Molecular Effects of Selected Copper Compounds on Novel Oncogenic Signaling Marker Expression in Cancer Cells

Abstract

This study evaluated the cytotoxic, apoptotic, and gene expression–modulatory effects of CuSO₄, CuCl₂, and CuCO₃ on L929 fibroblasts, DLD-1 colorectal cancer cells, and Capan-1 pancreatic cancer cells. Cells were exposed to concentrations ranging from 7.8 to 500 µM for 24 h. Cell viability was assessed by MTT assay, apoptosis/necrosis by double staining, and gene expression (EZH2, PGK1, HER2, USP28, AURKA, HPRT1, ER-α) by qPCR. At 500 µM, CuSO₄, CuCl₂, and CuCO₃ showed no significant cytotoxicity in L929 cells, with viability values of 99.81±9.4%, 90.69±5.58%, and 104.66±1.48%, respectively. In DLD-1 cells, limited cytotoxicity was observed, with viability remaining above 58% at most concentrations. In contrast, a pronounced, concentration-dependent cytotoxic effect was detected in Capan-1 cells. At 500 µM, viability decreased to 37.28±1.61% (CuSO₄), 36.28±3.29% (CuCl₂), and 31.34±4.93% (CuCO₃). At 250 µM, viability ranged between 42.66±3.44% and 53.50±4.11%, indicating sustained cytotoxic activity. Double staining analysis demonstrated that copper-induced cell death occurred predominantly via apoptosis. In Capan-1 cells, the apoptotic index reached up to 74.2% for CuCl₂ and showed a dose-dependent increase across all compounds, while necrotic rates remained comparatively low. qPCR results revealed significant modulation of cancer-related genes in Capan-1 cells. EZH2 expression was consistently downregulated, particularly in CuSO₄ and CuCO₃ groups. PGK1 and AURKA expression exhibited concentration-dependent changes, with peak upregulation observed at intermediate doses (notably 125–250 µM). HER2 and ER-α expression levels increased across most treatment groups, whereas USP28 expression was generally decreased, with compound-specific variations. HPRT1 expression showed a marked reduction and was nearly undetectable in CuCO₃-treated groups. In conclusion, CuSO₄, CuCl₂, and CuCO₃ exhibit selective and dose-dependent cytotoxic and pro-apoptotic effects in pancreatic cancer cells, while sparing healthy fibroblasts. These compounds also induce significant alterations in gene expression profiles associated with proliferation, metabolism, and tumor progression, highlighting their potential as candidates for targeted anticancer strategies.