotic pathways induced by 8m. This result was further supported by our flow cytometry data, which demonstrated that JNK1 siRNA, compared with the control siRNA, rescued 8m-induced apoptosis (Figure 4E).

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