Calcium hydroxide nanoparticles-induced oxidative stress and mitochondrial impairment drive genomic instability and programmed cell death in colorectal cancer cells

Abstract

The high aggressiveness, metastatic potential, and mortality of colorectal cancer, together with the limitations of conventional chemotherapy, highlight the urgent need for safer and more effective therapeutic alternatives. Nanotherapies offer promising advantages through improved bioavailability and tumor targeting. In particular, calcium hydroxide nanoparticles (Ca(OH)2NPs) possess unique physicochemical properties, yet their anticancer potential in colorectal cancer remains fully unexplored. This study was consequently undertaken to estimate the cytotoxic effects and underlying molecular mechanisms of Ca(OH)2NPs in human colorectal HCT-116 cancer cells, while also exploring their impact on the viability of normal human HFB4 melanocytes. Normal HFB4 and cancerous HCT-116 cells were exposed to serial two-fold concentrations of Ca(OH)2NPs ranging from 7.80 to 1000 mg/ml for 72 h, and cell viability was assessed using the MTT assay. Intracellular reactive oxygen species (ROS) generation and mitochondrial membrane potential were measured using 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) and rhodamine-123 staining, respectively. Genomic DNA damage was evaluated by the alkaline comet assay, whereas apoptosis induction was analyzed using DAPI staining and the chromatin diffusion assay. The expression levels of mitochondria- and apoptosis-related genes were quantified by quantitative real-time PCR (qRT-PCR). Results of the MTT assay demonstrated that Ca(OH)2NPs exerted significant, dose-dependent cytotoxicity on colorectal HCT-116 cancer cells, as evidenced by a markedly low IC50 value of 35.04 µg/ml and a substantial reduction in cell viability. In contrast, the viability of normal HFB4 melanocytes was only slightly affected, and only at the highest Ca(OH)2NPs concentration tested, as indicated by a comparatively high IC50 value of 190.80 µg/ml. The resulting selectivity index of 5.44 further supports the notable cytotoxicity of Ca(OH)2NPs toward HCT-116 colorectal cancer cells. Furthermore, treatment of HCT-116 cells with Ca(OH)2NPs at the IC50 concentration (35.04 µg/ml) led to a significant increase in intracellular ROS generation level, dramatic loss of mitochondrial membrane potential, and pronounced oxidative DNA damage, ultimately culminating in apoptotic cell death. qRT-PCR analysis also demonstrated significant downregulation of both the apoptotic p53 and the anti-apoptotic Bcl-2 gene expression, alongside significant upregulation of the mitochondrial ND3 gene expression. These molecular alterations support the involvement of mitochondrial dysfunction and apoptosis-related gene modulation in the observed Ca(OH)2NPs-induced cytotoxic effects. Conclusion: Ca(OH)2NPs demonstrate notable anticancer activity against human colorectal HCT-116 cancer cells, primarily through ROS-mediated oxidative stress, genomic DNA damage, mitochondrial dysfunction, and apoptosis induction. These findings highlight Ca(OH)2NPs as a potent nanotherapeutic candidate for colorectal cancer management. However, the current study was limited to in vitro experimental conditions, and the precise molecular mechanisms underlying these effects remain incompletely understood. Therefore, further mechanistic investigations, advanced in vitro and in vivo studies, and comprehensive biocompatibility and safety evaluations are required to validate their therapeutic potential and clinical applicability.