Dataset Information

Involvement of Glutathione Depletion in Selective Cytotoxicity of Oridonin to p53-Mutant Esophageal Squamous Carcinoma Cells.

ABSTRACT: Oridonin, a diterpenoid compound isolated from traditional Chinese medicine Rabdosia rubescens, has shown antitumor effects to esophageal cancer. However, its molecular mechanism is not fully understood, which limits its clinical application. In the present study, we used RNA-seq analysis to check the transcriptome changes after oridonin treatment and we found genes controlling the GSH-ROS system were up-regulated, namely SLC7A11, TXNRD1, TRIM16, SRXN1, GCLM, and GCLC. Furthermore, our data suggest that oridonin significantly increased the production of ROS in EC109 and TE1 cells, which can be inhibited by NAC. Interestingly, oridonin can dramatically reduce intracellular GSH levels in TE1 cells in a concentration and time-dependent manner. In addition, cell death caused by oridonin was strongly inhibited by GSH (1 mM), while GSSG (1 mM) had little effect. At the same time, we also found that oridonin showed selective cytotoxicity to esophageal squamous carcinoma cell with p53 mutation since mut-p53 cells had lower SLC7A11 expression, a component of the cystine/glutamate antiporter. We also found that ?-glutamyl cysteine synthetase inhibitor (BSO) synergizes with oridonin to strongly inhibit EC109 cells at a low dose. These results suggested that the antitumor effects of oridonin are based on its -SH reactivity and glutathione depletion. Esophageal squamous carcinoma cells with p53-mutation showed hypersensitivity to oridonin because of the suppression of SLC7A11 expression by p53 mutation.

SUBMITTER: Li Y

PROVIDER: S-EPMC6974803 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Involvement of Glutathione Depletion in Selective Cytotoxicity of Oridonin to p53-Mutant Esophageal Squamous Carcinoma Cells.

Li Yinchao Y Li Nana N Shi Jianxiang J Ahmed Tanzeel T Liu Hongmin H Guo Jiancheng J Tang Wenxue W Guo Yongjun Y Zhang Qi Q

Frontiers in oncology 20200115

Oridonin, a diterpenoid compound isolated from traditional Chinese medicine Rabdosia rubescens, has shown antitumor effects to esophageal cancer. However, its molecular mechanism is not fully understood, which limits its clinical application. In the present study, we used RNA-seq analysis to check the transcriptome changes after oridonin treatment and we found genes controlling the GSH-ROS system were up-regulated, namely <i>SLC7A11, TXNRD1, TRIM16, SRXN1, GCLM</i>, and <i>GCLC</i>. Furthermore, ...[more]

PMID: 32010620

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Project description:Background & aimsBarrett's esophagus (BE) can progress to dysplasia and esophageal adenocarcinoma (EAC), accompanied by mutations in TP53 that increase the stability of its product, p53. We analyzed BE tissues for messenger RNAs (mRNAs) that associate with BE progression and identified one that affects the stabilization of p53.MethodsWe obtained 54 BE samples collected from patients with high-grade dysplasia (HGD) or esophageal adenocarcinoma (EAC), from 1992 through 2015, and performed RNA sequence analyses, including isoform-specific analyses. We performed reverse-transcription polymerase chain reaction analyses of 166 samples and immunohistochemical analyses of tissue microarrays that contained BE tissues from 100 patients with HGD or EAC and normal esophageal squamous mucosa (controls). Proteins were expressed from transfected plasmids or knocked down with small interfering RNAs in BE cells and analyzed by immunoblots and in immunoprecipitation and ubiquitin ligase assays. Athymic nude mice bearing EAC xenograft tumors (grown from OE-33 cells) were given intraperitoneal injections of simvastatin; tumor growth was monitored and tumors were collected and analyzed by immunoblotting for levels of RNF128, p53, and acetylated p53.ResultsProgression of BE to HGD or EAC associated with changes in expression of mRNAs that encoded mucins and promoted inflammation and activation of ATM and the DNA damage response. As tissues progressed from BE to HGD to EAC, they increased expression of mRNAs encoding isoform 1 of RNF128 (Iso1) and decreased expression of Iso2 of RNF128. RNF128 is an E3 ubiquitin ligase that targets p53 for degradation. Incubation of BE cells with interferon gamma caused them to increase expression of Iso1 and reduce expression of Iso2. Iso1 was heavily glycosylated with limited ubiquitin ligase activity for p53, resulting in p53 stabilization. Knockdown of Iso1 in BE and EAC cells led to degradation of the mutant form of p53 and reduced clonogenic survival. In contrast, Iso2 was a potent ligase that reduced levels of the mutant form of p53 in BE cells. In BE cells, Iso2 was hypoglycosylated and degraded, via ATM and GSK3β-mediated phosphorylation and activation of the beta-TrCP1-containing SCF ubiquitin ligase complex. Simvastatin, which degrades the mutant form of p53, also degraded RNF128 Iso1 protein in BE cells and slowed growth of EAC xenograft tumors in mice.ConclusionsWe found that isoform 2 of RNF128 is decreased in BE cells, resulting in increased levels of mutant p53, whereas isoform 1 of RNF128 is increased in BE cells, further promoting the stabilization of mutant p53.

Project description:Colorectal cancer is the fourth leading cause of oncological-related deaths and the third most diagnosed malignancy, worldwide. The emergence of chemoresistance is a fundamental drawback of colorectal cancer therapies and there is an urgent need for novel plant-derived therapeutics. In this regard, other compounds are needed to improve the efficacy of treatment against colorectal cancer. Medicinal plants have been effectively used by traditional doctors for decades to treat various ailments with little to no side effects. Drimia calcarata (D. calcarata) is one of the plants used by Pedi people in South Africa to treat a plethora of ailments. However, the anticancer therapeutic use of D. calcarata is less understood. Thus, this study was aimed at evaluating the potential anticancer activities of D. calcarata extracts against human colorectal cancer cells. The phytochemical analysis and antioxidant activity were analysed using LC-MS, DPPH, and FRAP. The inhibitory effects and IC50 values of D. calcarata extracts were determined using the MTT assay. Induction of cellular apoptosis was assessed using fluorescence microscopy, the Muse® Cell Analyser, and gene expression analysis by Polymerase Chain Reaction (PCR). Water extract (WE) demonstrated high phenolic, tannin, and flavonoid contents than the methanol extract (ME). LC-MS data demonstrated strong differences between the ME and WE. Moreover, WE showed the best antioxidant activity than ME. The MTT data showed that both ME and WE had no significant activity against human embryonic kidney Hek 293 cell line that served as non-cancer control cells. Caco-2 cells demonstrated high sensitivity to the ME and demonstrated resistance toward the WE, while HT-29 cells exhibited sensitivity to both D. calcarata extracts. The expression of apoptosis regulatory genes assessed by PCR revealed an upregulation of p53 by ME, accompanied by downregulation of Bcl-2 and high expression of Bax after treatment with curcumin. The Bax gene was undetected in HT-29 cells. The methanol extract induced mitochondrial-mediated apoptosis in colorectal Caco-2 and HT-29 cells and WE induced the extrinsic apoptotic pathway in HT-29 cells. ME downregulated STAT1, 3, and 5B in HT-29 cells. The D. calcarata bulb extracts, therefore, contain potential anticancer agents that can be further targeted for cancer therapeutics.

Dataset Information

Involvement of Glutathione Depletion in Selective Cytotoxicity of Oridonin to p53-Mutant Esophageal Squamous Carcinoma Cells.

Publications

Involvement of Glutathione Depletion in Selective Cytotoxicity of Oridonin to p53-Mutant Esophageal Squamous Carcinoma Cells.

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