Norcantharidin Induced Colon Cancer LS-174T Cell Apoptosis And Its Mechanism

Objective: The human colon cancer LS-174T cells as the researchobject, explore norcantharidin induced colon cancer LS-174T function andmolecular mechanism of cell apoptosis,provide experimental basis for thetreatment of colon cancer.Methods: human colon cancer cell lines LS-174T cells in containing10%fetal bovine serum,100u/ml and100ug/ml penicillin streptomycinRPMI1640culture based on the temperature of37℃5%CO2incubator intraining,when the cells adherencing,take the logarithmic phase cell to test.Inthe following experiments:1Detecting the norcantharidin on LS–174T cell growth inhibition byMTT methodTake the logarithmic growth phase of LS–174T cells,to digest it intosingle cell suspension,adjust the final concentration of1x105cells/ml,vaccination in96-well culture plate, respectively,to join the massconcentration of5,10,20and40ug/ml norcantharidin, meanwhile set up blankgroup (DMSO solvent control group) and the negative control group (groupwithout drugs).Dosing role24,48,72h,20ulmtt per hole to join (5mg/ml), theincubator in continue to train4h,abandon the supernatant, Ascent enzymeD490value standard instrument detection,cell growth inhibition ratecalculation,to map the cell growth curve.2Cell morphology under fluorescence microscope observationThe logarithmic phase of LS–174T cells digest,vaccination in6orificeto climb,24h after being adherent cell growth,cultivate to culture,respectively,to join the mass concentration of10and40ug/ml norcantharidin,at the sametime a negative control group,and in the incubator to continue training after24h,rapidly under the fluorescent microscope, and taking photos.3Observe the changes of the ultrastructure of cells under transmission electron microscopy (tem).Collect colon LS–174T cells when they have effected by the0,10and40ug/ml norcantharidin after24hours,centrifuge for10min,along the wall tojoin the2.5%glutaraldehyde,4℃save,add1%osmic acid fixed4℃,usinggradient acetone dehydration,drenched in Epon618purified resin embedding,after polymerizationin the oven,with a Leica mechanism of ultra-thin slicedinto slices,after both uranium acetate and lead citrate staining,withtransmission electron microscope and photographed.4Flow cytometry AnnexinV-FITC/PI staining methodCollect the negative control group and drug group (5,10,20,40ug/ml)cells after24h, collecting cells,with heavy precooling PBS suspension cells,2000RPM centrifuge for10min,washing cell, add in300ul Binging Buffersuspension, add5ul AnnexinV-FITC after blending,avoid light,incubation atroom temperature for15min,5min before computer to join5ul PI,upflow celltest.5Detect the stat-3,survivin, Mcl-1,bax and protein expression byWestern blotCollect the control group and drug group (5,10,20,40ug/ml) cells after24h,add protein cracking fluid respectively,determine the protein concentrationby Bradford method, each sample hole join50ug protein,12%sds-pageprotein isolated,transferring protein onto PVDF membrane,37°C closed1h,join1%BSA1:500diluted survivin,stat–3,Mcl-1and bax polyclonalantibody,room temperature oscillation5h,after washing membrane,fluorescent two resistance (resistance to rabbit lgG) avoid light1h incubation,wash the membrane,glow.Results:1The inhibiting effect of norcantharidin on colon LS–174T cellDetermined by MTT results showed that norcantharidin have obviousgrowth inhibition on colon cancer LS–174T cell,and have the time-dosedependence,drug group of NCTD (5,10,20,40ug/ml) role after24h,theinhibition rate are (9.04±2.4)%,(18.5±3.5)%,(40.1±5.8)%,(58.2±2.7)%.Role after72h,inhibition rate are(18.14±7.4)%,(36.7±4.3)%,(49.9±3.6)%,(72.4±3.1)%(n=3, x±s).The maximum inhibition rate appear at40ug/ml acting72h.2The AO/EB observe cell morphologyNormal cell groups are uniform green dye;Drug group significantlyincreased with the increase of drug concentration in the apoptosis rate,10ug/ml drug group cell volume decreases, cytoplasm decreased, he nucleuspycnosis,chromatin concentration, apoptotic cells increased;40ug/ml druggroup of visible nucleus fragmentation,and apoptotic bodies,a typical "ghost"of the cell3Transmission electron microscopy (sem) observed the changes of theultrastructure of cellsThe negative control cells,The nuclei are large,dual-core and each has abig and obvious nucleolus,some slender projections on the cell surface,10ug/ml norcantharidin effect after24,Cell microvilli decreased,nuclearshrinkage,nuclear pyknosis,electron density increases.40ug/ml norcantharidineffect after24h,nuclear membrane rupture, nuclear chromatin outside into thecytoplasm.4Streaming AnnexinV-FITC/PI staining analysis cell apoptosis rateFlow cytometry instrument AnnexinV-FITC/PI staining method todetect apoptosis rate of drug group and the negative control group,the resultsshow that compared with negative control group,different drug concentrations(5,10,20,40ug/ml) NCTD effect after24h,cell apoptosis rate increasedsignificantly,and have a dose dependence,apoptosis rate exists obviousdifference between the different drug group (P <0.05).5Western blot method detection of stat3,survivin,Mcl-1and baxprotein expressionThe negative control group and drug intervention group (NCTD5,10,20,40ug/ml) after24h,detecting the expression of stat3,survivin,Mcl-1and baxprotein.Results showed that expression of stat3,survivin and Mcl-1reduced,the expression of bax increased,and the expression also increases with the inc- rease of drug concentration,show obvious dose relationship.Conclusions:1Norcantharidin has significant growth inhibition on colon LS-174Tcells, And rendering time-dose dependen time-dose relationship.2Norcantharidin can inhibit colon cancer by increasing apoptosis LS-174T cell growth.3With the increase of concentration of NCTD,the expression of stat3,survivin,Mcl-1reduced,the expression of bax protein increased,and also theincreases expression with the increase of drug concentration,show obviousdose relationship,its mechanism may be through the inhibition of stat3pathway.