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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

The effects of 8m on the viability of human colon cancer cells. (A) Chemical structure of 8m (N-((1H-benzo[d]imidazol-2-yl)methyl)-2-butylacridin-9-amine, C25N4H24). HCT116 cells (B) or SW480 cells (C) were incubated with the indicated concentrations of 8m for 48 h in 96-well plates, and cell viability was examined by the MTT assay. (D) HCT116 cells were treated with the indicated concentrations of 8m for 24 h, and apoptosis was detected by flow cytometry. (E) HCT116 cells were stained with DAPI and assessed by fluorescence microscopy. Apoptotic cells exhibited apoptotic bodies and chromatin condensation. Scale bar, 20 μm.
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fig1: The effects of 8m on the viability of human colon cancer cells. (A) Chemical structure of 8m (N-((1H-benzo[d]imidazol-2-yl)methyl)-2-butylacridin-9-amine, C25N4H24). HCT116 cells (B) or SW480 cells (C) were incubated with the indicated concentrations of 8m for 48 h in 96-well plates, and cell viability was examined by the MTT assay. (D) HCT116 cells were treated with the indicated concentrations of 8m for 24 h, and apoptosis was detected by flow cytometry. (E) HCT116 cells were stained with DAPI and assessed by fluorescence microscopy. Apoptotic cells exhibited apoptotic bodies and chromatin condensation. Scale bar, 20 μm.

Mentions: 8m was obtained from Associate Professor Chun-mei GAO of the Graduate School at Shenzhen, Tsinghua University (Shenzhen, China). Its molecular structure is shown in Figure 1A. Modified RPMI-1640 medium was purchased from HyClone (Logan, UT, USA). Fetal bovine serum (FBS) and Opti-MEM I reduced serum media were purchased from Gibco (Grand Island, NY, USA). Lipofectamine 2000, TRIzol Reagent and SlowFade® gold antifade mountant with DAPI were purchased from Invitrogen (Carlsbad, CA, USA). Annexin V-FITC (fluorescein isothiocyanate)/PI (propidium iodide) and 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-benzimidazol-carbocyanine iodide (JC-1) were purchased from eBioscience (San Diego, CA, USA). PrimeScript™ RT Master Mix (Perfect Real Time) kit and SYBR® Premix Ex Taq™ (Tli RNaseH Plus) kit were purchased from TaKaRa (Dalian, China). Short-interfering RNA (siRNA) was synthesized by GenePharma (Shanghai, China). Phosphor-SAPK/JNK (Thr183/Tyr185), SAPK/JNK, phosphor-p38, p38, phosphor-ERK, ERK, Bcl-2, Bid, cleaved caspase-9, cleaved caspase-8, cleaved caspase-7, cleaved caspase-3, and cleaved PARP were purchased from Cell Signaling Technology (Danvers, MA, USA). DR5 was obtained from Abcam (Cambridge, UK). β-Actin antibody, HRP-labeled Goat Anti-Rabbit IgG (H±L), HRP-labeled Goat Anti-Mouse IgG (H±L) and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) were purchased from Beyotime (Haimen, China). PVDF membrane, protease and phosphatase inhibitor cocktails were purchased from Roche (Mannheim, Germany). N-acetylcysteine (NAC), L-glutathione reduced (GSH) and 2′,7′-Dichloro-dihydrofluorescein diacetate (H2-DCFDA) were purchased from Sigma (St Louis, MO, USA).


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)

The effects of 8m on the viability of human colon cancer cells. (A) Chemical structure of 8m (N-((1H-benzo[d]imidazol-2-yl)methyl)-2-butylacridin-9-amine, C25N4H24). HCT116 cells (B) or SW480 cells (C) were incubated with the indicated concentrations of 8m for 48 h in 96-well plates, and cell viability was examined by the MTT assay. (D) HCT116 cells were treated with the indicated concentrations of 8m for 24 h, and apoptosis was detected by flow cytometry. (E) HCT116 cells were stained with DAPI and assessed by fluorescence microscopy. Apoptotic cells exhibited apoptotic bodies and chromatin condensation. Scale bar, 20 μm.
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getmorefigures.php?uid=PMC4561968&req=5

fig1: The effects of 8m on the viability of human colon cancer cells. (A) Chemical structure of 8m (N-((1H-benzo[d]imidazol-2-yl)methyl)-2-butylacridin-9-amine, C25N4H24). HCT116 cells (B) or SW480 cells (C) were incubated with the indicated concentrations of 8m for 48 h in 96-well plates, and cell viability was examined by the MTT assay. (D) HCT116 cells were treated with the indicated concentrations of 8m for 24 h, and apoptosis was detected by flow cytometry. (E) HCT116 cells were stained with DAPI and assessed by fluorescence microscopy. Apoptotic cells exhibited apoptotic bodies and chromatin condensation. Scale bar, 20 μm.
Mentions: 8m was obtained from Associate Professor Chun-mei GAO of the Graduate School at Shenzhen, Tsinghua University (Shenzhen, China). Its molecular structure is shown in Figure 1A. Modified RPMI-1640 medium was purchased from HyClone (Logan, UT, USA). Fetal bovine serum (FBS) and Opti-MEM I reduced serum media were purchased from Gibco (Grand Island, NY, USA). Lipofectamine 2000, TRIzol Reagent and SlowFade® gold antifade mountant with DAPI were purchased from Invitrogen (Carlsbad, CA, USA). Annexin V-FITC (fluorescein isothiocyanate)/PI (propidium iodide) and 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-benzimidazol-carbocyanine iodide (JC-1) were purchased from eBioscience (San Diego, CA, USA). PrimeScript™ RT Master Mix (Perfect Real Time) kit and SYBR® Premix Ex Taq™ (Tli RNaseH Plus) kit were purchased from TaKaRa (Dalian, China). Short-interfering RNA (siRNA) was synthesized by GenePharma (Shanghai, China). Phosphor-SAPK/JNK (Thr183/Tyr185), SAPK/JNK, phosphor-p38, p38, phosphor-ERK, ERK, Bcl-2, Bid, cleaved caspase-9, cleaved caspase-8, cleaved caspase-7, cleaved caspase-3, and cleaved PARP were purchased from Cell Signaling Technology (Danvers, MA, USA). DR5 was obtained from Abcam (Cambridge, UK). β-Actin antibody, HRP-labeled Goat Anti-Rabbit IgG (H±L), HRP-labeled Goat Anti-Mouse IgG (H±L) and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) were purchased from Beyotime (Haimen, China). PVDF membrane, protease and phosphatase inhibitor cocktails were purchased from Roche (Mannheim, Germany). N-acetylcysteine (NAC), L-glutathione reduced (GSH) and 2′,7′-Dichloro-dihydrofluorescein diacetate (H2-DCFDA) were purchased from Sigma (St Louis, MO, USA).

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