Abstract:

BACKGROUND: Studies have shown that disulfiram has anti-tumor activity, which can be combined with copper (Cu) ions to exert an anti-tumor effect on multiple tumors in vivo and in vitro, but the effect of disulfiram on osteosarcoma proliferation and apoptosis has not been clarified.

OBJECTIVE: To investigate the effect of disulfiram combined with Cu on osteosarcoma proliferation and apoptosis and the possible mechanism in vivo and in vitro.

METHODS: The study protocol was approved by the Animal Ethics Committee of Shanxi Medical University (approval No. 2017LL077). (1) In vitro study: diethyldithiocarbamate (DDTC)-Cu (0.5, 1, 2, 3, and 5 μmol/L) was configured with Cu and DDTC which is the transformation of disulfiram after absorbed by bodies. DDTC single drug (5 μmol/L), Cu single drug (5 μmol/L) and blank control groups were set. Osteosarcoma cell lines Saos-2 and MG-63 were treated with drugs, and cell counting kit-8 assay was used to detect the inhibitory effect of DDTC-Cu at different concentrations on the proliferation of Saos-2 and MG-63 cells. Changes in Saos-2 apoptosis were measured by AnnexinV-FITC/PI staining. (2) In vivo study: A total of 10 BALB/c-nu/nu female nude mice of 4 weeks old were randomly divided into DDTC-Cu group and control group. The mixture of Saos-2 cells and Matrigel (1:1 mixed, 400 μL per mouse) was injected subcutaneously into the right back of nude mice. Two weeks after inoculation, model mice were intraperitoneally injected dexamethasone (0.5 mg/kg once every other day) in the control group, and dexamethasone (0.5 mg/kg once every other day) and DDTC-Cu complex (10 nmol/g once every other day) in the DDTC-Cu group. Xenograft tumors in each group were measured at regular intervals and tumor growth curves were drawn. Five weeks after inoculation, the animals were sacrificed under anesthesia, and tumors were completely removed. Immunohistochemistry was used to detect the expression level of ki67 protein in tumor paraffin sections. The expressions of proteins related to cell proliferation and apoptosis and JNK pathway proteins were determined by western blot analysis.

RESULTS AND CONCLUSIONS: (1) In vitro study: The proliferation inhibition in the DDTC-Cu group was significantly stronger than that in the DDTC single drug, Cu single drug and blank control groups. Cell counting kit-8 results showed that DDTC-Cu inhibited osteosarcoma proliferation in a dose-dependent manner, with 50% inhibiting concentration of 0.337 μmol/L (Saos-2) and 0.487 μmol/L (MG-63) for 24 hours, respectively. The results of flow cytometry showed that DDTC-Cu promoted Saos-2 apoptosis in a dose-dependent manner. (2) In vivo study: The tumor volume and mass of the DDTC-Cu group were smaller than those of the control group. Immunohistochemical results showed that the expression level of ki-67 protein in the DDTC-Cu group was lower than that in the control group. Western blot results showed that the expression levels of p-JNK and c-jun in the DDTC-Cu group were up-regulated. To conclude, disulfiram combined with Cu inhibits proliferation and induces apoptosis of osteosarcoma in vitro and in vivo, and its mechanism may be related with activation of JNK signaling pathway.

Key words: disulfiram, cooper, osteosarcoma, proliferation, apoptosis, JNK pathway