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Safranal induces DNA double-strand breakage and ER-stress-mediated cell death in hepatocellular carcinoma cells.

ABSTRACT: Poor prognoses remain the most challenging aspect of hepatocellular carcinoma (HCC) therapy. Consequently, alternative therapeutics are essential to control HCC. This study investigated the anticancer effects of safranal against HCC using in vitro, in silico, and network analyses. Cell cycle and immunoblot analyses of key regulators of cell cycle, DNA damage repair and apoptosis demonstrated unique safranal-mediated cell cycle arrest at G2/M phase at 6 and 12?h, and at S-phase at 24?h, and a pronounced effect on DNA damage machinery. Safranal also showed pro-apoptotic effect through activation of both intrinsic and extrinsic initiator caspases; indicating ER stress-mediated apoptosis. Gene set enrichment analysis provided consistent findings where UPR is among the top terms of up-regulated genes in response to safranal treatment. Thus, proteins involved in ER stress were regulated through safranal treatment to induce UPR in HepG2 cells.

SUBMITTER: Al-Hrout A

PROVIDER: S-EPMC6240095 | biostudies-literature | 2018 Nov

REPOSITORIES: biostudies-literature

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Safranal induces DNA double-strand breakage and ER-stress-mediated cell death in hepatocellular carcinoma cells.

Al-Hrout Ala'a A Chaiboonchoe Amphun A Khraiwesh Basel B Murali Chandraprabha C Baig Badriya B El-Awady Raafat R Tarazi Hamadeh H Alzahmi Amnah A Nelson David R DR Greish Yaser E YE Ramadan Wafaa W Salehi-Ashtiani Kourosh K Amin Amr A

Scientific reports 20181116 1

Poor prognoses remain the most challenging aspect of hepatocellular carcinoma (HCC) therapy. Consequently, alternative therapeutics are essential to control HCC. This study investigated the anticancer effects of safranal against HCC using in vitro, in silico, and network analyses. Cell cycle and immunoblot analyses of key regulators of cell cycle, DNA damage repair and apoptosis demonstrated unique safranal-mediated cell cycle arrest at G2/M phase at 6 and 12 h, and at S-phase at 24 h, and a pro ...[more]

PMID: 30446676

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Project description:The incidence of chronic liver disease is constantly increasing, owing to the obesity epidemic. However, the causes and mechanisms of inflammation-mediated liver damage remain poorly understood. Endoplasmic reticulum (ER) stress is an initiator of cell death and inflammatory mechanisms. Although obesity induces ER stress, the interplay between hepatic ER stress, NLRP3 inflammasome activation and hepatocyte death signaling has not yet been explored during the etiology of chronic liver diseases. Steatosis is a common disorder affecting obese patients; moreover, 25% of these patients develop steatohepatitis with an inherent risk for progression to hepatocarcinoma. Increased plasma LPS levels have been detected in the serum of patients with steatohepatitis. We hypothesized that, as a consequence of increased plasma LPS, ER stress could be induced and lead to NLRP3 inflammasome activation and hepatocyte death associated with steatohepatitis progression. In livers from obese mice, administration of LPS or tunicamycin results in IRE1? and PERK activation, leading to the overexpression of CHOP. This, in turn, activates the NLRP3 inflammasome, subsequently initiating hepatocyte pyroptosis (caspase-1, -11, interleukin-1? secretion) and apoptosis (caspase-3, BH3-only proteins). In contrast, the LPS challenge is blocked by the ER stress inhibitor TUDCA, resulting in: CHOP downregulation, reduced caspase-1, caspase-11, caspase-3 activities, lowered interleukin-1? secretion and rescue from cell death. The central role of CHOP in mediating the activation of proinflammatory caspases and cell death was characterized by performing knockdown experiments in primary mouse hepatocytes. Finally, the analysis of human steatohepatitis liver biopsies showed a correlation between the upregulation of inflammasome and ER stress markers, as well as liver injury. We demonstrate here that ER stress leads to hepatic NLRP3 inflammasome pyroptotic death, thus contributing as a novel mechanism of inflammation-mediated liver injury in chronic liver diseases. Inhibition of ER-dependent inflammasome activation and cell death pathways may represent a potential therapeutic approach in chronic liver diseases.

Project description:Reptin/RUVBL2 is overexpressed in most hepatocellular carcinomas and is required for the growth and viability of HCC cells. Reptin is involved in several chromatin remodeling complexes, some of which are involved in the detection and repair of DNA damage, but data on Reptin involvement in the repair of DNA damage are scarce and contradictory. Our objective was to study the effects of Reptin silencing on the repair of DNA double-strand breaks (DSB) in HCC cells. Treatment of HuH7 cells with etoposide (25 ?M, 30 min) or ? irradiation (4 Gy) increased the phosphorylation of H2AX by 1.94 ± 0.13 and 2.0 ± 0.02 fold, respectively. These values were significantly reduced by 35 and 65 % after Reptin silencing with inducible shRNA. Irradiation increased the number of BRCA1 (3-fold) and 53BP1 foci (7.5 fold). Depletion of Reptin reduced these values by 62 and 48%, respectively. These defects in activation and/or recruitment of repair proteins were not due to a decreased number of DSBs as measured by the COMET assay. All these results were confirmed in the Hep3B cell line. Protein expression of ATM and DNA-PKcs, the major H2AX kinases, was significantly reduced by 52 and 61 % after Reptin depletion whereas their mRNA level remained unchanged. Phosphorylation of Chk2, another ATM target, was not significantly altered. Using co-immunoprecipitation, we showed an interaction between Reptin and DNA-PKcs. The half-life of newly-synthesized DNA-PKcs was reduced when Reptin was silenced. Finally, depletion of Reptin was synergistic with etoposide or ? irradiation to reduce cell growth and colony formation. In conclusion, Reptin is an important cofactor for the repair of DSBs. Our data, combined with those of the literature suggests that it operates at least in part by regulating the expression of DNA-PKcs by a stabilization mechanism. Overexpression of Reptin in HCC could be a factor of resistance to treatment, consistent with the observed overexpression of Reptin in subgroups of chemo-resistant breast and ovarian cancers.

Dataset Information

Safranal induces DNA double-strand breakage and ER-stress-mediated cell death in hepatocellular carcinoma cells.

Publications

Safranal induces DNA double-strand breakage and ER-stress-mediated cell death in hepatocellular carcinoma cells.

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