Induction Of Apoptosis By Furanodiene In HL60 Leukemia Cells Through Activation Of TNFR1 Signaling Pathway

Furanodiene, a natural product isolated from Curcuma wenyujin, has recently been reported to inhibit the growth of human hepatic cancer HepG2 cells by arrest of G2/M cell cycle and induction of mitochondrial pathway apoptosis. In present study, we investigated the growth inhibitory and the molecular mechanisms of furanodiene in human leukemia HL60 cells.Cell growth inhibitory effects of furanodiene were investigated in HL60 cells using trypan blue exclusion assay. The results showed that furanodiene （10-70μM） treatment inhibited the growth of HL60 cells in a time- and concentration- dependent manner, and the 50% inhibitory concentration (IC₅₀) was 32.7μM after 72h treatment. To elucidate whether furanodiene induced apoptosis in HL60 cells, flow cytometry analysis, AO/EB staining assay and agarose gel electrophoresis assay were used to confirm the apoptotic characterizations in furanodiene-treated HL60 cells. Flow cytometry analysis showed that furanodiene stimulation increased the percentage of SubG1 phase in HL60 cells, but did not arrest cell cycle at G0/G1, S as well as G2/M phase. In AO/EB fluorescence staining assay, morphological changes of apoptosis were found in furanodiene-treated HL60 cells. Apoptotic cells counting showed that furanodiene treatment resulted in an increase in the percentage of apoptotic cells in a concentration-dependent manner. In DNA agarose gel electrophoresis experiment, DNA laddering, a marker of apoptosis was detected in furanodiene-treated HL60 cells. Taking together, we conclude that furanodiene induces apoptosis of HL60 cells.Caspases have been shown to play a crucial role in the initiation and execution of apoptosis. In order to investigate the molecular mechanisms of apoptosis induced by furanodiene in HL60 cells, we examined the activation of caspases using Western blotting analysis. The activation of caspase-3. -8, -9 and PARP was detected after furanodiene treatment. Hence, we suppose that furanodiene induces apoptosis of HL60 cells by the way of the activation of caspases signaling. Moreover. Bid. a substrate of caspase-8, was also activated by furanodiene. However, other Bcl-2 family proteins, including Bcl-2, Bax and Bcl-xL, were not influenced by furanodiene stimulation. Therefore, we demonstrate that furanodiene activates caspase-3 through both intrinsic and extrinsic pathways, and the activation of caspase-9 resultes from Bid activation in caspase-8 signal.Caspase-8 is a prominent effector of death receptor, which is necessary for death receptor-mediated apoptosis. To further characterize the apoptotic pathway activated by furanodiene, we detected the expression of various kinds of death receptors. The results showed that furanodiene treatment induced an obvious upregulation of TNFR1 expression, but did not influence the expression of Fas, DR4 and DR5. In addition, using enzyme-linked immunosorbent assay （ELISA）, an increased secretion of TNFαin the cell culture supernatants could be detected when HL60 cells were stimulated with furanodiene. Taking together, we conclude that TNFα-TNFR1 signaling system may contribute to apoptosis of HL60 cells induced by furanodiene. To confirm the activation of TNFR1 signaling system, the formation of TNFR1 complex was analyzed by using immunoprecipitation of TNFR1 with TRRF1/2 and RIP in furanodiene-treated HL60 cells. The results showed that furanodiene treatment upregulated the expression of TRAF2 and RIP in the TNFR1 immunoprecipitate. Therefore, furanodiene could induce the formation of TNFR1 complex, which could induce apoptosis by activating caspase-8. Further evidence for the function of TNFR1 signaling in furanodiene-caused apoptosis of HL60 cells came from our observation that TNFR1-Fc chimera strongly blocked furanodiene-induced apoptosis in HL60 cells. Taking together, TNFα-TNFR1 signaling system plays a crucial role in furanodiene-induced apoptosis of HL60 cells.Generation of reactive oxygen species （ROS） contributes to mitochondrial apoptosis by acting as an apoptotic signaling molecule. ROS production was detected by flow cytometric analysis using DCFH-DA as a fluorescence probe. The results indicated that furanodiene treatment had no effects on the ROS production in HL60 cells. Furthermore, the antioxidants, NAC and CAT, had no effects on furanodiene-induced apoptosis and the activation of caspase-3 and PARP. From those findings. we conclude that the apoptosis of HL60 cells induced by furanodiene is ROS-independent.In summary, we demonstrate that furanodiene inhibits the growth of HL60 cells via induction of apoptosis. Furanodiene-induced apoptosis in HL60 cells is mediated by activating TNFR1 signaling pathway.