A Study On The Effect And Mechanism Of Nimotuzumab Combined With Chemotherapeutic Agents On Human Colon Cells And The PI3K/Akt Pathway

IntroductionColorectal cancer(CRC) is the one of the most common cancers in the world.In the year 2000,900,000 new cases of colorectal cancer were diagnosed worldwide, causing 490,000 deaths.Colorectal cancer accounts for 10-15%of the newly diagnosed cancer cases each year.In our country,especially in big cities,the yearly incident of colorectal cancer increases by 4.2%on the average.While the early stage colorectal cancer is generally curable with surgery,the unresectable metastatic disease accounting for 40～50%of the total such diseases is unexceptionally fatal,to which adjuvant therapy and palliative care are available methods that can help to improve efficacy and patients' life quality.Adjuvant and palliative treatments for colorectal cancer mainly involve fluorouracil(5-Fu)-based chemotherapy,irinotecan(CPT-11) and oxaliplatin(L-OHP) chemotherapy.However,when the patients become refractory to these cytotoxic agents,there are essentially no options of established treatment of demonstrated efficacy.There is a clear need for new and improved therapies for this kind of disease.The last few years have witnessed a growing interest and significant advances in the use of targeted therapy for colorectal cancer.Epidermal growth factor receptor(EGFR) belongs to the ErbB/HER family of receptors that can bind its ligand.Ligand binding induces autophosphorylation and the formation of homo or heterodimers with other members of the family.Receptor phosghorylation mediated the activation of the mitogen-activated protein(MAP) kinase,the phosphatidylinositol 3 kinase(PI3K)/Akt,the phospholipase C gamma (PLC-c)/protein kinase C(PKC) and the signal transducer and activator of transcription(STAT) pathways,which in turn regulate cell's transformation, proliferation and survival.PI3K/Akt signaling is associatedwith both cell proliferation and apoptosis,and plays a major role not only in tumor growth but also in potential response of a tumor to cancer chemotherapy.Specific inhibition of the activation of PI3K/Akt signaling may overcome the resistance to chemotherapy of cancer cells.Therefore,combination of inhibitorwith various cytotoxic drugs enhance the effectiveness of the treatments,and suggested that the PI3K/Akt can be considered as the potential target for tumor therapy.An overexpressing of EGFR (about 25～80%) has always been found in patients with colorectal cancer and was shown to be associated with a poor prognosis and resistance to conventional therapies. The studies in recent years have mainly focused on the application of anti-epidermal growth factor receptor in oncology field.Cetuximab,a chimeric IgG1 monoclonal antibody targeting EGFR,has been approved of satisfactory effects on patients with EGFR-expressing metastatic colorectal cancer when used alone or in combination with chemotherapy or radiotherapy in clinic,having effectively improved the median survival time of patients with advanced colorectal cancer.Couples of clinic trials showed that it can inhibit tumor cell proliferation.However,no relation between cetuximab therapeutic effect and EGFR expression of tumor was found;what's more, EGFR expression isn't a maker of determining whether or not cetuximab treatment is practicable oapplication and response prediction.Nimotuzumab(h-R3),a humanized monoclonal antibody(MAb)(IgG1 isotype) that recognizes an epitope located in the extracellular domain of human EGFR,is widely recognized for its high affinity,high specificity and prolonged half-life.The antibody blocks EGF binding to the receptor and inhibits its intrinsic tyrosine kinase activity.In a preclinical study,h-R3 showed marked antiproliferative,proapoptotic and antiangiogenic effects on tumors overexpressing EGFR.A Phase-â…¡clinical study has demonstrated the activity of combination with irinotecan in patients with irinotecan-refractory metastatics colorectal cancers.Similarly,a study,conducted in Canada designed to evaluate the activity of irinotecan plus h-R3 in patients with colorectal cancer refractory to irinotecan,found that h-R3 can reverse irinotecan resistance,when combined with iriinotecan can improve survival time.Given that little is known of the action of h-R3 plus others chemothraputic drugs.To evaluate the effect and mechanism,we conducted a study on human colorectal cells expressing different levels of EGFR and explored the activity of PI3K/Akt pathway in these cells.Objective:To investigate the inhibitory proliferation effect of nimotuzumab combined with chemotherapeutic drugs on human colon cell lines expressing different level of EGFR and whether the responses are correlated with the level of EGFR expression,and to study the possible molecular mechanisms by detecting the molecules associated with cell signaling pathway.Methods:Chapter One:Inhibitory effect of nimotuzumab combined with chemotherapeutic drugs on human colon cell lines in vitro1.Flow cytometric analysis of surface EGFR expression of LoVo and Colo205.2.Cells were cultured in RPMI 1640 containing 10%fetal calf serum.Exponentially growing cells were chosen for experiment.Cells were divided into 4 groups:control group,nimotuzumab-treated group,chemotherapeutic drugs-treated group(5-fluorouracil, irinotecan and oxaliplatin) and combination treatment group.Cells were incubated with nimotuzumab(6.25,12.5,25,50,75,100μg/ml) alone,CPT-11(6.25,12.5,25,50,100,200μg/ml)along,5-Fu(0.25,0.5,1,2,4,8μg/ml)alone and L-OHP(0.125,0.25,0.5,1,2,4μg/ml) alone,and with nimotuzumab plus other agents(50μg/ml) for 24,48 and 72 hours respectively.3.The inhibitory of cells were detected by MTT assay after treatment with the inhibitory rates of cells calculated according to OD value by the following equation: Rate of growth inhibition(%) =(1 - Atreated/Acontrol)×100%.4.To groups treated with nimotuzumab(50μg/ml),flow cytometry was applied to analyze cell cycle and apoptosis after cells were collected.Chapter Two:Inhibitory mechanism of nimotuzumab combined with chemotherapeutic drugs on human colon cell lines in vitro1.The proteins were extracted from the cells treated with nimotuzumab alone or without it.Western Blot was performed for protein expression quantification of PTEN,PI3K,AKT,p-Akt(Ser⁴⁷³),c-Raf,GSK3-βand phospho-GSK-3β2.RNA was extracted from cells treated with nimotuzumab alone or without it.PCR was performed for gene expression quantification of PTEN,PI3K,AKT,Raf-1,and GSK3-β.Statistical analysis:All data were analyzed with SPSS 13.0 statistical software. Unless otherwise stated,data were expressed as mean±S.D.The inhibitory rates were analyzed by Repeated Measure followed by LSD multiple comparison test. Statistical comparisons of multiful-group were calculated by One-way ANOVA followed by LSD multiple comparison test.Two samples were compared using the independents-Samples t Test.P-Values were considered to be significant at＜0.05.Results: Chapter One:Inhibitory effect of nimotuzumab combined with chemotherapeutic drugs on human colon cell lines in vitro1.Surface EGFR expression in human colon cell lines differs in EGFR status:our flow cytometric analysis demonstrated that LoVo cells express high levels of EGFR on the surface,whereas Colo205 cells showed a low level of surface EGFR expression.2.Nomotuzumab treatment alone did not decrease LoVo and Colo205 cells viability inshort- or long-term cytotoxicity tests(MTT),regardless of the concentration used. Chemotheputic drugs(5- fluorouracil,irinotecan and oxaliplatin) alone inhibited cell proliferation time- and dose-dependently(P=0.000).Combination of nimotuzumab and Chemotheputic drugs(5-Fu,CPT-11) enhanced inhibition of proliferation in LoVo and Colo205 cells(P=0.000,0.002).These effect also studied in Colo205 treated with nimtuzumab plus L-OHP(P=0.003),not in LoVo(P=0.126).3.An increase of LoVo and Colo205 cells at the G0/G1 phase of the cell cycle was observed 24h after nimotuzumab treatment(P=0.025,0.001),with a simultaneous decrease of cells at the S phase when compared to controls.Nimotuzumab induced an increase of two kinds of cells apoptosis after 48h when compared to controls (P=0.012,0.000).Chapter Two:Inhibitory mechanism of nimotuzumab combined with chemotherapeutic drugs on human colon cell lines in vitro1.The decreases of PI3K,p-AKT(Ser⁴⁷³) and p-GSK-3βprotein expression were seen after treated with nimotuzumab;the up-regulation of PTEN was found after treated with nimotuzumab;there were not significant difference in protein expression of Akt, c-Raf and GSK-3βbetween two groups.2.The decreases of PI3K gene expression were seen after treated with nimotuzumab; the up-regulation of PTEN gene expression was found after treated with nimotuzumab;AKT,Raf-1 and GSK-3βgene expression had not invenstigated significant difference in two groups.Conclusion:Nomotuzumab treatment alone did not decrease LoVo and Colo205 cells viability in short- or long-term cytotoxicity test;5-Fu and CPT-11 alone induced inhibition of proliferation in LoVo and Colo205 cells,when combined with nimotuzumab could enhance cytotoxicity of both.However,similar results were not found in LoVo when treated with nimotuzumab plus L-OHP.The mechanisms of additive effect were possibly related to the blockage of cell cycle progress and the augmentation of apoptosis by affecting PI3K/Akt cell signaling pathway.