Research On The Mechanism Of Colon Cancer Chemo-sensitivity Through The P21^WAF1/CIP1 Signaling Pathway

Objective:To detect the protein expression of p21WAF1\CIP， p53, MDM2and theubiquitination of p21WAF1\CIPin stage III colon cancer tumor tissues byimmunohistochemical staining technology, and then study the relationship of thep21WAF1\CIPexpression with prognosis and survival the during the follow-upobservation in the postoperative adjuvant chemotherapy colon cancer patients.Further to detect the effects of chemotherapeutics (5-FU, L-OHP, CPT-11, CDDP)on the cellular proliferation inhibition and drug sensitivity, and to detect the proteinexpression of p21WAF1/CIP1, p53and MDM2in P21cell signal pathway in HCT-116human colon cancer cell line. Preliminary discuss the mechanism of p21WAF1\CIPpathway regulating the chemo-sensitivity in colon caner.Methods:This study is divided into two parts; One part: clinical immunohistochemicalstaining: collect our postoperative pathologic stage for Dukes C colon cancer patientsfrom1th Jan,2007to31th Dec,2010.And then selecting20cases from those patiensrandomly.Follow ing up the selecting20cases to1th May,2014. Inclusion criteria:the postoperative pathological diagnosis Ⅲ colon cancer patients; Postoperativeadjuvant chemotherapy patients; KPS score80points or more, or ZPS score3pointsor less. Exclusion criteria: the preoperative chemotherapy patients; Patients withsevere systemic disease or other primary tumor; Women during pregnancy; The other.Pathological specimens collected by double blind method. Detecting the expressionof p21WAF1/CIP1,p53, MDM2and ub of tumor tissue by immunohistochemical staining.Follow up with the selecting20cases to1th May,2014to evaluate of DFS. The otherpart is human colon cancer cells in vitro experiment: HCT-116Western blot methodto detect p21WAF1/CIP1, p53, MDM2expressed in colon cancer cells. Determinedby MTT method to detect chemotherapy drugs (5-FU, DDP, LOHP, CPT-11)impact on colon cancer cell proliferation inhibition and drug sensitivity analysis;Annexin V-FITC/PI (flow cytometry) detecting various effective impact on colon cancer cell apoptosis rate.Results:1. clinicopathological immunohistochemical study: stage Ⅲ colon cancerpatients with cancers p21WAF1/CIP1and p53expression has an obvious positivecorrelation, the correlation coefficient r=0.995, p=0.00, was statistically significant;And is associated with pathologic type, tubular adenocarcinoma than other types ofhigh expression, statistically significant (p=0.012); A negative of p21WAF1/CIP1positive expression: p=0.032, risk than HR=1.592, there is statistical significance,p21WAF1/CIP1has a tendency to extend the postoperative chemotherapy in patientswith long-term Disease Free Survival.2. human colon cancer cells HCT-116for5-Fu, L-OHP, CPT-11andCDDP chemotherapy drug sensitivity test:Determined by MTT method to detect5-Fu, L-OHP, CPT-11and CDDPchemotherapy drugs of HCT-116proliferation inhibition: in each of thechemotherapy drugs on human colon cancer cells IC50concentration of HCT–116.The results have no significant differences (P>0.05) L-OHP group comparingwith the CPT-11group. The results have no significant difference (P>0.05) CPT-11comparing with5-Fu group.The results have no significant difference (P>0.05)5-Fu group comparing with CDDP group.The results have no significant difference(P>0.05) CDDP group comparing with L-OHP group,.FCM double fluorescence detection of5-Fu, L-OHP, CPT-11and CDDPchemotherapy drugs of HCT-116cells apoptosis: CDDP group total apoptosis rate is7.5%, the early apoptosis rate is1.40%.L-OHP group apoptosis rate is32.5%, theearly apoptosis rate is11.21%. CPT-11groups of HCT-116cell apoptosis rate is47.4%, total early apoptosis rate is24.6%.5-Fu group of HCT-116cell apoptosisrate is27.9%, total early apoptosis rate is5.71%.3.5-Fu, L-OHP, CPT-11and CDDP chemotherapy drugs on the mechanismof action of human colon cancer cells HCT-116: L-OHP, CPT-11,5-Fu, andCDDP chemotherapy drugs on P21and P53in HCT-116cells (DO-1), theinfluence of MDM2protein expression (western blot method) western blot, raised theexpression of P21protein, decrease the expression of MDM2protein, with the function of p53protein is first (48h) enhance the p53protein expression, fade out (72h) after the protein expression.Conclusion:1. clinicopathological immunohistochemical study: stage Ⅲ colon cancerpatients with carcinoma tissue p21WAF1/CIP1and p53expression has an obviouspositive correlation, and is associated with pathologic type, tubular adenocarcinomathan other types of high expression, and has a tendency to extend the postoperativechemotherapy in patients with long-term survival.2. human colon cancer cells HCT-116for5-Fu, L-OHP, CPT-11andCDDP chemotherapy drug sensitive experiment: each can drugs can significantlyinhibit cell proliferation in vitro cultivation of HCT-116, and has obviouscytotoxicity, but no significant statistical differences between the chemotherapydrugs.3.5-Fu, L-OHP, CPT-11and CDDP chemotherapy drugs on the mechanismof action of human colon cancer cells HCT-116: increase the expression of P21protein, decrease the expression of MDM2protein, is to enhance the function of P53protein P53protein expression, weakened after the protein expression.In a word: colon cancer cells to chemotherapy drugs sensitivity associated withp21signaling pathways activated, chemotherapy drugs by raising p21and P53protein expression, inhibition of MDM2protein expression, thus inducing tumor cellapoptosis, and lengthen Ⅲ chemotherapy postoperatively in patients with coloncancer survival trend.