Effects Of Gamma-Secretase Inhibitor (GSI) Block On The Pathway Of Notch1in Drug-resistant Colon Carcinoma Cell Line

Background and Objective：Colon carcinoma is one of the most common tumor in the digestivesystem. Metastasis and recurrence mainly due to drug resistance caused bychemotherapy. The resistance of tumor cells after standard chemotherapy isstill the problem of clinical treatment. The Notch signaling pathway is anevolutionarily conserved cell interaction, signal transduction pathways. Itaffects cell differentiation, development and apoptosis. In recent years, it isfound that Notch signaling pathway is closely related with the occurrenceand development of many tumors. Notch abnormal expression is detected inmany tumor cell lines.Notch pathway plays an important role in themaintenance of tumor phenotype. γ-secretase enzyme is an importantregulator of notch signaling pathway. GSI blocks the third step of digestionreaction of Notch1, resulting in that the NOTCH1intracellular domain(NICD) can not be released, and thus affect the proliferation anddifferentiation of cells. The study is to establish oxaliplatin-resistant coloncancer sw480/L-OHP, evaluate expression of Notch1and effects of GSIblock on the pathway of Notch1in drug-resistant colon carcinoma cell line. Methods:Part1Establishment and characterization of resistant colon cancer cellssw480/L-OHP.1. Oxaliplatin resistant colon cancer cell line sw480/L-OHP wasestablished in vitro by continuous exposed to oxaliplatin (L-OHP) of lowand gradually increased concentrations.2. Resistance index of the drug-resistant cell lines to L-OHP wasdetermined by CCK8assay.3. The mRNA expressions of notch1, cyclinD1and survivin genes insw480/L-OHP were detected by using reverse transcriptase polymerasechain reaction (RT-PCR).4. The expressions of NOTCH1were detected by Western blot.Part2Effects of gamma-Secretase Inhibitor (GSI) block on thepathway of Notch1in drug-resistant colon carcinoma cell line.Then sw480/L-OHP were divided into4groups,including: GroupA(control group),Group B(L-OHP alone group),Group C(GSI alone group)and Group D(GSI conbine with L-OHP group).1. Inhibition ratios of groups were determined by CCK8assay.2. Apoptosis ratios were measured by flow cytometry.3. The mRNA expressions of notch1,hes1,cyclinD1and survivingenes were detected by RT-PCR.4. The expressions of NOTCH1, NICD and HES1were detected by Western blot.Results:Part1Establishment and characterization of resistant colon cancer cellssw480/L-OHP.1. Oxaliplatin resistant colon cancer cell line sw480/L-OHP wasestablished successfully.2. The L-OHP resistance index（RI）of sw480/L-OHP cells to sw480was16.36.3. The mRNA expressions of notch1, survivin and cyclinD1insw480/L-OHP cell line were up regulated.4. NOTCH1was overexpressed in protein level.Part2Effects of gamma-Secretase Inhibitor (GSI) block on thepathway of Notch1in drug-resistant colon carcinoma cell line.1. GSI alone may inhibite growth of sw480/L-OHP, GSI conbine withL-OHP inhibite growth of which much more obviously.2. In GSI groups,mRNA expressions of hes1, survivin and cyclinD1were dowm regulated.3. NICD and HES1were inhibited in protein level.While Notch1remained unchanged.Conclusion:Blockade of Notch Signaling by GSI hindered from NICD to release,inhibited expression of Hes1and growth of sw480/L-OHP.GSI enhanced the sensitivity of sw480/L-OHP cell line to L-OHP.The results of the currentstudy provide evidence of a synergistic relation between GSI and L-OHP inboth inhibition of cells growth and induction of apoptosis．...