| Lung cancer is the leading cause of cancer-related death both worldwide and inChina, principlely categorized into non-small cell lung cancer (NSCLC) and smallcell lung cancer (SCLC). NSCLC occupies around80%of total lung cancers.Thelatest statistical data showed that about1.6088million cancer cases and1.3784million cancer deaths are estimated to have occurred in2008world-wide. But the5-years survival rate of lung cancer is still10%in china poorer than16%in developedcountry.In clinical we usually treated the EGFR mutation patients with EGFR-TKI, andtreated the ALK fusion gene patients with ALK-TKI (Crizotinib was approved by theUnited States FDA on Aug26,2011). Based on the development of the lung cancermolecular aberration (driver mutation)-based personalized treatment cansignificantly benefit patients. However, the single targeted therapy will eventuallyappear resistance, and the resistance mechanisms and strategy of novel drugs toovercome resistance is also under development. In addition to targeted therapy againstmutant EGFR and rearranged ALK, chemotherapy containg platinum is still thecornerstone for the advanced lung cancers with wild type RGFR and ALK, and thoseafter EGFR or ALK-TKI resistance. Therefore, looking for effective new target andtargeted drugs in the pathways of apoptosis and/or cell cylcle may be of greatimportance in translational medicine of lung cancer.“Resisting cell death†is one of the hallmarks of malignant cancer cells. Functioning of the molecules in the apoptosis pathway may play roles during theentire process of cancer development and evolution. Molecules in the apoptoticpathway can be classified as regulators and effectors. The process of apotosis can bedivided into stages of activation and execution. To promote the pro-apototic activationprocess is one of the strategies of cancer treatment.In this study, we try to observe the apoptotic signal molecules, including(Survivin BIRC5), CASP8, CASP9, C-PARP1, PARP1, BCL2, CASP3, C-CASP3,and XIAP nine apoptosis related molecules, in patients with lung cancer and adjacentnon-cancerous tissues based on tissue microarray immunohistochemical.And analysisof the differential expression levels of apoptotic molecules and the relationship withclinical prognostic parameters were conducted. One goal is to look for potentiallydrugable apoptotic pathway molecular target to teat lung cancer. In in vitro study, weuse apoptosis promoter CASP3activator cystatin winter peptide zymogen activation(procaspase-activating compound1, PAC-1, can promote the CASP3into C-CASP3)combined with CISplatin to test in lung cancer cell lines, and to identify the bestfunction pattern and potential mechanisms of the targeting drugs (PAC-1) andchemotherapy.Chaper1Expression levels of the apoptotic signal molecules PARP1andCASP3, etc., and their clinical significance in tissue microarray of lung cancerMethods144cases of formalin-fixed paraffin-embedded tissues of NSCLC were collectedduring2003through2006. These tissues were made into tissue micaroarray (TMA)with tissue microarayer of Beecher InstruMents. In the TMA block26cases of pairedcancer versus adjacent non-cancerous tissues (lung parenchyma with5~7cm awayfrom the edge of tumor mass) were included. Immunohistochemistry (IIHC) wereperformed to analyse the expression level of CASP3, CASP8, CASP9, PARP1,Cleaved CASP3(C-CASP3), Cleaved PARP1(C-PARP1),XIAP,BIRC5(Survivin)and BCL2. In each sample the staining intensity (negative=0,modest=1, intermediate=2and strong=3) and positive celllproportion (0%=0,1%~10%=1,11%~50%=2,51%~80%=3,81%~100%=4) were recoeded. IHCscore for each sample was calculated by multiplying the intesnsity score with theproportion score, within a range of0through12. The median value of all scores wasused as cut-off value to separate high-expression from low-expression groups. Results1. In the evaluable25cases of paired cancer versus adjacent tissues, paired sample ttest showed that C-PARP1were not detected in apired samples, CASP8was notsignificantly differentially expressed between cancer and adjacent tissues (P>0.05),other molecules CASP3, CASP9, PARP1, C-CASP3, XIAP, BIRC5(Survivin) andBCL2were all expressed in cancers at a higher level than that in adjacent tissues(P<0.01).2. In the analysis of nonpaired139cases of cancers versus25adjacent tissues,non-parametric rank test showed that difference of C-PARP1exrepssion between thetwo groups were not significant (P>0.05), CASP8was significantly down-regulated incancer tissues in comparison to adjacent tissues (P<0.05), other molecules CASP3,CASP9, PARP1, C-CASP3, XIAP, BIRC5(Survivin) and BCL2were all expressed incancers at a higher level than that in adjacent tissues (P<0.01).4. Relations of apoptotic molecules with clinical, pathological and survival factorswere analyzed. In the bi-variate correlation analysis C-PARP1was primarily foundsignificantly correlated to overall survival (OS)(P<0.05). C-CASP3and CASP8wereexpressed in non-squamous cell carcinoma (non-SCC) at higher levels than that inSCC (P<0.05). BCL2, BIRC5(Survivin) and PARP1were expressed in squamous cellcarcinoma (SCC) at higher levels than that in non-SCC (P<0.05). PARP1andBIRC5(Survivin) also associated with male and smoking factors (P<0.05).Kaplan-Meir survival curve test showed that whether in all patients or in stage III-IVpatients, C-PARP1was significantly associated with OS (P<0.05), suggestingC-PARP1might be a prognostic factor. In all patients with all stages, pN, Tumor sizeand disease stage were all statiscally associated with OS (P<0.05).Multi-variate Cox regression model showed that disease stage, histology andC-PARP1were significantly associated with OS, and C-PARP1might server as aindependent prognostic factor for these lung cancer patients.ConclusionsProtein expression analysis in paired and non-paired cancer versus adjacent tissuesconfirmed that apoptotic molecules CASP3, PARP1, CASP8, CASP9, XIAP,BIRC5(Survivin) and BCL2were significantly upregulated in cancer tissues.suggesting in these lung cancer patients, CASP3, BCL2, BIRC5(Survivin)and PARP1 could be taken as potential treatment targets.BCL2, BIRC5(Survivin) and PARP1were expressed in squamous cell carcinoma(SCC) at higher levels than that in non-SCC. PARP1and BIRC5(Survivin) alsoassociated with male and smoking factors, suggesting that BIRC5(Survivin) andPARP1might be potential treatment target for these sub-group of patients.Kaplan-Meir survival curve test and multivariate Cox ergression model bothsuggested that C-PARP1might be a independent prognostic factor for NSCLC.Chaper2In vitro anti-cancer effects of CIS-plantinum and/or PAC-1in differentlung cancer cell linesMethodsNon-small cacner cell lines of H1299(homozygous partial deletion of the TP53),A549(KRAS mutation), PC9(EGFR mutation), H1650(EGFR mutation) and H1975(EGFR mutation) were taken as cell models, colorometric MTT (3-(4,5-cimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) tests were used to test invitro anti-cancer effects of CIS-plantinum (CIS), PAC-1and CIS combined withPAC-1. Western blotting was performed to analyze the protein expression levels ofCASP3, CASP9, C-CASP3, PARP1, C-PARP1, XIAP and BCL2, whereby ACTBused as internal control. Flow cytometry was perfromed to analyze the cell cyclechanges in each experimental groups. Immunofluorescent cell chemistry was carriedout to test the CASP3, C-PARP1and activated C-CASP3in cells under fluorescentmicroscopy.Results1. By colorimetric MTT tests, it was found that single agent CIS-plantinum couldinhibit the growth of H1299, A549, PC9, H1650and H1975cells at a certain doserange with IC50values of1.9～11.2μM for these cells. PAC-1could also inhibit thegrowth of H1299, A549, PC9, H1650and H1975cells at a certain dose range withIC50values of7.4～14.8μM for these cells. For combination tests, only in H1299cells it was found that concurrent CIS+PAC-1or CIS→PAC-1sequential combinationcould have synergistic effects of inhibiting cell growth, while in other4cel lines,combination of the two drugs mostly presented antagonistic effects (CI>1.0). 2. Flow cytometry results showed that in all5cell lines, PAC-1could block cellsat atG1phase and CIS could block cells at G2/M phase. In H1299cells CIS→PAC-1sequentrial combination had strong pro-apoptotic effects.3. Western blotting results showed that in H1299cells, PAC-1and CIS+PAC-1andCIS→PAC-1could promote the activation of CASP3into C-CASP3. Single agentPAC-1could partially inhibit C-PARP1prduction in H1299and PC9cells, but not inH1975ells.4. Immunofluorescent cytochemistry showed that in all cell lines of H1299, A549,H1650, H1975and PC9, PAC-1couled promote the scizzering of CASP3intoactivative forms of C-CASP3;CIS couled promote the scizzering of PARP1intoactivative forms of C-PARP1and it showed relatively stronger effects of inducingC-CASP3formation in H1299cells.ConclusionsIn vitro experiments preliminarily showed that in EGFRmut (PC9, H1650, H1975) orKRASmut (A549) cells, PAC-1showed antagonistic effects when combined withCIS-platinum. However, in H1299, PAC-1could play a synergistic role incombination with CIS-platinum, which might result specifically from pomotingformations of active C-CASP3。... |
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