The Effect Of Cinobufacini Injections On The Proliferation And Expression Of Topoâ…¡ In Hepatocarcinoma HepG-2Cells

Hepatocarcinoma(HHC) is one of the most common malignant tumorworld-widely. HHC belongs to chemotherapy nonsensitive cancer,clinical reports showthat the partial response to chemotherapy of HHC is less than20%up to now,and theefficacy of targeted antineoplastic drug on HHC,such as sorafenib doesn’t improvedsignificantly therapeutic efficacy in HHC.So the efficient drug against HHC isanxiously expected all the time.Cinobufacini injections(CINO) is a traditional Chineseantineoplastic drug.Clinical data showed it inhibit tumor growth in multiways.What’smore,it has impressive effect of immune promotion TopoisomeraseⅡ(TopoⅡ) plays animportant roles in DNA replication,transcription and recombination,and acts as keyantineoplastic target in clinical practice.Objective:In this project,we observe the effect of CINO on the proliferation andexpression of TopoⅡin HepG-2cells to illustrate the inhibiting mechanism of CINO inHHC.Methods:1.CCK-8method was used to analyse proliferation of HepG-2cells after12h,24h, and48h impactence of CINO on the cells with different concentrationsrespectively.The experiment was repeated three times.2.FCM technique was used to analyse apoptosisn of HepG-2cells after24h and48h impactence of CINO on the cells with different concentrations respectively.Theexperiment was repeated three times.3.FCM technique was used to analyse cycle of HepG-2cells after24h impactenceof CINO on the cells with different concentrations respectively.The experiment wasrepeated three times.4.RT-PCR was used to analyse the mRNA expression of TopoⅡ in HepG-2cellsafter48h impactence of CINO on the cells with different concentrations respectively.The experiment was repeated three times. 5.Western Blot was used to analyse the protein expression of TopoⅡ in HepG-2cells after24h impactence of CINO on the cells with different concentrationsrespectively.The experiment was repeated three times.Results:1.The CCK-8analysis shows that CINO inhibits significantly HepG-2cells proliferation in dose and time dependent ways.2.The FCM analysis shows that CINO promotes significantly HepG-2cellsapoptosis in dose and time dependent ways.3.The FCM analysis shows that CINO blocks significantly the HepG-2cells cyclein S phase in dose dependent ways,compares with the control,P<0.05.4.Rt-PCR analysis shows that CINO decreases significantly the mRNA expressionof TopoⅡ in HepG-2cells in dose dependent ways.5.Western Blot analysis shows that CINO decreases significantly the proteinexpression of TopoⅡ in HepG-2cells in dose dependent waysConclusion:Cinobufacini injection inhibits significantly HepG-2cellsproliferation,promotes the cells apoptosis in dose dependent ways;blocks the cells cyclein S phase;decreases the mRNA and protein expression of TopoⅡ in dose dependentways,is a promising medicine on HHC treatment.