In Vitro And In Vivo Inhibitory Effect Of Cucurbitacin B On The Growth Of Hepatoma Cancer Cells

IntroductionSignal Transducer and Activator of Transcription(STAT)constitutes a family of signal transduction proteins that are activated in the cytoplasm by the binding of extracellular polypeptides to transmembrane receptors.In addition to physiological functions,increasing evidences are supporting an important role for STATs in tumorigenesis,especially for STAT-3.It has been reported to be constitutively active in different types of malignant tumours.Activation of STATs results in the expression of genes that control critical cellular functions such as cell proliferation,differentiation and development.Cucurbitacin B treatment downregulated both STAT-3 activity and inhibited cell proliferation in different types of malignant tumours.Until now there is no detailed report of Cucurbitacin B on the inhibitory effect of hepatoma cells and its possible mechanisms.To evaluate the in vitro and in vivo antitumor effect and the mechanisms of Cucurbitacin B on human hepatoma cell line HepG-2,we treated the cells with different doses of the Cucurbitacin B for different time.The MTT assay, flow cytometry,fluorescent microscopy,western blot,nude mouse xenoplastic transplantation were used.Methods1.MTT assay investigating Cell proliferation5×10~3 HepG-2 cells(100μ1)/well were grown in 96-well plate for 24 h and the cells were further incubated with different concentrations of Cucurbitacin B in RPMI-1640 supplemented with 10%FBS medium and solvent control for 24 h,48 h and 72 h.At least three reproducible experiments were performed with three replicate wells per concentration.2.Cell morphology observationHepG-2 cells are treated with 0.01μM,0.1μM,1μM Cucurbitacin B for 24 h, Morphologic changes were evaluated under light microscope.3.Fluorescent microscopyHepG-2 cells(5×10~5)were grown on coverslips placed into six-well plate and allowed to attach overnight for 24 hours.Cells were treated with the indicated concentrations of Cucurbitacin B.After 24 hours,Cells were washed in PBS and fixed by methanol and ethanoic acid(3/1,v/v)at 4℃for 15 minutes.Preparations were stained with Hoechst 33258(5μg/ml final concentration)for 30 min in the dark. Microscopic observations were performed and photographed using a fluorescence microscope.4.Flow cytometryHepG-2 cells(1×10~6)were seeded in six-well plates and allowed to attach overnight.Exponentially growing cells were treated with the indicated concentrations of Cucurbitacin B and were harvested by 0.25%trypsin at the indicated time.The cells were fixed in 70%ethanol overnight at 4℃.Before DNA analyses,The cells were treated with 50μg/ml RNase,and stained with 100μg/ml propidium iodide in the dark. Flow cytometry analyses were carried out on a FACScalibur instrument using the ModFit program.5.Annexin V-FITC/PI staining assayApoptosis was detected with an Annexin V-FITC/Propidium iodide(PI)detection kit.5,000 cells per sample were analyzed with a FACScalibur flow cytometer.6.Western blotWhole cell Proteins were extracted,loaded at 100μg per lane on 10%(w/v) SDS-PAGE.Protein bands were transferred to NC membranes,blocked in 5%nonfat milk.Membranes were probed with Polyclone antibody against PhosPho-STAT3 which was then incubated with secondary antibody.Immunoreactive bands were visualized by ECL.7.Antitumor activity in the nude mouse tumor xenograft modelHepG-2 cells were harvested,resuspended in NS,and injected subcutaneously (s.c.)into the right and left flank(5×10~6 cells per flank)of female nude mice.When tumors reached about 300mm~3,inoculate to another nude mice.Repeat twice,then inoculate 50 mice,when tumors reached about 100mm~3,picked up 36 mice, randomized(6 animals per group)and dosed(i.v.)either with Cucurbitacin B(27.5 or 55 or 110μg/kg/d)in NS or with an equal volume of vehicle control,5-FU was served as positive control.The tumor volumes were determined by measuring the length(1) and the width(w)and calculating the volume.8.Statistical analysisThe software SPSS13.0 was used in the statistical analysis.Results were expressed as mean value±standard deviation.The t-test were for evaluating statistical significance,where a P value less than 0.05(P＜0.05)denotes statistical significance.Results1.Effect of Cucurbitacin B on HepG-2 cell viabilityHuman hepatoma cell line HepG-2 were treated with 0.1-100μM of Cucurbitacin B for 24-72 hrs and the cell viability was measured by MTT assay as indicated in the methods.The treatment of human hepatoma cell line HepG-2 with Cucurbitacin B resulted in a dose-and time-dependent inhibition of cell viability.2.Cell morphology observationHepG-2 cells were treated with 0.01μM,0.1μM,1μM Cucurbitacin B for 24 h, morphologic changes were evaluated under light microscope.Control cells showed typical morphology,part of the Cells after 0.01μM treatment from their typical morphology to round-shaped cells,brightly,cell number decreased,debris can be observed,all the cells after 1μM treatment from their typical morphology to round-shaped cells,brightly,cell number decreased obviously,more debris can be observed.3.Nuclear morphological changesAfter cells were treated with 0μM,0.01μM,0.1μM Cucurbitacin B for 24 h,marked morphological changes of cell apoptosis such as condensation of chromatin, nuclear fragmentations and apoptotic bodies were found clearly using Hoechst 33258 staining.Apoptotic cells gradually increased in dose-and time-dependent manner in HepG-2 cells.4.Effect of Cucurbitacin B on HepG-2 cell cycle and apoptosisThe percentage of S phase cells was increased in a dose and time-dependent manner.24 h exposure of HepG2 cells to 0.1μM,1μM,10μM Cucurbitacin B caused a significant increase in the population of S phase cells(from 30.3%to 42.0%). Treating HepG2 cells with 10μM of Cucurbitacin B for 0,4,8 and 24 h resulted in similar changes.There appeared increase in the proportion of apoptotic cells in samples treating with 1μM and 10μM of Cucurbitacin B compared with control5.Protein expressions of phospho-STAT3 in HepG-2 cell line detected by western blotProtein expressions of phospho-STAT3 in HepG-2 cells decreased as concentration(≥1μM)and time(≥4 h)increase.6.Cucurbitacin B inhibits transformed HepG-2 tumor growth in nude miceCompared with 532±104 mm~3 of control group,the average tumor volume of 27.5,55,110μg/kg/d Cucurbitacin B group were 436±82 mm~3(P＜0.05),309±109 mm~3(p＜0.01)and 202±33mm~3(p＜0.01).In vivo experiment showed that Cucurbitacin B suppressed tumor growth in a dose dependent manner.DiscussionSeveral studies were published pointing out that Cucurbitacin B exhibits inhibitory activities against numerous human cancer cell lines and tumour xenografts. In the present study,we examined the inhibitory effect of Cucurbitacin B on human hepatoma cell line HepG-2.Cucurbitacin B exhibited growth inhibitory activity of human hepatoma cell line in a dose-and time-dependent manner as can be seen by MTT assay and in vivo test.In order to elucidate the mechanism by which Cucurbitacin B inhibited HepG-2 cell growth,we performed cell-cycle analysis,cell morphology observation,cell nuclear morphology observation and Western Blot analysis.Cucurbitacin B induced caused cell cycle arrest in a dose-and time-dependent manner and induced cell apoptotis.Marked morphological changes of cell apoptosis including condensation of chromatin,nuclear fragmentation and apoptotic bodies were observed clearly.Protein expressions of phosphorylated STAT3 in HepG-2 cells can be suppressed by Cucurbitaein B.In conclusion,the results of the present study indicate that Cucurbitacin B effectively inhibited viability of human hepatoma cells by cell cycle arrest and apoptosis.Its molecular mechanism by which Cucurbitacin B induce HepG-2 cells cell cycle arrest and apoptosis may concern with its effect of inhibitting the activity of STAT3.ConclusionsOur results have demonstrated that Cucurbitacin B can inhibit HepG-2 cell proliferation through cell cycle arrest and induced cell apoptosis,suggesting that Cucurbitacin B may provide a potential alternative for treating HCC.