Lipoic acid induces p53-independent cell death in colorectal cancer cells and potentiates the cytotoxicity of 5-fluorouracil

Dörsam, Bastian; Göder, Anja; Seiwert, Nina; Kaina, Bernd; Fahrer, Jörg

doi:10.1007/s00204-014-1434-0

Cited by 39 publications

(34 citation statements)

References 59 publications

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“…To this end, 1 × 10 3 cells per milliliter were mixed with 5% low melting point agarose, transferred onto agarose-coated slides, and allowed to settle. The alkaline Comet assay was then performed as reported (49,50). In each experiment, at least 50 cells were scored per sample using the software Comet Assay IV (Perceptive Instruments).…”

Section: Methodsmentioning

confidence: 99%

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PARP-1 protects against colorectal tumor induction, but promotes inflammation-driven colorectal tumor progression

Dörsam

Seiwert

Foersch

et al. 2018

Proc. Natl. Acad. Sci. U.S.A.

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Colorectal cancer (CRC) is one of the most common tumor entities, which is causally linked to DNA repair defects and inflammatory bowel disease (IBD). Here, we studied the role of the DNA repair protein poly(ADP-ribose) polymerase-1 (PARP-1) in CRC. Tissue microarray analysis revealed PARP-1 overexpression in human CRC, correlating with disease progression. To elucidate its function in CRC, PARP-1 deficient (PARP-1) and wild-type animals (WT) were subjected to azoxymethane (AOM)/ dextran sodium sulfate (DSS)-induced colorectal carcinogenesis. Miniendoscopy showed significantly more tumors in WT than in PARP-1 mice. Although the lack of PARP-1 moderately increased DNA damage, both genotypes exhibited comparable levels of AOM-induced autophagy and cell death. Interestingly, miniendoscopy revealed a higher AOM/DSS-triggered intestinal inflammation in WT animals, which was associated with increased levels of innate immune cells and proinflammatory cytokines. Tumors in WT animals were more aggressive, showing higher levels of STAT3 activation and cyclin D1 up-regulation. PARP-1 animals were then crossed with -methylguanine-DNA methyltransferase (MGMT)-deficient animals hypersensitive to AOM. Intriguingly, PARP-1/MGMT double knockout (DKO) mice developed more, but much smaller tumors than MGMT animals. In contrast to MGMT-deficient mice, DKO animals showed strongly reduced AOM-dependent colonic cell death despite similar -methylguanine levels. Studies with PARP-1 cells provided evidence for increased alkylation-induced DNA strand break formation when MGMT was inhibited, suggesting a role of PARP-1 in the response to -methylguanine adducts. Our findings reveal PARP-1 as a double-edged sword in colorectal carcinogenesis, which suppresses tumor initiation following DNA alkylation in a MGMT-dependent manner, but promotes inflammation-driven tumor progression.

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Section: Methodsmentioning

confidence: 99%

“…To inactivate cellular MGMT, cells were incubated with the potent MGMT inhibitor O 6 -benzylguanine (20 μM; Sigma Deisenhofen) for 2 h before TMZ addition. Cells were then harvested and processed for the alkaline Comet assay as described (49,50).…”

Section: Methodsmentioning

confidence: 99%

PARP-1 protects against colorectal tumor induction, but promotes inflammation-driven colorectal tumor progression

Dörsam

Seiwert

Foersch

et al. 2018

Proc. Natl. Acad. Sci. U.S.A.

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“…Cell death induction was analyzed by Annexin-V/PI staining as described previously. 59 Briefly, cells grown in six-well plates were incubated with or without CDT for 48 h in the absence or presence of inhibitors. Attached and detached cells were harvested, washed with PBS and processed for Annexin-V/PI staining in Annexin-V-binding buffer (10 mM HEPES, pH 7.4, 140 mM NaCl, 2.5 mM CaCl 2 , 0.1% BSA) containing Annexin-V labeled with Alexa Fluor 488 (ratio 1 : 20; Miltenyi Biotech, Gladbach, Germany).…”

Section: Methodsmentioning

confidence: 99%

“…Cell cycle distribution and subG1 population were assessed as previously described. 59 Briefly, HCT116 cells were treated with increasing doses of CDT or exposed to IR. After 24 h, adherent cells were detached by trypsin incubation and pooled with cells floating in the cell culture supernatant.…”

Section: Methodsmentioning

confidence: 99%

AKT2 suppresses pro-survival autophagy triggered by DNA double-strand breaks in colorectal cancer cells

et al. 2017

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DNA double-strand breaks (DSBs) are critical DNA lesions, which threaten genome stability and cell survival. DSBs are directly induced by ionizing radiation (IR) and radiomimetic agents, including the cytolethal distending toxin (CDT). This bacterial genotoxin harbors a unique DNase-I-like endonuclease activity. Here we studied the role of DSBs induced by CDT and IR as a trigger of autophagy, which is a cellular degradation process involved in cell homeostasis, genome protection and cancer. The regulatory mechanisms of DSB-induced autophagy were analyzed, focusing on the ATM-p53-mediated DNA damage response and AKT signaling in colorectal cancer cells. We show that treatment of cells with CDT or IR increased the levels of the autophagy marker LC3B-II. Consistently, an enhanced formation of autophagosomes and a decrease of the autophagy substrate p62 were observed. Both CDT and IR concomitantly suppressed mTOR signaling and stimulated the autophagic flux. DSBs were demonstrated as the primary trigger of autophagy using a DNase I-defective CDT mutant, which neither induced DSBs nor autophagy. Genetic abrogation of p53 and inhibition of ATM signaling impaired the autophagic flux as revealed by LC3B-II accumulation and reduced formation of autophagic vesicles. Blocking of DSB-induced apoptotic cell death by the pan-caspase inhibitor Z-VAD stimulated autophagy. In line with this, pharmacological inhibition of autophagy increased cell death, while ATG5 knockdown did not affect cell death after DSB induction. Interestingly, both IR and CDT caused AKT activation, which repressed DSB-triggered autophagy independent of the cellular DNA-PK status. Further knockdown and pharmacological inhibitor experiments provided evidence that the negative autophagy regulation was largely attributable to AKT2. Finally, we show that upregulation of CDT-induced autophagy upon AKT inhibition resulted in lower apoptosis and increased cell viability. Collectively, the findings demonstrate that DSBs trigger pro-survival autophagy in an ATM- and p53-dependent manner, which is curtailed by AKT2 signaling.

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“…Cell cycle distribution and subG1 population were assessed as previously described (37). Briefly, Caco-2 and HCT116 cells were treated with N-OH-PhIP (5 and 2.5 μM, respectively) in the absence or presence of the ATR inhibitor (5 μM) for 48 h. Detached cells and trypsinized adherent cells were pooled, harvested by centrifugation and washed twice in PBS.…”

Section: Methodsmentioning

confidence: 99%

DNA damage response curtails detrimental replication stress and chromosomal instability induced by the dietary carcinogen PhIP

Mimmler¹,

Schmidt²,

Kraus³

et al. 2016

Nucleic Acids Research

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PhIP is an abundant heterocyclic aromatic amine (HCA) and important dietary carcinogen. Following metabolic activation, PhIP causes bulky DNA lesions at the C8-position of guanine. Although C8-PhIP-dG adducts are mutagenic, their interference with the DNA replication machinery and the elicited DNA damage response (DDR) have not yet been studied. Here, we analyzed PhIP-triggered replicative stress and elucidated the role of the apical DDR kinases ATR, ATM and DNA-PKcs in the cellular defense response. First, we demonstrate that PhIP induced C8-PhIP-dG adducts and DNA strand breaks. This stimulated ATR-CHK1 signaling, phosphorylation of histone 2AX and the formation of RPA foci. In proliferating cells, PhIP treatment increased the frequency of stalled replication forks and reduced fork speed. Inhibition of ATR in the presence of PhIP-induced DNA damage strongly promoted the formation of DNA double-strand breaks, activation of the ATM-CHK2 pathway and hyperphosphorylation of RPA. The abrogation of ATR signaling potentiated the cell death response and enhanced chromosomal aberrations after PhIP treatment, while ATM and DNA-PK inhibition had only marginal effects. These results strongly support the notion that ATR plays a key role in the defense against cancer formation induced by PhIP and related HCAs.

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Lipoic acid induces p53-independent cell death in colorectal cancer cells and potentiates the cytotoxicity of 5-fluorouracil

Cited by 39 publications

References 59 publications

PARP-1 protects against colorectal tumor induction, but promotes inflammation-driven colorectal tumor progression

PARP-1 protects against colorectal tumor induction, but promotes inflammation-driven colorectal tumor progression

AKT2 suppresses pro-survival autophagy triggered by DNA double-strand breaks in colorectal cancer cells

DNA damage response curtails detrimental replication stress and chromosomal instability induced by the dietary carcinogen PhIP

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