skip to main content
OSTI.GOV title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: Inhibition of autophagy enhances DNA damage-induced apoptosis by disrupting CHK1-dependent S phase arrest

Abstract

DNA damage has been shown to induce autophagy, but the role of autophagy in the DNA damage response and cell fate is not fully understood. BO-1012, a bifunctional alkylating derivative of 3a-aza-cyclopenta[a]indene, is a potent DNA interstrand cross-linking agent with anticancer activity. In this study, BO-1012 was found to reduce DNA synthesis, inhibit S phase progression, and induce phosphorylation of histone H2AX on serine 139 (γH2AX) exclusively in S phase cells. Both CHK1 and CHK2 were phosphorylated in response to BO-1012 treatment, but only depletion of CHK1, but not CHK2, impaired BO-1012-induced S phase arrest and facilitated the entry of γH2AX-positive cells into G2 phase. CHK1 depletion also significantly enhanced BO-1012-induced cell death and apoptosis. These results indicate that BO-1012-induced S phase arrest is a CHK1-dependent pro-survival response. BO-1012 also resulted in marked induction of acidic vesicular organelle (AVO) formation and microtubule-associated protein 1 light chain 3 (LC3) processing and redistribution, features characteristic of autophagy. Depletion of ATG7 or co-treatment of cells with BO-1012 and either 3-methyladenine or bafilomycin A1, two inhibitors of autophagy, not only reduced CHK1 phosphorylation and disrupted S phase arrest, but also increased cleavage of caspase-9 and PARP, and cell death. These results suggest that cellsmore » initiate S phase arrest and autophagy as pro-survival responses to BO-1012-induced DNA damage, and that suppression of autophagy enhances BO-1012-induced apoptosis via disruption of CHK1-dependent S phase arrest. - Highlights: • Autophagy inhibitors enhanced the cytotoxicity of a DNA alkylating agent, BO-1012. • BO-1012-induced S phase arrest was a CHK1-dependent pro-survival response. • Autophagy inhibition enhanced BO-1012 cytotoxicity via disrupting the S phase arrest.« less

Authors:
; ; ;  [1]; ;  [2];  [1]
  1. Institute of Cellular and Organismic Biology, Academia Sinica, Taipei 115, Taiwan, ROC (China)
  2. Institute of Biomedical Sciences, Academia Sinica, Taipei 115, Taiwan, ROC (China)
Publication Date:
OSTI Identifier:
22439796
Resource Type:
Journal Article
Resource Relation:
Journal Name: Toxicology and Applied Pharmacology; Journal Volume: 278; Journal Issue: 3; Other Information: Copyright (c) 2014 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved.; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; ALKYLATING AGENTS; APOPTOSIS; CLEAVAGE; CROSS-LINKING; DNA; DNA DAMAGES; INDENE; INHIBITION; MICROTUBULES; PHOSPHORYLATION; SERINE; SYNTHESIS; TOXICITY

Citation Formats

Liou, Jong-Shian, Wu, Yi-Chen, Yen, Wen-Yen, Tang, Yu-Shuan, Kakadiya, Rajesh B., Su, Tsann-Long, and Yih, Ling-Huei, E-mail: lhyih@gate.sinica.edu.tw. Inhibition of autophagy enhances DNA damage-induced apoptosis by disrupting CHK1-dependent S phase arrest. United States: N. p., 2014. Web. doi:10.1016/J.TAAP.2014.04.028.
Liou, Jong-Shian, Wu, Yi-Chen, Yen, Wen-Yen, Tang, Yu-Shuan, Kakadiya, Rajesh B., Su, Tsann-Long, & Yih, Ling-Huei, E-mail: lhyih@gate.sinica.edu.tw. Inhibition of autophagy enhances DNA damage-induced apoptosis by disrupting CHK1-dependent S phase arrest. United States. doi:10.1016/J.TAAP.2014.04.028.
Liou, Jong-Shian, Wu, Yi-Chen, Yen, Wen-Yen, Tang, Yu-Shuan, Kakadiya, Rajesh B., Su, Tsann-Long, and Yih, Ling-Huei, E-mail: lhyih@gate.sinica.edu.tw. Fri . "Inhibition of autophagy enhances DNA damage-induced apoptosis by disrupting CHK1-dependent S phase arrest". United States. doi:10.1016/J.TAAP.2014.04.028.
@article{osti_22439796,
title = {Inhibition of autophagy enhances DNA damage-induced apoptosis by disrupting CHK1-dependent S phase arrest},
author = {Liou, Jong-Shian and Wu, Yi-Chen and Yen, Wen-Yen and Tang, Yu-Shuan and Kakadiya, Rajesh B. and Su, Tsann-Long and Yih, Ling-Huei, E-mail: lhyih@gate.sinica.edu.tw},
abstractNote = {DNA damage has been shown to induce autophagy, but the role of autophagy in the DNA damage response and cell fate is not fully understood. BO-1012, a bifunctional alkylating derivative of 3a-aza-cyclopenta[a]indene, is a potent DNA interstrand cross-linking agent with anticancer activity. In this study, BO-1012 was found to reduce DNA synthesis, inhibit S phase progression, and induce phosphorylation of histone H2AX on serine 139 (γH2AX) exclusively in S phase cells. Both CHK1 and CHK2 were phosphorylated in response to BO-1012 treatment, but only depletion of CHK1, but not CHK2, impaired BO-1012-induced S phase arrest and facilitated the entry of γH2AX-positive cells into G2 phase. CHK1 depletion also significantly enhanced BO-1012-induced cell death and apoptosis. These results indicate that BO-1012-induced S phase arrest is a CHK1-dependent pro-survival response. BO-1012 also resulted in marked induction of acidic vesicular organelle (AVO) formation and microtubule-associated protein 1 light chain 3 (LC3) processing and redistribution, features characteristic of autophagy. Depletion of ATG7 or co-treatment of cells with BO-1012 and either 3-methyladenine or bafilomycin A1, two inhibitors of autophagy, not only reduced CHK1 phosphorylation and disrupted S phase arrest, but also increased cleavage of caspase-9 and PARP, and cell death. These results suggest that cells initiate S phase arrest and autophagy as pro-survival responses to BO-1012-induced DNA damage, and that suppression of autophagy enhances BO-1012-induced apoptosis via disruption of CHK1-dependent S phase arrest. - Highlights: • Autophagy inhibitors enhanced the cytotoxicity of a DNA alkylating agent, BO-1012. • BO-1012-induced S phase arrest was a CHK1-dependent pro-survival response. • Autophagy inhibition enhanced BO-1012 cytotoxicity via disrupting the S phase arrest.},
doi = {10.1016/J.TAAP.2014.04.028},
journal = {Toxicology and Applied Pharmacology},
number = 3,
volume = 278,
place = {United States},
year = {Fri Aug 01 00:00:00 EDT 2014},
month = {Fri Aug 01 00:00:00 EDT 2014}
}
  • No abstract prepared.
  • Jaridonin, a novel diterpenoid from Isodon rubescens, has been shown previously to inhibit proliferation of esophageal squamous cancer cells (ESCC) through G2/M phase cell cycle arrest. However, the involved mechanism is not fully understood. In this study, we found that the cell cycle arrest by Jaridonin was associated with the increased expression of phosphorylation of ATM at Ser1981 and Cdc2 at Tyr15. Jaridonin also resulted in enhanced phosphorylation of Cdc25C via the activation of checkpoint kinases Chk1 and Chk2, as well as in increased phospho-H2A.X (Ser139), which is known to be phosphorylated by ATM in response to DNA damage. Furthermore,more » Jaridonin-mediated alterations in cell cycle arrest were significantly attenuated in the presence of NAC, implicating the involvement of ROS in Jaridonin's effects. On the other hand, addition of ATM inhibitors reversed Jaridonin-related activation of ATM and Chk1/2 as well as phosphorylation of Cdc25C, Cdc2 and H2A.X and G2/M phase arrest. In conclusion, these findings identified that Jaridonin-induced cell cycle arrest in human esophageal cancer cells is associated with ROS-mediated activation of ATM–Chk1/2–Cdc25C pathway. - Highlights: • Jaridonin induced G2/M phase arrest through induction of redox imbalance. • Jaridonin increased the level of ROS through depleting glutathione in cell. • ATM–Chk1/2–Cdc25C were involved in Jaridonin-induced cell cycle arrest. • Jaridonin selectively inhibited cancer cell viability and cell cycle progression.« less
  • Methamphetamine (METH) is an amphetamine-like psychostimulant that is commonly abused. Previous studies have shown that METH can induce damages to the nervous system and recent studies suggest that METH can also cause adverse and potentially lethal effects on the cardiovascular system. Recently, we demonstrated that DNA damage-inducible transcript 4 (DDIT4) regulates METH-induced neurotoxicity. However, the role of DDIT4 in METH-induced cardiotoxicity remains unknown. We hypothesized that DDIT4 may mediate METH-induced autophagy and apoptosis in cardiomyocytes. To test the hypothesis, we examined DDIT4 protein expression in cardiomyocytes and in heart tissues of rats exposed to METH with Western blotting. We alsomore » determined the effects on METH-induced autophagy and apoptosis after silencing DDIT4 expression with synthetic siRNA with or without pretreatment of a mTOR inhibitor rapamycin in cardiomyocytes using Western blot analysis, fluorescence microscopy and TUNEL staining. Our results showed that METH exposure increased DDIT4 expression and decreased phosphorylation of mTOR that was accompanied with increased autophagy and apoptosis both in vitro and in vivo. These effects were normalized after silencing DDIT4. On the other hand, rapamycin promoted METH-induced autophagy and apoptosis in DDIT4 knockdown cardiomyocytes. These results suggest that DDIT4 mediates METH-induced autophagy and apoptosis through mTOR signaling pathway in cardiomyocytes. - Highlights: • METH exposure increases DDIT4 expression in cardiomyocytes. • DDIT4 mediates METH-induced autophagy and apoptosis in cardiomyocytes. • DDIT4 silencing protects cardiomyocytes against METH-caused autophagy and apoptosis.« less
  • Natural flavonoids have diverse pharmacological activities, including anti-oxidative, anti-inflammatory, and anti-cancer activities. In this study, we investigated the molecular mechanism underlying the action of 5-methoxyflavanone (5-MF) which has a strong bioavailability and metabolic stability. Our results show that 5-MF inhibited the growth and clonogenicity of HCT116 human colon cancer cells, and that it activated DNA damage responses, as revealed by the accumulation of p53 and the phosphorylation of DNA damage-sensitive proteins, including ataxia-telangiectasia mutated (ATM) at Ser1981, checkpoint kinase 2 (Chk2) at Thr68, and histone H2AX at Ser139. 5-MF-induced DNA damage was confirmed in a comet tail assay. We alsomore » found that 5-MF increased the cleavage of caspase-2 and -7, leading to the induction of apoptosis. Pretreatment with the ATM inhibitor KU55933 enhanced 5-MF-induced {gamma}-H2AX formation and caspase-7 cleavage. HCT116 cells lacking p53 (p53{sup -/-}) or p21 (p21{sup -/-}) exhibited increased sensitivity to 5-MF compared to wild-type cells. 5-MF further induced autophagy via an ERK signaling pathway. Blockage of autophagy with the MEK inhibitor U0126 potentiated 5-MF-induced {gamma}-H2AX formation and caspase-2 activation. These results suggest that a caspase-2 cascade mediates 5-MF-induced anti-tumor activity, while an ATM/Chk2/p53/p21 checkpoint pathway and ERK-mediated autophagy act as a survival program to block caspase-2-mediated apoptosis induced by 5-MF. - Graphical abstract: Display Omitted Highlights: > 5-MF inhibits the proliferation of HCT116 colon cancer cells. > 5-MF inhibits cell cycle progression and induces apoptosis. > Inhibition of autophagy triggers 5-MF-induced apoptosis. > Inhibition of ERK signaling blocks 5-MF-induced autophagy but activates apoptosis. > Treatment with 5-MF in combination with an ERK inhibitor may be a potential therapeutic strategy in human colon cancer.« less
  • Inhibitors of the G{sub 2} DNA damage checkpoint can selectively sensitize cancer cells with mutated p53 to killing by DNA-damaging agents. Isogranulatimide is a G{sub 2} checkpoint inhibitor containing a unique indole/maleimide/imidazole skeleton identified in a phenotypic cell-based screen; however, the mechanism of action of isogranulatimide is unknown. Using natural and synthetic isogranulatimide analogues, we show that the imide nitrogen and a basic nitrogen at position 14 or 15 in the imidazole ring are important for checkpoint inhibition. Isogranulatimide shows structural resemblance to the aglycon of UCN-01, a potent bisindolemaleimide inhibitor of protein kinase C{beta} (IC{sub 50}, 0.001 micromol/L) andmore » of the checkpoint kinase Chk1 (IC{sub 50}, 0.007 micromol/L). In vitro kinase assays show that isogranulatimide inhibits Chk1 (IC{sub 50}, 0.1 {micro}mol/L) but not protein kinase C{beta}. Of 13 additional protein kinases tested, isogranulatimide significantly inhibits only glycogen synthase kinase-3{beta} (IC{sub 50}, 0.5 {micro}mol/L). We determined the crystal structure of the Chk1 catalytic domain complexed with isogranulatimide. Like UCN-01, isogranulatimide binds in the ATP-binding pocket of Chk1 and hydrogen bonds with the backbone carbonyl oxygen of Glu{sup 85} and the amide nitrogen of Cys{sup 87}. Unlike UCN-01, the basic N15 of isogranulatimide interacts with Glu{sub 17}, causing a conformation change in the kinase glycine-rich loop that may contribute importantly to inhibition. The mechanism by which isogranulatimide inhibits Chk1 and its favorable kinase selectivity profile make it a promising candidate for modulating checkpoint responses in tumors for therapeutic benefit.« less