Tubeimoside â…  Induces Apoptosis Of Human Choriocarcinoma Cells Through Mitochondrial Damage Pathway

Chorionic carcinoma called choriocarcinoma for short, is an highly malignant gestational trophoblastic neoplasia. Chemotherapy is the main clinical therapy. However, in recent years, with the increase of multidrug- resistant, side effect and recurrence rate, chorioepithelioma is difficult to cure, the clinic cure rate is decreased significantly. So the most important thing is to seek for anti-chorioepithelioma drug, which is safe, small side-effect and effective . Anticancer chemotherapies eliminate cancer cells mainly through the induction of apoptosis. Mitochondria play important roles in the intrinsic pathways that trigger apoptosis. tubeimoside I (TBMS1), a triterpenoid saponin isolated from Bolbostemma paniculatum (Maxim) Franquet (Cucurbitaceae), showed potent antitumor effects. This anticancer activity has been linked to its ability to directly trigger mitochondrial pathway. However, the detailed mechanisms and intracellular targets of TBMS1 in inducing cell death remain to be elucidated. Whether TBMS1 is effective on choriocarcinoma is still unknown. Therefore, to study the Effect of Tubeimoside I on Choriocarcinoma cells through Mitochondria-apoptosis pathway can provide some fundamental experimental evidence for new strategy to treat chorioepielioma.We treated the choriocarcinoma cells with different concentrations of TBMS1. Then we used MTT assay, flow cytometry, DNA ladder and Hoechest staining to detect the effect of TBMS1 on cells apoptosis. We used flow cytometry and confocal fluorescence microscopy to observe the change of mitochondrial transmembrane potential(?Ψm). Further more changes of mRNA and protein of mitochondria pathway signaling molecules were detected with RT-PCR, western blot analysis and dual-luciferase reporter plasmid transfection. Our study showed that:(1) MTT experiment results showed that different concentrations of TBMS1 could inhibit the proliferation of Bewo and JEG-3 cells in a time and dose dependent manner.(2) Flow cytometry results showed that TBMS1 treatment led to marked cell apoptosis, proliferation inhibition, significant arrest in phase G0/G1(Bewo) and G2/M(JEG-3) of cell cycle, statistical decrease in mitochondrial transmembrane potential (?Ψm) and significant increase in the levels of intracellular Ca²⁺.(3) DNA ladder essay results showed that TBMS1 treatment led to cells DNA damage, induced cells apoptosis.(4) After treatment JEG-3 cells with TBMS1, we observed the morphological changes of apoptosis by fluorescence microscopy after staining with Hoechest-33342.(5) Immunofluorescence results showed that the movement of Cytochrome c from the mitochondria into the cytoplasm by confocal fluorescence microscopy after incubation with Cyt c antibody and JC-1.(6) Western-blotting results showed that after treatment JEG-3 cells with TBMS1, caspase-3 expression was enhanced. Furthermore, TBMS1 induced the up-regulation of Bax expression, down-regulation of Bcl-2 expression, inhibition of nuclear factor NF-kappaB function and impacted the phosphorylation of p38, ERK1/2 and Akt.(7) Dual-luciferase reporter plasmid transfection results showed that TBMS1 markedly inhibited NF-kappaB promoter and down-regulated NF-kappaB expression.Taken together, TBMS1 is an efficient apoptosis-inducing agent for choriocarcinoma cells, which exerts its effects, at least partially, by the induction of mitochondrial dysfunction and regulation of p38/MAPK, ERK1/2 and PI3K/Akt signaling pathways.