Abstract
Background: This study examined the effect of calcium-sensing receptor (CaSR) antagonism on human osteosarcoma cells and investigated the underlying molecular mechanisms of this effect through transcriptome sequencing. Methods: Human osteosarcoma cell lines MG-63 and Saos-2 were treated with different concentrations (0.1-10 µM) of the CaSR antagonist NPS-2143. Cell Counting Kit-8 (CCK-8) assays were used to detect the effect of CaSR antagonism on the viability of the cells. RNA sequencing was performed on cells treated with five µM NPS-2143 for 24 hours, followed by bioinformatic analysis to identify differentially expressed genes and enriched pathways. qRT-PCR was conducted to validate key genes. Results: CCK-8 assays showed that at low concentrations (0.1 and one µM), NPS-2143 had no significant effect on MG-63 and Saos-2 cell viability. At higher concentrations (five µM and 10 µM), the viability of MG-63 and Saos-2 cells was significantly reduced. Five µM was therefore selected for subsequent experiments. RNA sequencing revealed distinct gene expression profiles in NPS-2143-treated cells compared to controls. A total of 927 differentially expressed genes (DEGs) were identified in Saos-2 cells (378 upregulated, 549 downregulated), and 59 DEGs were identified in MG-63 cells (33 upregulated, 26 downregulated). Reactome and KEGG pathway enrichment analyses indicated significant enrichment of cholesterol and steroid biosynthesis-related pathways. Transcriptome sequencing showed that NPS-2143 modulated the expression of genes in cholesterol and steroid synthesis pathways. Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) confirmed that NPS-2143 promoted the expression of the cholesterol and steroid synthesis pathway genes, CYP51A1, DHCR24, LSS, and MSMO1 in MG-63 and Saos-2 cells. Discussion: The inhibitory effect of NPS-2143 on MG-63 and Saos-2 osteosarcoma tumor cell viability was confirmed. CaSR antagonism significantly up-regulated genes involved in cholesterol and steroid biosynthesis, including CYP51A1, DHCR24, LSS, and MSMO1. These genes encode key enzymes in the cholesterol synthesis pathway, and their upregulation may lead to cholesterol overproduction. This may, in turn, lead to the formation of oxysterols, which are known to induce inflammation and cytotoxicity. These findings suggest a potential metabolic mechanism through which CaSR antagonists influence osteosarcoma cell viability. Although further validation is warranted, our results provide preliminary evidence implicating cholesterol biosynthesis as a mechanistic target in osteosarcoma and underscore the exploratory value of CaSR antagonists as metabolic regulators in cancer research.