Study On The Anti-hepatoblastoma Mechanism Of Dichloromethane Extract Of Fresh Earth Fritillaria

Hepatoblastoma(HB)is the commonest hepatic malignancy in children,its inci-dence is 1.2-1.5per million and most of them are under 5 years old.The major clinic-al therapy for HB are surgery and chemotherapy.The minority of the HB patients are diagnosed at an early stage that could treated with a complete resection(CR).Adjuva-nt chemotherapy is need to be applied if there is any rich factor detected after CR.For the patients that the tumor could not be resected completely,neoadjuvant chemothera-py would degrade its staging to a resectable one.Advanced stage of HB is threated with chemotherapy and best support cure.The overall survival was significantly improved due to the progresses in antineoplastic drugs and surgery.Chermotherapy not only promote the possibility of CR,but also improve the survival of patients with unresectable or metastatic tumor.But the side effects and adverse events of conventional chemo-therapy limited the improvement of clinical efficacy.The strategy now for chemotherapy is the management of dose of chemo drugs and the development of new antineoplastic drugs,which would decrease the adverse events and finally improve the prognosis.There are plentiful drugs in Traditional Chinese Medicine that have been proved with antineoplastic properties.Tubeimu[Bolbostemma paniculatum(Maxim.)Fran-quet.TBM]has been used to treat varied of diseases including malignancy for long.Resear-ches confirmed that the saponins in TBM inhibits various of cancer cells,and even induce them to apoptosis and cell cycle arrest.The substancesin the ichlorometh-ane extracted fresh Tubeimu(ETBM)are different from the extracts that did by previous researches.ETBM was proved to inhibit growth and metastasis of triple negative breast cancer both in vitro and in vivo which indicate that ETBM is a potential candidate for development of new antineoplastic drug.Hence,we applied ETBM in our study to investigate the mechanism and efficacy both in vitro and in vivo hoping to find new drug for the tragedy.We observed the morphologic changes in the HepG2 cell line,and applied cell proliferation assay,Western blotting,RT-PCR,immunofluorescence analysis,Flow cytometry analysis and xenograft to investigate the mechanism of ETBM in inhibiting HB.In our study,ETBM inhibits the growth of HepG2 cell line in a dose-depend manner and the IC 50 is 113.10�12.13?g/ml.In the cytoplasm of the cells that treated by ETBM there are a lot of vacuoles observed by optical microscope and autophago-somes and autolysome were observed by transmission electron microscope(TEM).Western blotting indicate the change in the expression of autophagy marker proteins including LC3 ? and p62(P<0.05).Further,immunofluorescence analysis tells the distribution and expression changes of them.There results betrayed that ETBM induced autopha-gy in the HepG2 cell line in a dose-depend manner.Flow cytometry analysis in our study for the detection of apoptosis and cell cycle arrest revealed that there was no evidence of early apoptosis or cell cycle arrest no matter 12 hours or24 hours ETBM in treating HepG2 in two concentrations.There is no apoptosis or cycle arrest accompanied when autophagy taking place.We hypothese that there might autophagic cell death(ACD)occur in the inhibition.To vernfy the hypothesis,we applied PIK-? to inhibit the autophagy induced by E TBM in the HepG2 cell line and found that there are more vacuoles either PIK-? applied alone or in combination with ETBM.Nevertheless,neither autophagosome nor autolysome could be observed by TEM.And more than that,the expression and distribution of LC3 ? and p62 was alter back to the level of blank control.According to the criterna of the Nomenc-lature Committee on Cell Death(NCCD)there were autophagic cell death induced by ETBM in the HepG2 cell line and ACD is the underlying mechani-sm.The HepG2 cell line was reported to be poor with tumorigenicity.We suspended the cells into PBS and PBS-Matrigel solution to compare the tumorigenicity.There were nearly 16.7%of PBS alone while 83.3%in the PBS-Matrigel solution.We grouped the established xenograft randormly and treated them with oxaliplatin,low dose of ETBM and high dose of ETBM respectively for four weeks.The highest inhibition rate was 56.73%in the oxaliplatin group,the inhibition rates of the ETBM groups were 34.93%and 37.88%(P>0.05).The body weight of the mice kept declin-ing of the oxalipatin group while there were no difference among the ETBM and control groups.These results betray that,ETBM inhibits the growth of HepG2 in vivo with good tolerance.The inhibition rate could be improved if there is an enhancement in dose that applied to the xenograft without obvious toxicity.There were ten major substances was identified in ETBM in our study.Interestingly,some of them have no cytotoxicity against tumor cell according to previous literatures.Stigmasterol and cucurbitacin E was reported to possess anti-cancer properties.Members of cucurbitacin family proved to induce autophagy in cancers.In our study,autophagy occurred,but whether induced by single antineoplastic substance or synergistic effect and the details still need further studies.Above all,ETBM inhibits the growth of HB both in vitro and in vivo with good tolerances.ETBM induces ACD in the HepG2 cell line to exert the inhibition.