Study Of Effect Of Flavonol From Euphorbia Humifusa Willd On Anticancer And Its Mechanism

Cervial cancer is the second common malignant tumor in woman. Treatmen ofcervical cancer consists of operation, radiotherapy and chemotherapy with poor prognosisat present. Research and development of natural-derived agents have attracted moreattention and some drugs have been used for therapy of cervical cancer in clinic.Euphorbia Humifusa Willd(EHW), as a tradition Chinese herbs, contains manytyapes of active compounds. It has obvious anti-inflammation and immune regulatoryeffect. In previous study, we used the aqueous extract of Euphorbia humifusa to treat U14cervica cancer bearing mice. The result showed that tumor growth in mice was inhibitedand immune level was enhanced significantly. Whereas, it is still unknown how theflavonol from EHW show the effect of anti-tumor as a main active compounds, especiallyits pharmacologic mechanism on cervical cancer. In this study, Euphorbia Humifusa Willdflavonol(EHWF) was extracted and pured and followed by a series of analysis. Finally,anti-tumor mechanisms were evaluated through treatment of U14cervica cancer bearingmice and Hela cells with the EHWF in vivo and in vitro.EHWF was extracted by organic solvent combined with column chromatography.EHWF was obtained as a brown powder with rate of1.41%, content of0.14mg/g.In vivo experiments, U14cervica cancer bearing mice were randomly divided intoEHWF low dose group (100mg/kg), EHWF high dose group (200mg/kg), the controlgroup and CTX group randomly. The effects of EHWF on U14cervical cancer bearingmice and its likely mechanism were studied by using hematoxylin eosin stain (HE),immunohistochemistry SP method, Enzyme Linked Immunosorbent Assay (ELISA), Flowcytometry (FCM), fluorescence staining, Reverse Transcription-poly Merase ChainReaction (RT-PCR) as well as Western blot method. Regulation of the immune function,inhibition of cell cycle and induction of cell apoptosis were also reseached. The resultswere as following:After treatment of tumor mice with EHWF for14days, EHWF treatment by gavagewith dose of100mg/kg and200mg/kg significantly inhibited the tumor growth of U14 tumor-bearing mice (p<0.05), and the tumor inhibition rate reach to40.17%and55.06%,respectively. EHWF decreased the positive cell numbers which expression of gene ofmutant p53, Bcl-2, CerbB-2and Ki-67proteins in tumor tissues (p<0.05). Vital signs wereimproved obviously without liver and kidney injury as well. Cell cycle analysis showedthat EHWF increased the numbers of tumor cells in G0/G1phage (p<0.01), and appearedapoptotic peak of Sub-G1at rate of20.11%(high dose group), which demonstrated thatEHWF can arrest the cell cycle at G0/G1phase in apoptosis. Meanwhile, the expression ofgene Bcl-2were downreguated (p<0.01), while the expression of Bax, Fas, FasL,caspase-8and caspase-3were upregulated compared with the control group (p<0.01) atthe level of description and translation, which indicated that the mechanisms of apoptosismay be regulated through the triggering of death-receptor pathway and themitochondrial-dependent pathway with result of activation of caspase-3. Duing theinhibition of tumor cells, the expression p16upregulation and C-myc and PKCdownrugulation indicated that gene C-myc and PKC and p16played a key roles in theprocessing of apoptosis induced by EHWF.The thymus index and the spleen index of U14tumor-bearing mice increasedsignificantly after treatment of EHWF(high dose group) in U14mice(p<0.05). The activityof NK cells increased (p<0.01) and the ability for splenocyte proliferation increased too(p<0.01). Moreover, the number of CD4+T lymphocytes subsets (p<0.01) increased as wellas the ratio of CD4+/CD8+T cells in peripheral blood after treatment of EHWF (p<0.01).EHWF can inhibited the growth of tumor by promoting the level of IL-2and TNF-α inserum (p<0.05).In Hela cell experiments in vitro, the IC50values of EHWF were212.46μg/mL in48h. Not only cells growth could be inhibited, but also activities of Caspase-8and Caspase-3were significantly increased. The typical morphological ultrastructure characteristics ofapoptosis appeared under the electron microscopic in Hela cells.Cell cycle test showedthat EHWF can influence on DNA synthesis and replication in tumor cells. The tumorcells were blocked in phase of G0/G1, and normal cells division were inhibited. So theinhibition of cells proliferation induces the apoptosis in tumor cells.EHWF treatment of Hela cells increased the number of apoptotic cells, and the apoptotic rate at dose of400μg/mL reached to36.62%by Annexin V-FITC. DNA“ladder”zone in the agarose gel electrophoresis appeared after treatment of EHWF in Hela cells.EHWF can also arrested cell cycle in G0/G1phase. The expression of gene of Bcl-2andBax and and CyclinD1and p16changed after treatment of EHWF (p<0.01), whichindicated that the mechanism of apoptosis was associated with the death recptor and themitochondrial pathway directedly, as well as the cell cycle.In our research, U14cervica cancer bearing mice and Hela cell were acted asexperimental material in vitro and in vivo. Effect of EHWF was systematically evaluatedthrough analysis of cytokines, proteins, ultrasructures and apoptosis by methods of SP,ELISA, MTT, TEM, western blot, FCM, RT-PCR after treatment of EHWF. The antitumor effects of EHWF and its mechanisms were researched deeply, which provided atheoretical basis for utilization of natural medicines in cervical cancer therapy.