Accelerating effect of TNF-α on the Rhus verniciflua-induced growth inhibition and apoptosis in human osteosarcoma cells

Previously, a flavonoid fraction, which consisted mainly of protocatechuic acid, fustin, fisetin, sulfuretin, and butein, here named RCMF [Rhus verniciflua Stokes (RVS) chloroform-methanol fraction], was prepared from a crude acetone extract of RVS which is traditionally used as a food additive and as an herbal medicine. In the present study, we investigated the effects of TNF-α on RCMF-induced growth inhibition and apoptosis induction using human osteosarcoma (HOS) cells. The results from tritium uptake and MTT assays showed that TNF-α treatment itself (10 ng/ml) did not induce any cytotoxicity, but it actively accelerated RCMF-mediated cytotoxicity of HOS cells. RCMF-induced cytotoxicity and its facilitation by TNF-α was verified to be apoptotic, based on the increased DNA fragmentation and low fluorescence intensity in nuclei after propidium iodide (PI) staining of HOS cells. This speculation was further demonstrated by monitoring the Annexin V/PI double staining which could discriminate the difference between apoptotic and necrotic deaths. Collectively, our findings indicate that TNF-α accelerates RCMF-induced cytotoxicity in HOS cells.