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Carbonic anhydrase 9 confers resistance to ferroptosis/apoptosis in malignant mesothelioma under hypoxia.

ABSTRACT: Hypoxia and acidity provide microenvironment for selection under evolutionary pressure and proliferation in cancer cells. Carbonic anhydrases (CAs) are a superfamily of metalloenzymes present in all life kingdoms, equilibrating the reactions among CO2, bicarbonate and H+. CA9, a membrane-associated ?-CA, has been a drug target for various cancers. Whereas iron is essential not only for cancer cells but also for all the lives on earth, little is known on the association among hypoxia, iron metabolism, extracellular acidity and redox regulation. Malignant mesothelioma (MM), an aggressive tumor with poor prognosis, is an intriguing model in that asbestos-associated pathogenesis includes excess iron environment during carcinogenesis. Re-analysis of rat asbestos-induced MM model revealed an inverse association between high CA9 expression and survival. Here we used human MMs to identify the molecular events surrounding CA9 from the viewpoint of iron metabolism. CA9 expression was significantly higher in MM cells than in MeT-5A mesothelial cells, which was further amplified under hypoxia (1%O2) with increased catalytic Fe(II). CA9 suppression by inhibitors (S4 and U104) decreased viability and migration of MM cells, accompanied by overexpression of TFRC, IREB1/2 and FPN1(SLC40A1) and by downregulation of FTH/FTL. This expressional pattern was similar to that of erastin-induced ferroptosis in the same cells. Furthermore, we observed mitochondrial fission and enhanced autophagy with increased catalytic Fe(II) in both mitochondria and lysosomes after CA9 inhibition, accompanied by increased peroxides, mitochondrial O2- and lipid peroxidation. The eventual cell death was significantly inhibited by deferoxamine, ferrostatin-1 and Z-VAD-FMK, suggesting a mixed cell death of ferroptosis and apoptosis. Therefore, CA9 plays a role in equilibrating among hypoxia, iron metabolism and redox regulation in MM cells.

SUBMITTER: Li Z

PROVIDER: S-EPMC6831888 | biostudies-literature | 2019 Sep

REPOSITORIES: biostudies-literature

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Carbonic anhydrase 9 confers resistance to ferroptosis/apoptosis in malignant mesothelioma under hypoxia.

Li Zan Z Jiang Li L Chew Shan Hwu SH Hirayama Tasuku T Sekido Yoshitaka Y Toyokuni Shinya S

Redox biology 20190810

Hypoxia and acidity provide microenvironment for selection under evolutionary pressure and proliferation in cancer cells. Carbonic anhydrases (CAs) are a superfamily of metalloenzymes present in all life kingdoms, equilibrating the reactions among CO<sub>2</sub>, bicarbonate and H<sup>+</sup>. CA9, a membrane-associated α-CA, has been a drug target for various cancers. Whereas iron is essential not only for cancer cells but also for all the lives on earth, little is known on the association amon ...[more]

PMID: 31442913

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Project description:BackgroundHypoxia is a hallmark of the solid tumor microenvironment and is associated with poor outcomes in cancer patients. The present study was performed to investigate mechanisms underlying the hypoxia-induced phenotypic changes using human malignant mesothelioma (HMM) cells.MethodsHypoxic conditions were achieved by incubating HMM cells in the air chamber. The effect of hypoxia on phenotype changes in HMM cells was investigated by performing in vitro clonogenicity, drug resistance, migration, and invasion assays. Signaling pathways and molecules involved in the more aggressive behaviors of HMM cells under hypoxia were investigated. A two-tailed unpaired Student's t-test or one-way ANOVA with Bonferroni post-test correction was used in this study.ResultsHypoxic conditions upregulated hypoxia-inducible factor 1 alpha (HIF-1?) and HIF-2? in parallel with the upregulation of its target, Glut-1, in HMM cells. In vitro clonogenicity of HMM cells was significantly increased in hypoxic conditions, but the proliferation of cells at a high density in hypoxia was lower than that in normoxic conditions. The expression levels of HIF-2? and Oct4 were increased in hypoxic HMM cells. The percentage of cells with high CD44 expression was significantly higher in HMM cells cultured in hypoxia than those cultured in normoxia. Hypoxia significantly enhanced the resistance of HMM cells to cisplatin, which occurred through cytoprotection against cisplatin-induced apoptosis. While cisplatin treatment decreased the ratio of Bcl-2 to Bax in normoxic condition, hypoxia conversely increased the ratio in HMM cells treated with cisplatin. Hypoxia increased the mobility and invasiveness of HMM cells. Epithelial to mesenchymal transition was promoted, which was indicated by the repression of E-cadherin and the concomitant increase of vimentin in HMM cells.ConclusionsThe data illustrated that hypoxic conditions augmented the aggressive phenotypes of HMM cells at the biological and molecular levels. The present study provides valuable background information beginning to understand aggressiveness of HMM in tumor microenvironments, suggesting that a control measure for tumor hypoxia may be an effective therapeutic strategy to reduce the aggressiveness of cancer cells in HMM patients.

Project description:AIM:To study the expression of carbonic anhydrase (CA) 9 in human hepatocellular carcinoma (HCC) cells. METHODS:We studied CA9 protein, CA9 mRNA and hypoxia-inducible factor-1 alpha (HIF-1?) protein levels in Hep3B cells exposed in different parallel approaches. In one of these approaches, HCC cells were exposed to extreme in vitro hypoxia (24 h 0.1% O(2)) without or with interleukin (IL)-1, IL-6, tumor necrosis factor-alpha (TNF-?) and transforming growth factor-beta (TGF-?) stimulation for the same hypoxic exposure time or exposed to normoxic oxygenation conditions without or with cytokine stimulation. RESULTS:The tumour cell line analysed showed a strong hypoxic CA9 mRNA expression pattern in response to prolonged severe hypoxia with cell-line specific patterns and a marked induction of CA9 protein in response to severe hypoxia. These results were paralleled by the results for HIF-1? protein under identical oxygenation conditions with a similar expression tendency to that displayed during the CA9 protein expression experimental series. Continuous stimulation with the cytokines, IL-1, IL-6, TNF-? and TGF-?, under normoxic conditions significantly increased the carbonic anhydrase 9 expression level at both the protein and mRNA level, almost doubling the CA9 mRNA and CA9 and HIF-1? protein expression levels found under hypoxia. The findings from these experiments indicated that hypoxia is a positive regulator of CA9 expression in HCC, and the four signal transduction pathways, IL-1, IL-6, TNF-? and TGF-?, positively influence CA9 expression under both normoxic and hypoxic conditions. CONCLUSION:These findings may potentially be considered in the design of anti- cancer therapeutic approaches involving hypoxia-induced or cytokine stimulatory effects on expression. In addition, they provide evidence of the stimulatory role of the examined cytokine families resulting in an increase in CA9 expression under different oxygenation conditions in human cancer, especially HCC, and on the role of the CA9 gene as a positive disease regulator in human cancer.

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Carbonic anhydrase 9 confers resistance to ferroptosis/apoptosis in malignant mesothelioma under hypoxia.

Publications

Carbonic anhydrase 9 confers resistance to ferroptosis/apoptosis in malignant mesothelioma under hypoxia.

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