Induction of apoptosis by furanodiene in HL60 leukemia cells through activation of TNFR1 signaling pathway

Objective Curcuma wenyujin,named Ezhu in Chinese,a traditional Chinese medicine,which has been shown to possess anticarcinogenic activity and used for the treatment of tumor in China,for example,hepatic cancer and leukemia.β-elemene,a component of Curcuma wenyujin,was largely reported as a main active component for anti-tumor effect of Curcuma wenyujin.However,furanodiene,another main component of Curcuma wenyujin,was seldom investigated for its anti-tumor activities.In the present study,we aim to investigate the effect of furanodiene on human leukemia HL60 cells and to study the accurate molecular mechanisms of its action.Methods Trypan blue exclusion experiment was used for cell growth inhibition assay.The apoptotic characterizations were assessed by flow cytometry analysis,AO/EB staining assay and agarose gel electrophoresis assay.The expression of apoptosis-related proteins and the release of cytochrome c from mitochondrial were detected by western blotting,and the mRNA levels of TNF-α and TNFR1 were probed by RT-PCR.Formation of TNFR1 complex was analyzed by using immunoprecipitation of TNFR1 with TRRF1/2 and RIP.Results Furanodiene(10-100 μM)treatment inhibited the growth of HL60 cells in a concentration-dependent manner.The effect of furanodiene on HL60 cells was associated with the induction of apoptosis,which was characterized by DNA fragmentation,cleavage of poly(ADP-ribose)polymerase(PARP),caspase-3,caspase-8 and caspase-9.In the Bcl-2 family proteins,Bid protein(a substrate of caspase-8)was activated by furanodiene,but Bcl-2,Bax and Bcl-xL proteins were not inuenced by furanodiene stimulation.Furanodiene elicited cytochrome c release from mitochondria into the cytosol.Moreover,furanodiene treatment caused the upregulation of TNFR1,the formation of TNFR1 complex and an obvious production of TNF-α in HL60 cells.The soluble TNFR1 receptor effectively inhibited furanodiene-induced apoptosis.Conclusions Furanodiene inhibited the growth of HL60 leukemia cells via induction of apoptosis.Furanodiene-induced apoptosis in HL60 cells is mediated by upregulation of TNF receptor 1 as well as induction of TNF-α production to activate TNFR1 signaling pathway.Our research provides insight into the molecular mechanisms on furanodiene-induced cell death,and may aid to the development of furanodiene as a new anti-tumor agent.

Potential Anti-cancer Activity of Furanodiene

Objective： To study the isolated from the essential oil VIVO anti-tumor activities of furanodiene of the rhizome of Curcuma wenyujin （C15H200）, a primary sesquiterpene compound YH Chen et C. Ling（Wen Ezhu）, in vitro and in Methods： In vitro MTT assay was used to further study the effects of time and dosage on anti-proliferation of furanodiene against the sensitive Hela, Hep-2, HL-60, U251 cells, based on the cytotoxic effects of furanodiene on 12 human malignant tumor cell lines with the essential oil of Wen Ezhn as control., and the half-inhibitory concentration （IC50） was observed. In vivo uterine cervix （U14） tumor cell was selected and the conventional assay method of anti-tumor activity was employed. Furanodiene liposome was administered intraperitoneally, and tumor-inhibitory rate, thymus and spleen indexes were observed. Results： The inhibitive effects on cell proliferation were shown in all of the twelve cell lines and the cytotoxic effects of furanodiene against Hela, Hep-2, HL-60, U251 cells were observed after 12 h of administration, the effect could last for at least 48 h in a dose dependent manner, and the IC50 values were 0.6, 1.7, 1.8, 7.0μg/ml, respectively. Furanodiene was also found to show inhibitive effects on the proliferation of uterine cervix （U14） tumor induced in mice. The tumor inhibition rates were 36.09% （40 mg/kg）, 41.55% （60 mg/kg）, 58.29% （80 mg/kg）, respectively. Conclusion： Furanodiene is one of primary anti-cancer active components in the essential oil of Wen Ezhu, and also a very effective agent against uterine cervix cancer, and has protection effect on the immune function.