Curcumin Inhibits Proliferation Of Human Colon Adenocarcinoma Cell Line HCT116through Down-regulating Notch1Signaling And Its Mechanism

ObjectiveThe first part to test Notch1expression in human colorectal adenocarcino-ma, The second part to discuss its clinical significance.To reaserch themechanism of curcumin effects the cell proliferation of human colorectaladenocarcinoma cell line HCT116of wildtype-P53in vitro.MethodsUse immunohistochemical the EnVision method detects100cases ofcolorectal carcinoma and30cases of adjacent normal colorectal tissues paraffinsections of Notch1expression,determine its relationship with human colorectaladenocarcinoma. The experimental cells are divided into blank control, solventcontrol and20μmol/L,40μmol/L curcumin group, RNA interference Notch1groups are divided into blank control,negative control, interference of Notch1,20μmol/L curcumin, and interfere Notch1adds20μmol/L curcumin group(dualsuppression group).Cell morphology was observed in optical microscope, MTTand flow cytometry to observe the proliferation and cell cycle distribution,Western blot was used to detect the expression of related protein expression.ResultsNotch1expresses in both human colorectal adenocarcinoma and adjacentnormal tissue,the positive rates were78%and40%,the difference wasstatistically significant(P <0.01),futher statistical results shows that it was relatedto tumor location,differentiation degree and Dukes stage,but not to gender, age,tumor size and lymph node metastasis.(P﹥0.05).The experimental cellswere added in different treatment factors after24h, large floating cells of20μmol/L,40μmol/L curcumin groups can be seen in visual observation ascompared with the control group under light microscope, the edge of adherentcells were round and its number are significantly reduce. MTT experiment wasobserved that with the increase of curcumin concentration and action time, theabsorbance decreased and the growth inhibition rate increased graduallycompared with the control group.Flow cytometry showed that curcumin grouphas G1block, the percentage of S phase of40μmol/L curcumin group hasminimum.Western blot showed the expression of Notch1protein content ofblank control and solvent control groups were higher, its expression decreasedafer with20μmol/L curcumin and40μmol/L,and it decreased more with theincrease of curcumin dose.Expression of cyclin proteins CyclinD1and CDK4protein content showed the same trend with Notch1. While the P53and P21areopposite, the expression content of blank control and solvent control groupsare less,20μmol/L and40μmol/L curcumin group increases gradually. Thesame experiments were performed RNA interference Notch1to comparison,MTT showed that20μ mol/L curcumin group was decreased significantlycompared with the control group, the intervention group was also decreased buthigher than20μ mol/L curcumin group.20μ mol/L curcumin plus Notch1groupwas the lowest interference. Flow cytometry showed that interference groupalso has G1phase arrest, but the effect is smaller than20μ mol/L curcumingroup, dual suppression group in S phase of the smallest proportion. Westernblot showed that the expression of Notch1protein was higher in control group,the expression of20μ mol/L curcumin group was decreased, the interventiongroup and20μ mol/L curcumin adds interfere Notch1group were not detected.the expression of cycle related protein CyclinD1and CDK4have the sametrend.the expression of P53and P21protein protein increased gradually.theresult of intereference Notch1group suggested that down-regulation Notch1was only one part of the pharmacologic effects of curcumin,it is also related toother unknown pathways. ConclusionsNotch signaling pathway may exists as an oncogene in the development ofhuman colorectal adenocarcinoma.Curcumin can inhibit the proliferation ofwildtype-P53human colorectal adenocarcinoma cell line HCT116and itsmechanism might through the down-regulation of Notch1, up-regulation of P53and P21and then down-regulation of CyclinD1and CDK4, thereby inhibiting cellcycle activity.