The chemotherapeutic agent N-acetyl-L-cysteine (NAC) decreases insulin-like growth factor I receptor (IGF-IR) and epidermal growth factor receptor (EGFR) levels on colorectal cancer cell lines

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The purpose of this study was to investigate potential molecular mechanisms underlying the anti-cancer effects of the thiol-containing antioxidant N-acetyl-L-cysteine (NAC). The insulin-like growth factor I receptor (IGF-IR) and epidermal growth factor receptor (EGFR) are overexpressed in colon tumors and contribute to autocrine growth, resistance to apoptosis and angiogenesis. NAC has been shown to have anti-proliferative, anti-angiogenic and anti-metastatic properties both in vitro and in vivo. It also augments the efficacy of chemotherapeutic agents in a mouse model of colorectal cancer. However the molecular mechanisms underlying these effects are poorly defined. We investigated if NAC had any effect on expression levels of IGF-IR and EGFR on a panel of colorectal cancer cell lines (HT29, SW480, KM12SM and LoVo). Methods: We analyzed the effect of NAC on expression of IGF-IR and EGFR at a range of concentrations and a range of timepoints by a combination of western blotting and flow cytometry. The effects of a range of other antioxidants were also tested. Semi-quantitative RT-PCR and reporter assays were performed to determine if NAC had any effect on the transcription of the growth factor receptors. The effect of NAC on cellular proliferation was also assessed. Results: Treatment with NAC produced a dose-dependent decrease in IGF-IR and EGFR levels in the four cell lines. The decrease in receptor levels was detectable at 10mM NAC with almost total ablation of receptor expression at concentrations > 20mM. The downregulation began immediately and was maximal between 8 and 24 hours. Following NAC removal receptor levels began to recover immediately, with normal levels returning within 48 hours. Reduced glutathione (GSH) − which is formed from NAC − also downregulated levels of IGF-IR and EGFR in a dose- and time-dependent manner. However, GSH synthesis is not necessary for NAC’s effect. Of the other antioxidants tested, only those containing a free –SH group were capable of downregulating the growth factor receptors. Semi-quantitative RT-PCR and reporter assays indicate that NAC’s effect on EGFR and IGF-IR expression is mediated at the transcriptional level. NAC inhibited growth factor-induced proliferation of the colorectal cancer cells. Conclusion: Treatment of colorectal cancer cell lines with NAC results in a dose- and time-dependent decrease of IGF-IR and EGFR. This effect occurs at the level of transcription. Other –SH containing antioxidants have a similar effect. NAC-mediated growth factor receptor downregulation inhibits the proliferation of colorectal cancer cell lines. These data demonstrate a potential molecular mechanism underlying NAC’s anti-cancer properties.