Expression Of ABCG2 In Lung Cancer And Its Effect On The Biological Behavior Of Lung Cancer Cell Line

Backgroud and objectiveLung cancer is the leading cancer killer in both men and women especially in industrial city while its morbidity and mortality still continue to rise.The surest way to defeat it is to prevent it from ever happening.Since the detection,treatment and prognosis will not be optimistic without clear mechanism and effective therapy,it is urgent to explore etiopathogenisis and potent means of prevention and cure.The ATP-binding cassette transporter protein ABCG2,a new member of the supeffamily of ABC transporter proteins,also known as mitoxantrone resistance protein,ABCP,and Breast cancer resistance protein(BCRP),was identified in a MCF-7 breast cancer cell subline that selected for resistance to doxorubicin.The Human Genome Nomenclature Committee recently changed the gene symbol to ABCG2.Structurally ABCG2 contains a single NH2-terminal cytosolic nucleotide-binding domain(NBD)-an ATP binding cassette involved in ATP hydrolysis,and six transmembrane domains(TMD) involved in drug binding and efflux follow.so it is proposed to be a halftransporter that may be function as a homodimer or a tetramer bridged by disulphide bonds or other formations.Based on the structure it plays an important role in prevention host against extraneous matter and drug resistance.Recent evidence suggests that ABCG2 as the transporter is responsible for the Hoechst 33342 dye efflux pattern in cells.Side population(SP) cells are enriched for stem cell activity and are defined by their ability to efficiently efflux Hoechst 33342.SP cell can be isolated by which can rapidly efflux the Hoechst dye to produce a characteristic SP profile based on fluorescence-activated flow cytometric analysis. ABCG2 was subsequently characterized as a novel stem cell transporter and a promising(tumor) stem cell maker.The importance of ABCG2 in chemotherapy and pharmacokinetics and the recent discoveries of ABCG2 role in stem cell regulation encourage further investigation of the relationship between this transporter and lung cancer,which might provide a new discovery to the develeopment and a new strategy for the prevention and cure.Methods1.The quantitative study of ABCG2 expression in lung cancer(1) By immunohistochemical method(S-P) the expression of ABCG2 protein was detected in specimens of 86 lung cancer including 42 squamous carcinoma(Sq Ca), 31adenocarcinoma(AC),5 large cell carcinoma(LC) and 8 small cell carcinoma(SC), 33 with regional lymphatic metastasis,and 24 normal lung tissue far from the focus.(2)By hybridization in situ the expression of ABCG2 mRNA was deteceted in specimens of 86 lung cancer including 42 Sq Ca,31AC,5 large cell carcinoma LC and 8 small cell carcinoma SC.(3)By image quantitive analysis,the gray scale of the target cell was measured and positive unit(PU)of ABCG2 protein or mRNA was calculated.2.The effect of ABCG2 silence on the biological behaviors of lung cancer cell line in vitro (1)Morphological feature of GLC-82 and A549 was observed by inverted microscope and HE staining of cell slide and tissue section.The expression of ABCG2 protein and mRNA GLC-82 and A549 was detected and compared by immunocytochemstry, hybridization in situ,Quantitative reverse transcriptase polymerase chain reaction (RT-QPCR),western blot and Hoechst33342.(2)4 RNAi vector,1 positive control vector and 1 negative control vector were separately tranfected into GLC-82 with lipofectamin2000 to establish the stable ABCG2 silencing lung caner cell line with G418.The ABCG2 expression in transfectant was determined by RT-PCR or RT-QPCR,immunocytochemistry and Western blot.(3) MTT assay,colony formation assay,cell cycle distribution was used to assess growth ability in vitro.Scratching experiment was applied to compare the migration speed.The resisitance to cisplatin was assessed by MTT.Morphological feature of ABCG2 slienced GLC-82 was observed by light microscope and electron microscope.3.The effect of ABCG2 silence on tumorgenesis of lung cancer cell line in vivo The process of tumorgenesis was observed by nude eye and Whole-body optical images and the proliferation change of GLC-82 after ABCG2 silencing was compared. The histological morphology was observed by HE staining tissue section.Results1.ABCG2 monoclonal antibody shows specific plasma membrane staining,prominent staining was observed in AC and SqC of primary focus and lymphatic metastasis,the apical membrane of the bronchial layer and the epithelium of mucus glands..It was not present in LC and SC.(1) in primary focus PU difference of ABCG2 protein was found among the four type of lung cancer(F=15.857,P＜0.001).The differnce was also found between AC or and the others(P＜0.05).the PU in AC exceed the one in SqC(P＜0.01).(2) the PU of ABCG2 protein in normal lung tissue was higher than in lung cancer (t=5.158,P＜0.001).Significant difference of PU was found among normal lung tissue(F=22.470,P＜0.001) and each type of lung cancer.The difference between normal lung tissue and each type(P＜0.05) besides AC(P＞0.05) was significant.(3) in lymphatic metastasis PU difference of ABCG2 protein was found among the four type of lung cancer(F=7.804,P=0.001).The differnce was also found between AC or and the others(P＜0.05).the PU in AC exceed that in SqC(P＜0.01). Significant difference of PU was not found between primary focus and lymphatic metastasis(t=1.388,P=0.169).and the separate differnce was either not found between primary focus and lymphatic metastasis of each type(P＞0.05).(4) the PU of ABCG2 protein was not associated with sex,old,metastasis and TNM stage(P＞0.05),but associated with differentiation(F=65.385 P＜0.001).the PU was higher when the cell is fully differentiated.2.ABCG2 mRNA was expressed in the cell plasma of AC and SqC,and it was not found in LC and SC.(1) PU difference of ABCG2 mRNA was found among the four type of lung cancer (F=12.155,P＜0.001).The differnce was also found between AC or SqC and the others(P＜0.05).PU in AC exceed one in SqC(P＜0.01).(2) the PU of ABCG2 mRNA was not associated with sex,old,metastasis and TNM stage(P＞0.05),but associated with differentiation(F=57.895 P＜0.001).The PU was higher when the cell is fully differentiated.3.Comparision between GLC-82 and A549(1)cells of GLC-82 and A549 have visible difference in size and appearance.The majority of GLC-82 is epitheliod cell lined like a gland,and scattered spindle cell is the main point of A549. (2)Positive stainning of ABCG2 protein was detected in the plasma membrane of GLC-82 and A549.Quantitative analysis show PU of GLC-82 was higher than that of A549(t=7.860,P＜0.001).(3)Positive signal of ABCG2 mRNA existed at the plasma of GLC-82 and A549. Quantitative analysis show PU of GLC-82 was higher than that of A549(t=8.958,P＜0.001).(4) ABCG2 mRNA expression in A549 was 32%of that in GLC-82 byRT-QPCR.(5)Much more ABCG2 protein was detected in GLC-82 than in A549 by western blot t=20.839,P＜0.001)(6)Nucleus staining of hoechst33342 in GLC-82 was dimmer than in A549. Quantitative analysis show PU of GLC-82 was lower than that of A549(t=17.223, P＜0.001)4.10 clone of RNAi,lclone of positive clone(GLC-82/P) and lclone of negative clone(GLC-82/N) were purified after transfection.(1)in positive control the expression of GAPDH mRNA was 19%of that of GLC-82 (t=11.490,P＜0.001) by RT-QPCR.(2)in clone 1 and clone 2 the expression of GAPDH mRNA was 20.11%and 27.59% of that of GLC-82/N.the interfering ratio was 79.99%and 72.49%.(3) The difference of PU of ABCG2 protein expression among GLC-82,clone 1 (GLC-82/R) and GLC-82/N was detected by immunocytochemistry and quantitative analysis(F=242.536,P＜0.001).There was no difference between GLC-82 and GLC-82/N(P＞0.05).Compared with GLC-82 and GLC-82/N,PU of ABCG2 protein expression decreased(P＜0.01).(4) The difference of ABCG2 protein expression among GLC-82,clone 1 (GLC-82/R) and GLC-82/N was detected by western blot(F=77.190,P＜0.001). There was no difference between GLC-82 and GLC-82/N(P＞0.05).Compared with GLC-82 and GLC-82/N,ABCG2 protein expression decreased(P＜0.01).(5) Nucleus staining of hoechst33342 in GLC-82/R was stronger than in GLC-82/N. (t=35.107,P＜0.001)5.To observe the change of the biobehavior after ABCG2 silence in GLC-82 in vitro(1)GLC-82/R showed a significantly slow proliferation compared with GLC-82 and GLC-82/N by MTT assay in vitro from day3(F=91.333,P＜0.001).(2) The percent of cell numbers in G2,S and G1/G2 show no difference among GLC-82,GLC-82/R and GLC-82/N(P＞0.05) by flow cytometry,compared with GLC-82 and GLC-82/N,the percent of cell numbers in G1 decreased in GLC-82/R (F=5.208,P=0.49).(3) The ratio of colony formation in GLC-82/R was lower than that of GLC-82 and GLC-82/N(F=31.869,P＜0.001).(4)No interaction was found between group and time(F=0.511,P=0.780).The difference of the healing rate was not found among GLC-82,GLC-82/R and GLC-82/N(F=0.511 P＞0.05).(5) No differnce had been observed by microscope and electron microscope.(6)IC50 difference was found among GLC-82,GLC-82/R and GLC-82/N (F=32.828,P=0.001).Compared with GLC-82,the resisitance index of GLC-82/R and GLC-82/N decreased to 0.314 and 0.5896.To observe the process of tumorgenesis of GLC-82,GLC-82/R and GLC-82/N and compare the proliferative ability in vivo.(1)Compared with the tumor from GLC-82 and GLC-82/N,the tumor from GLC-82 grow slowly from day 17 after injection(F=26.102,P＜0.001).(2)The tumor from GLC-82 is smaller(F=5.920,P=0.013) and lighter(F=4.114, P=0.038) than that from GLC-82 and GLC-82/N.(3)The gross appearance of the tumors was gray and nodose.Histologically the pleomorphic cancer cells grow infiltrative widespreadly with hemorrhage and necrosis,and surrounded by slim fibrous connective tissue,no obvious glandular differentiation is seen on light macroscopy.Tumor cells with monocaryon and dikaryon,polykaryocyte and multinucleated giant cells were visible.There was nuclear pleomorphism with numerous bizarre mitoses.Histology and morphology of tumor cell were similar in the three group.Conclusion1.ABCG2 might be taken as a marker to diagnose the type and differenciated extent of lung caner.2.The expression of ABCG2 was not involved in sex,old,mestastasis and TNM stage of lung cancer,but associated with the extent of differciate,which indicated that ABCG2 might be play a role in drug resistant of lung cancer.3.The expression of ABCG2 was higher in GLC-82 than in A549 in terms of mRNA and protein expression of ABCG2 and its function.4.we established the stable lung cancer cell line of ABCG2 silence,which will provide an ideal cell model for the ulterior experiment.5.Since ABCG2 was associated with proliferattion and drug resistance of lung caner, deep research into the correlation between ABCG2 and lung cancer will provide a new clue for carcinogenesis,development and treatment.New point1.To study the expression of ABCG2 protein and mRNA from the quantitative point for the first time2.To establish the lung cancer cell line of ABCG2 silence,to provide an ideal cell model for the reserch of lung cancer in vitro and establish the foundation of animal model.3.For the first time systematically to study the effect on the biological behavior of ABCG2 silence in lung cancer cell line.