NCTD promotes C-28 methyl ester of 2-cyano-3, 12-dioxoolen-1, 9-dien-28-oic acid (CDDO-Me)-mediated anti-osteosarcoma in osteosarcoma cells

Abstract

Objective To explore whether demethylcantharidin (NCTD) could promote C-28 methyl ester of 2-cyano-3, 12-dioxoolen-1, 9-dien-28-oic acid (CDDO-Me)-mediated anti-osteosarcoma and related molecular mechanisms. Methods U-2OS and Saos-2 osteosarcoma cells were treated with single or dual drugs. Viability of cells was measured by cell counting kit-8 (CCK-8), and trypan blue staining count was used to measure the cell mortality. The anti-osteosarcoma effects of NCTD combined with CDDO-M were tested, and the molecular mechanisms were studied by Western blotting. Results The results of CCK-8 showed that the half maximal inhibitory concentration (IC50) value of CDDO-Me was 1.1 μmol/L in U-2OS cells, and the IC50 values were 0.70, 0.30, and 0.03 μmol/L after treatment with 3, 10, and 30 μmol/L NCTD, decreased by 37%, 81%, and 99%, respectively. In the Saos-2 cells, it was also observed that NCTD had a significant enhancement effect on CDDO-Me-mediated cell inhibition. After treatment of U-2OS and Saos-2 cells with 10 μmol/L NCTD single drug, 0.5 μmol/L CDDO-Me single drug, or combination for 24 h, the results of trypan blue staining counts showed that in U-2OS cells, NCTD and CDDO-Me alone induced 25% and 36% cell death respectively, and the combination of NCTD and CDDO-Me could induce about 79% cell death, which was significantly more potent than single drug (P=0.000). In the Saos-2 cells, NCTD and CDDO-Me induced 19% and 43% of cell death respectively, while the combination of NCTD and CDDO-Me resulted in approximately 82% of cell death, which was also significantly more potent than monotherapy (P=0.000). Western blotting results showed that in U-2OS and Saos-2 cells, the combination of NCTD and CDDO-Me could stimulate the activation of cysteinyl aspartate-specific protease (Caspase)-3, resulting in the majority of poly adenosine diphosphate-ribose polymerase (PARP) cleavage, and promote the activation of apoptosis signal. Conclusion NCTD promotes the killing effect of CDDO-Me on osteosarcoma cells by up-regulating the apoptosis signal, and promotes CDDO-Me-mediated anti-osteosarcoma. Key words: Osteosarcoma; C-28 methyl ester of 2-cyano-3, 12-dioxoolen-1, 9-dien-28-oic acid; Demethylcantharidin; Apoptosis