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Elucidation of Mechanisms of Topotecan-Induced Cell Death in Human Breast MCF-7 Cancer Cells by Gene Expression Analysis.

ABSTRACT: Topotecan is a clinically active anticancer agent for the management of various human tumors. While the principal mechanism of tumor cell killing by topotecan is due to its interactions with topoisomerase I and formation of DNA double-strand breaks, recent studies suggest that mechanisms involving generation of reactive free radicals and induction of oxidative stress may play a significant role in topotecan-dependent tumor cell death. We have shown that topotecan generates a topotecan radical following one-electron oxidation by a peroxidase-hydrogen peroxide system which reacts with reduced glutathione and cysteine, forming the glutathiyl and cysteinyl radicals, respectively. While little is known how these events are involved in topotecan-induced tumor cell death, we have now examined the effects of topotecan short (1 h) and long (24 h) exposure on global gene expression patterns using gene expression microarray analysis in human breast MCF-7 cancer cells, a wild-type p53 containing cell line. We show here that topotecan treatment significantly down-regulated estrogen receptor alpha (ER?/ESR1) and antiapoptotic BCL2 genes in addition to many other p53-regulated genes. Furthermore, 8-oxoguanine DNA glycosylase (OGG1), ferredoxin reductase (FDXR), methionine sulfoxide reductase (MSR), glutathione peroxidases (GPx), and glutathione reductase (GSR) genes were also differentially expressed by topotecan treatment. The differential expression of these genes was observed in a wild-type p53-containing breast ZR-75-1 tumor cell line following topotecan treatment. The involvement of reactive oxygen free radical sensor genes, the oxidative DNA damage (OGG1) repair gene and induction of pro-apoptotic genes suggest that reactive free radical species play a role in topotecan-induced tumor cell death.

SUBMITTER: Sinha BK

PROVIDER: S-EPMC7379903 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Elucidation of Mechanisms of Topotecan-Induced Cell Death in Human Breast MCF-7 Cancer Cells by Gene Expression Analysis.

Sinha Birandra K BK Tokar Erik J EJ Bushel Pierre R PR

Frontiers in genetics 20200717

Topotecan is a clinically active anticancer agent for the management of various human tumors. While the principal mechanism of tumor cell killing by topotecan is due to its interactions with topoisomerase I and formation of DNA double-strand breaks, recent studies suggest that mechanisms involving generation of reactive free radicals and induction of oxidative stress may play a significant role in topotecan-dependent tumor cell death. We have shown that topotecan generates a topotecan radical fo ...[more]

PMID: 32765594

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Project description:BACKGROUND:Functional erythropoietin (EPO) signaling is not specific only to erythroid lineages and has been confirmed in several solid tumors, including breast. Three different isoforms of erythropoietin receptor (EPOR) have been reported, the soluble (EPOR-S) and truncated (EPOR-T) forms acting antagonistically to the functional EPOR. In this study, we investigated the effect of human recombinant erythropoietin (rHuEPO) on cell proliferation, early gene response and the expression of EPOR isoforms in the MCF-7 breast cancer cell line. MATERIALS AND METHODS:The MCF-7 cells were cultured with or without rHuEPO for 72 h or 10 weeks and assessed for their growth characteristics, expression of early response genes and different EPOR isoforms. The expression profile of EPOR and EPOR-T was determined in a range of breast cancer cell lines and compared with their invasive properties. RESULTS:MCF-7 cell proliferation after rHuEPO treatment was dependent on the time of treatment and the concentration used. High rHuEPO concentrations (40 U/ml) stimulated cell proliferation independently of a preceding long-term exposure of MCF-7 cells to rHuEPO, while lower concentrations increased MCF-7 proliferation only after 10 weeks of treatment. Gene expression analysis showed activation of EGR1 and FOS, confirming the functionality of EPOR. rHuEPO treatment also slightly increased the expression of the functional EPOR isoform, which, however, persisted throughout the 10 weeks of treatment. The expression levels of EPOR-T were not influenced. There were no correlations between EPOR expression and the invasiveness of MCF-7, MDA-MB-231, Hs578T, Hs578Bst, SKBR3, T-47D and MCF-10A cell lines. CONCLUSIONS:rHuEPO modulates MCF-7 cell proliferation in time- and concentration-dependent manner. We confirmed EGR1, FOS and EPOR as transcription targets of the EPO-EPOR signaling loop, but could not correlate the expression of different EPOR isoforms with the invasiveness of breast cancer cell lines.

Project description:BackgroundWe characterized an endocrine disruptor from ground corncob bedding material that interferes with male and female sexual behavior and ovarian cyclicity in rats and stimulates estrogen receptor (ER)-positive and ER-negative breast cancer cell proliferation. The agents were identified as an isomeric mixture of tetrahydrofurandiols (THF-diols; 9,12-oxy-10,13-dihydroxy-octadecanoic acid and 10,13-oxy-9,12-dihydroxyoctadecanoic acid). Synthetic THF-diols inhibited rat male and female sexual behavior at oral concentrations of 0.5-1 ppm, and stimulated MCF-7 human breast cancer cell proliferation in vitro.ObjectivesBecause THF-diols are derived from lipoxygenase and cyclooxygenase pathways, we suspected that these compounds may regulate cell proliferation by modulating specific enzymatic sites involved in linoleic acid metabolism including phospholipase A(2) (PLA2), lipoxygenases (LOX-5 and LOX-12), cyclooxygenases (COX-1 and COX-2), and closely coupled enzymes including aromatase (AROM).MethodsMCF-7 human breast cancer cells were treated with inhibitors for PLA2 (quinacrine), lipoxygenases (LOX-5 and LOX-12; baicalein, REV-5091, nordihydroguaiaretic acid), cyclooxygenases (COX-1, COX-2, indomethacin), and AROM (formestane). The effects of these enzyme inhibitors on cell proliferation in response to THF-diols or estradiol (E(2)) were assessed. THF-diol modulation of the expression (RNA and protein) of these enzymes was also evaluated by quantitative real-time PCR (QPCR) and Western blot analyses.ResultsThe enzyme inhibition and gene expression (RNA and protein) studies identified PLA2, LOX-5, LOX-12, COX-2, and perhaps AROM as likely sites of THF-diol regulation in MCF-7 cells. COX-1 was not affected by THF-diol treatment.DiscussionTHF-diol stimulation of MCF-7 cell proliferation is mediated through effects on the expression of the PLA2, COX-2, LOX-5, and LOX-12 genes and/or their respective enzyme activities. The products of these enzymes, including prostaglandins, hydroxyeicosatetraenoic acids (HETEs) and hydroxyoctadecenoic acids (HODEs), are well-established mitogens in normal and malignant cells. Therefore, it is likely that these compounds are involved in the mechanism of action of THF-diols in breast cancer cells. Although the formestane inhibition studies suggested that AROM activity might be modulated by THF-diols, this was not confirmed by the gene expression studies.

Dataset Information

Elucidation of Mechanisms of Topotecan-Induced Cell Death in Human Breast MCF-7 Cancer Cells by Gene Expression Analysis.

Publications

Elucidation of Mechanisms of Topotecan-Induced Cell Death in Human Breast MCF-7 Cancer Cells by Gene Expression Analysis.

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