Investigation Of OGTCT-induced Apoptosis And The Molecular Mechanisms In Human Large-cell Lung Cancer H460 Cells

BACKGROUND AND OBJECTIVEAccording to the statistics of WHO,lung cancer is the most dangerous cancer all over the world,which mortality tops the list of all kinds of tumor.And china has become a country that has the largest number of lung cancer patient.According to histological type,lung cancer can be divided two types:small cell lung cancer (SCLC) and non-small cell lung cancer(NSCLC).NSCLC accounts for more than 85%of lung cancer.Because NSCLC become not suitable for surgery after metastasis,and not sensitive to radiotherapy,therefore,it is particularly important for development and research of chemotherapeutic drugs.The classifications of frequently-used drugs for lung cancer chemotherapy are five categories as bellows:directly damage DNA drugs,inserted DNA template drugs,affecting nucleic acid synthesis drugs,repressing microtubule assembly drugs and impacting protein synthesis drugs.Although the above drugs can induce apoptosis of lung cancer cells,but strong toxicity side effect and easily leading to drug resistance cause frequently failure of chemotherapy.Therefore developments of drug which has minor toxicity,less side effect,ability to overcome multi-drug resistance,and able to start apoptosis signal transduction pathway,are the urgent problems to be settled.In this study,reasons that H460,A549,H1792,H226 and WI38 were selected as experimental cells to investigate mechanisms of growth-inhibition in NSCLC cells are as follows:1) These four kinds of NSCLC cells were derived from adenocarcinoma,large cell carcinoma,squamous cell carcinoma,covering three subtypes of NSCLC;2) These four kinds of NSCLC cells can be used seperately in detection on the cellular level in the field of lung cancer study as model cell;3) To date,investigation of mechanism of small molecular organotin-induced apoptosis in NSCLC cells remains relative few.4) WI38 is a diploid fibroblast cell line which derived from human embryonic lung tissue and frequently used as control cell in the study of lung cancer.Thus the above cell lines are available for screening small molecular compound,and make in-depth study of its mechanism aim to provide a theoretical basis for development of chemotherapeutic drug of NSCLC.Chemical genetics is a new means that utilizing chemical tools for investigating biological system,and it could promote the developments of new drugs in the following two important aspects:on the one hand,identifying gene or protein which plays an important role in formation of a disease,so as to provide targets for the new drug screening;The other hand is finding small molecular compound which act with a gene or protein,so as to provide lead compounds for developing new drugs.One of the key of chemical genetics researches is to have a large number of compounds with different structures for screening.The emerging combined chemistry is the core technology accessing to numerous small molecular compounds.The principle of the combined chemistry is to add different structural units into the same chemical reaction system,and to synthesize systematically numerous compounds utilizing permutations and combinations of these structural units.Over the years,the chemical genetics has been widely applied in the field of cell apoptosis research:Professor Yuan Junying from Harvard Medical School lead the research team carrying out the investigation named as "using small molecules to study on the signal transduction in the process of cell death:from Bcl-2 to the programmed necrosis";Professor Wang Shaomeng from the cancer research center of U.S.Michigan University launched study named as "rationally design small molecular antagonist to regulate key cell necrosis and protein-protein interactions"; Additionally,the U.S.National Cancer Institute(NCI) launched a new plan in 1997, the application chemical genetics to research of cancer treatment,namely the use of cell permeable small molecular compound to trigger death of tumor cell.The above examples fully prove that chemical genetics method provides a reliable platform for research of cell apoptosis.In this platform,utilizing small molecular compound with a certain biological activity as a tool to investigate signal transduction in the process of cell apoptosis has many advantages:small molecular compounds to enter cells easily,easy to cause changes in the cellular phenotype,its role in a dose-dependent relationship,applicable to multi-proteins family as well as not cause adaptive changes in cells,etc.In addition,these small molecular compounds often serve as "lead compounds" to further drug development and research.In recent years,our team has been engaged in construction the "library" of small organic compounds,at present,the library already contains more than 60 species of small molecular organotin.Studies have shown that some organotin have anti-tumor activities which are already confirmed in MCF-7,WiDr and P388 cells. Most of these studies still remained in the experimental phase of growth inhibition. Some studies indicated that organotin could block cell cycle of tumor cell.However, the molecular mechanisms of inhibiting growth and inducing apoptosis remain unclear.So investigation of molecular mechanism of apoptosis induced by small molecular organotin is of great significance.Based on the above considerations,aims of this research are as follows: applying chemical genetics methods,to investigate In vitro anti-tumor activity of OGTCT in H460,A549,H1792,H226 cells,and then,to explore molecular mechanisms of OGTCT-induced apoptosis in H460 cells.This study can also provide a more complete theoretical basis for the development of new chemotherapeutic agents for the treatment of lung cancer.STUDY CONTENTS1 Identification growth inhibition to NSCLC,H460/TaxR and WI38 of OGTCT.2 Exploration of apoptosis induction in H460 cells by OGTCT.3 Analysis of the influences of OGTCT on cycle distribution and p53,p21,PCNA protein levels in H460 cells.4 To analyze changes of apoptosis-related factors and the important apoptosis pathways activated by OGTCT in H460 cells. METHODS1.Cell growth inhibition Detection:1.1 MTT assay for cell viability,by which OGTCT was screened initially;1.2 Select the current clinically used anticancer drug(Paclitaxel) as control drug, using MTT assay for growth inhibition to tumor cells and normal cells,and make comparison to OGTCT.2.Methods for apoptosis identification:2.1 Observation of cell morphological changes and apoptosis bodies by Phase Contrast Microscope;2.2 Observation of nuclear morphological changes by Hoechst33258 staining combined with fluorescence microscopy;2.3 LDH(Lactate dehydrogenase) assay to determine cell toxicity induced by OGTCT;2.4 Trypan blue dye exclusion assay to confirm whether the cells treated by OGTCT undergo necrosis;2.5 Detection of apoptosis rates by Nucleosome ELISA assay kits.3.Detection of changes in cell cycle distribution and related regulatory proteins expression:3.1 Analysis of the changes in cell cycle distribution by Flow cytometry;3.2 ELISA methods for analysis of expression changes in p53/p21 proteins associated to regulation of cell cycle;3.3 Detection of expression and distribution of PCNA by immuno-fluorescence combined with Laser Scanning Confocal Microscopy(LSCM);3.4 Confirmation of the changes in p53 expression by Western blot.4.Detection changes of Ca²⁺ concentration in H460 cells by immuno-fluorescence probe combined with LSCM.5.Methods for determining whether caspases are activated by OGTCT:5.1 Analyzing caspase-3,-8 activities by ApoAlert caspase-3,-8 colorimetric assay kits; 5.2 Analyzing caspase-9/6 activity by ApoAlert caspase-9/6 fluoremetric assay kits;5.3 Identification the influences of caspase-3,-8,-6,-9 on apoptosis induced by OGTCT with caspase inhibitors.6.To analyze distribution and expression of anti-apoptotic protein Bcl-2 by Immuno-fluorescence combined with LSCM.7.Detection expression changes of Cytochrome C(Cyto-C) by Western blot.8.Detection changes of expression in Fas/FasL proteins by ELISA kits.RESULTS1.Detected results of anti-tumor activity of OGTCT(MTT assay for cell viability)1.1 Influences of OGTCT on viability of NSCLC,H460/TaxR and WI38 cell:Within 48h,OGTCT reduced significantly viabilities of NSCLC cells (H460,A549,H1792,H226) and drug-resistant cell H460/TaxR in a dose-dependent manner;but at the same concentration and time,viabilities of WI38 cell were much higher than that of NSCLC and H460/TaxR cells.1.2 Results of positive drug comparison test:Within 48h,paclitaxel reduced viabilities of NSCLC,H460/TaxR and WI38 cells in a dose-dependent manner.But compared to OGTCT,paclitaxel inhibited obviously growth of WI38 cells,and its growth inhibition to H460/TaxR was not extremely significant.The results indicated:OGTCT had two advantages compared with paclitaxel, OGTCT had a minor toxicity to normal cell WI38,and its growth inhibition to drug-resistant cell H460/TaxR was particularly obvious.2.Results of apoptosis induced by OGTCT in H460 cells2.1 Cell morphological changes observed under Phase Contrast Microscope: Treated with 0.1%DMSO,cell morphology has no obvious change;Exposed to 0.56μM/L OGTCT for 48h,morphological changes are as follows:cell body shrinkage,obvious cytoplasmic vacuolization,karyopyknosis,refractive index decreased,membrane blebbing,formed apoptotic bodies,and cells showed typical morphological characteristics of apoptosis.2.2 Results of detection cell nuclear fragmentation by Hoechst33258 staining combined with Fluorescence Microscopy:There had no obvious change in cell nuclear morphology between DMSO group and control group,and cell nuclei showed a uniform light blue;When H460 cells were treated with 0.56μM/L OGTCT for 48h,typical apoptotic features such as cell shrinkage,chromatin condensation and marginalization,emerging nuclei fragmentation,apoptotic bodies clearly visible occurred.2.3 Detected results of activity of LDH:When H460 cells were respectively incubated in 0.14,0.28μM/L OGTCT for 48h,compared with the control group,there was no significant change of activity of LDH in culture medium(p>0.05,n=3);While LDH activity in culture medium containing 0.56μM/L OGTCT was significantly enhanced(p<0.01,n=3).2.4 Results of Trypan blue dye exclusion assay:Cell necrosis rate of 0.14 and 0.28μM/L test groups were extremely low, compared with the control group;While the necrosis rate of 0.56μM/L test group increased obviously(p<0.01,n=3).2.5 Results of nucleosome ELISA assay:When H460 cells were respectively treated with 0.14,0.28,0.56μM/L OGTCT for 6,12,24 and 48h,compared with the control group,OGTCT induced effectively apoptosis of H460 cells in dose- and time-dependent manners.3.Influence of OGTCT on H460 cell cycle and assay of related proteins3.1 Influence of OGTCT on H460 cell cycle distributionAfter changes of DNA were analyzed,we found that cell cycles of H460 cells witch treated by OGTCT for 24-48h were blocked at G₀/G₁ phase in a time-dependent manner.Furthermore,H460 cells could not enter S phase. However,OGTCT has a minor effect on M phase.3.2 Distribution and expression of p53/p21 protein·p53/p21 ELISA assay The levels of p53 and p21 proteins were up-regulated by OGTCT and increased significantly with the extension of time,reached the maximum at 24h,since then, the levels of p53 and p21 gradually reduced.·Western blot assay confirmed that OGTCT significantly up-regulated the expression of p53 protein(p<0.01,n=3).3.3 Immuno-fluorescence combined with LSCM detection for expression and distribution of PCNA protein in H460 cells:Expression level of PCNA in H460 cells was significantly reduced after treated with 0.56μM/L OGTCT for 48h(p<0.05,n=3).4.Research findings of OGTCT-induced apoptosis in H460 cells and its related molecular mechanisms4.1 Detection changes and distribution of Ca²⁺ in H460 cells by immuno-fluorescence probe combined with LSCM:Calcium concentration was significantly enhanced in H460 cells treated with 0.56μM/L OGTCT for 48h(p<0.001,n=3).4.2 To detect expressions of protein Bcl-2 in H460 cells by immuno-fluorescence assay combined with LSCM:When cells were treated with OGTCT 0.56μM/L for 48h,in comparison to control group,the expression of protein Bcl-2 was down-regulated significantly(p<0.01,n=3).4.3 Assay results of activities of caspases:·ApoAlert caspase-3 colorimetric assay showed a higher light absorption at 405 nm compared to control group(0.982 vs 0.246,p<0.001) by OGTCT 0.56μM/L in H460 cells at 48 h.·ApoAlert caspase-8 colorimetric assay showed a higher light absorption at 405 nm compared to control group(0.787 vs 0.132,p<0.001) by OGTCT 0.56μM/L in H460 cells at 48 h.·ApoAlert caspase-9/6 fluoremetric assay showed that fluorescence intensity of caspase-9 at 460nm was dramatically up-regulated from 24.45 to 185.44 by OGTCT 0.56μM/L(p<0.001),in other words,its activity increased by 6.58 folds. And activity of capase-6 increased by 0.82 fold with the fluorescence intensity enhanced from 18.36 to 33.55(p<0.01).·Detection influences of caspases inhibitors on apoptosis induced by OGTCT: Compared to 0.56μM/L OGTCT treatment group,pan-caspase inhibitor (Z-VAD-FMK) completely blocked the apoptosis induced by OGTCT,caspase-3 inhibitor(Z-DEVD-FMK),caspase-8 inhibitor(Z-IETD-FMK) and caspase-9 inhibitor(Z-LEHD-FMK) significantly reversed the OGTCT-induced apoptosis in H460 cells.And caspase-6 inhibitor(Z-VEID-FMK) reversed partially the OGTCT-induced apoptosis.4.4 Detection expression of Cytochrome C(Cyto-C) by Western blot assay Compared with the control group,the level of Cyto-C was significantly up-regulated by 0.56μM/L OGTCT at 48h in cytoplasm of H460 cells (p<0.01,n=3).4.5 ELISA assay detected expressions of Fas/FasL in H460 cells treated with OGTCT:4.5.1 Results of Fas/FasL ELISA assayThe expression of Fas protein was up-regulated by OGTCT and significantly increased with the extension of time,reached the maximum at 24h,since then,the level of Fas reduced to some extent.However,the expressions of mFasL and sFasL did not change significantly(p>0.05,n=3).4.5.2 Influences of Fas inhibitor(anti-Fas Ab,ZB4) on apoptosis induced by OGTCT in H460 cellsCells were treated with 0.56μM/L OGTCT after ZB4 pre-treatment for 48h, MTT and ELISA assay indicated:apoptosis induced by OGTCT and proliferation inhibition were reversed by ZB4.CONCLUSIONS1 OGTCT reduced significantly viabilities of NSCLC cells and drug-resistant cell H460/TaxR in a dose-dependent manner;OGTCT had two advantages compared with paclitaxel:OGTCT had a minor toxicity to normal cell WI38,and it could overcome the multi drug-resistance of tumor cells.2 OGTCT could effectively induce apoptosis in H460 cells.It may well exert it's function of apoptosis induction through the following factors:up-regulation of levels of p53 and p21 proteins,reducing expression of PCNA,blocking cell cycle at G₀/G₁ phase,down-regulation expression level of Bcl-2,induction release of Ca²⁺ and Cyto-C into cytosol,activations of capase-3,-8,-9,-6.3 Fas signaling pathway and mitochondrial signaling pathway might jointly participate in the process of apoptosis induced by OGTCT in H460 cells.