Study Of Expression And Effect Of BCRP In Human Esophageal Squamous Cell Carcinoma

Esophageal squamous cell carcinoma(ESCC) is a common cancer in China with high incidence,which threaten the health of people seriously.Although great progress in the diagnosis and therapy of EC have been made in the last decades,the cure rate of EC has not been improved yet and 5 year survival rate still remained around 20%-30%. Recurrences occur frepuently in EC patients,which are the leading cause of death,and the main cause of recurrences is the remnant of cancer cell.Metastasis and drug resistance is the main cause of remnant of cancer cell.Metastasis was adjusted by bionomics of cancer,and drug resistance was adjusted by drug resistance gene.As a multi-drug resistance(MDR) protein,breast cancer resistance protein(BCRP) is a marker of tumor stem cell.So BCRP not only effect the drug resistance of cancer,but also effect the bionomics of cancer,including invasion activity,proliferation activity and oncogenicity.To study the effect of BCRP in cancer biological characteristics, metastasis,recurrences and drug resistance is owned important significance on caner early diagnosis,therapy and Prognostic in clinically,which indicated in some cancer.To our knowledge,there is no report about the effect of BCRP in ESCC's biological characteristics,neither report about the relationship between BCRP and ESCC's metastasis,recurrences and drug resistance now.So it is necessary to study the expression and effect of BCRP in ESCC.According to the mentioned above,we performed the following experiments.Fistly, we investigated the expression of BCRP in esophageal cancer samples and explored its correlation with clinical and pathological characters.Then we established ESCC cell line. Based on above work,we investigated the effect of BCRP in ESCC's drug resistance and the relationship between BCRP and ESCC's biological characteristics.We also explored the possible mechanism of this effect.Purpose a new theoretical basis of early diagnosis and therapy for esophageal cancer.Part1:Expression of BCRP in ESCC and its possible implicationsObjective:To determined the expression of BCRP in ESCC,and to investigate the association of BCRP with ESCC.Methods:Western-blot and immunohistochemical technique were used to detect the expression level of protein of BCRP in 50 pairs of fresh esophageal cancer tissues and according para-cancer normal tissues.Paraffin-embedded sections were obtained from 30 patients with ESCC for immunohistochemical studies to detect the expression level of protein of BCRP.Implications between the expression of CBP and clinical and pathological characters was Analyzed.Results:1.The expression levels of BCRP by Western-blot in cancer tissues were 1.22-fold(P＜0.05) higher than those in para-cancer normal tissues respectively.The decreased expression of CBP gene in lung cancer tissues was significantly associated with TNM stage(P＜0.05)and lymph node metastasis(P＜0.05);2.The positive rate of BCRP in cancer and normal tissue was 50%and33.75%respectively and the difference was very significant(P＜0.01) by immunohistochemical;3.The increased expression of BCRP gene in esophageal cancer tissues was significantly associated with histology differentiated stage (P＜0.05),survival time and recurrences(P＜0.05).Conclusions:Increased expression of BCRP in esophageal cancer is tumor specific, contributed to survival time and recurrences in ESCC,may be considered as a supplementary predictor prognostic.Part 2:Establishment of ESCC continuous cell line which expressed BCRPObjective:To establish ESCC continuous cell line which expressed BCRP,in order to study its effects of BCRP on the biological behavior of esophageal cancer.Methods:To establish a multi drug-resistant cell line,ESCC continuous cell line cells were cultured with intermittent gradual-increasing-concentration of cisplatin(cDDP) and hydroxycamptotbecine(HCT).Detect proportion of BCRP positive cells of ESCC by FCM,and detect BCRP protein by Western-blot and cell immunofluorescence.Detection known to cisplatin resistance from the key role of glutathione-S-transferase(GST), pulmonary resistance protein(LRP),P-gp and MRP1 expression to identify whether resistant to cDDP was originated by BCRP.Results:Established SHEC-4-D-0.3,SHEC-4-D-0.75 and SHEC-4-D-1.0 which resistant to cDDP.The proportion of BCRP positive cells increased one by one(SHEC-4 vs SHEC-4-D-0.3(0.75,1.0),p＜0.01),but not statistically significant between SHEC-4-D-0.3,SHEC-4-D-0.75and SHEC-4-D-1.0.Western-blot showed that protein expression level of SHEC-4-D-0.3 and SHEC-4-D-0.75,SHEC-4-D-1.0 increased to SHEC-4.Immunofluorescence showed SHEC-4 has no fluorescence cell,however,a great deal of fluorescence cells was found in SHEC-4-D-0.3/0.75/1.0.Expression of LRP in SHEC-4 and the three resistance cells with a significant difference(p＜0.05),and GST in the four cell lines were increased in statistical significance(p＜0.01).Conclusions:Established ESCC continuous cell line which expressed BCRP by chemotherapy drugs.The up regulation mechanism of BCRP by Cisplatin may be cisplatin resistant cells at the same time high expression of BCRP and other resistance proteinPart 3:Effect of BCRP to drug resistant of ESCCObjective:To study the relationship between ESCC continuous cells' drug resistant with BCRP,to explore the effect of BCRP to drug resistant of ESCC.Methods:1.Effect of expression of BCRP in ESCCs' drug resistant:SHEC-4, SHEC-4-D-0.3,SHEC-4-D-0.75 and SHEC-4-D-1.0 was cultured with different concentration of cDDP,5-Fu,Carboplatin,HCT and Daunorubicin(DNR) for 48 hours,then draw curve of cell inhibition ratio and calculate cell resistant index(RF) in order to evaluate their resistance to chemotherapy drugs.3.Drug resistant of ESCCs changed by use inhibitor of BCRP and GST:ESCCs were cultured with dipyridamole (specific inhibitor of BCRP) and Ethacrynic acid(specific inhibitor of GST),then cultured with different concentration of cDDP,5-Fu,Carboplatin,HCT and Daunorubicin(DNR) for 24 hours,draw curve of cell inhibition ratio and calculate cell resistant index(RF) and reverse index(RI) in order to evaluate their resistance to chemotherapy drugs,compared with ESCCs which not cultured with inhibitor.Result:SHEC-4-0.3 cDDP,SHEC-4-D-0.75,SHEC-4-1.0 pairs cDDP,carboplatin,DNR, HCT resistance index increased,while the 5-FU resistance index lower.After joining the dipyridamole,the RI of SHEC-4- cDDP-0.3,SHEC-4-D-0.75,SHEC-4-D-1.0 to daunorubicin and I hydroxycamptothecin lower,and on other drugs RF no significant impact.In SHEC-4—0.3 cDDP,SHEC-4-D-0.75,SHEC-4—1.0,RF dropped by join Ethacrynic acid,but no significant effect on the other drugs.Conclusion:The expression of BCRP in esophageal cancer is an important reason for drug resistance.PartⅣ:The Effect of BCRP on biological characteristics of esophageal squamous cell carcinomaObjective:To study the effect of BCRP in biological characteristics of esophageal squamous cell carcinoma cell line. Methods:Detect the similarities and differences of esophageal cancer cell lines SHEC-4—0.3 cDDP,SHEC-4-D-0.75,SHEC-4—1.0 cDDP SHEC-4's biological characteristics.Include:A:The effect of BCRP in biological characteristics of esophageal squamous cell carcinoma cell line in vitro:1.Cell morphology;2.Immunohistochemical staining of CK and Ki67 expression3.Detection of cell growth:Growth curve,flat colony formation rate,the rate of adherent cells,the cell cycle;4.Experimental of cell migration5.Transwell cave invasive experimentalB:The effect of BCRP in biological characteristics of esophageal squamous cell carcinoma cell line in vivo:1.Tumorigenicity in nude mice inoculated experiment:Different concentrations of ECCs will be inoculated in Balb/C mice back subcutaneous,observed in nude mice transplanted tumor cells and the necessary amount of time to observe the size of a tumor;2.The routine pathological of tumor tissue and cultured tumor cells again.3.FCM to tumor tissue in the expression of BCRPResult:A.The effect of BCRP in biological characteristics of esophageal squamous cell carcinoma cell line in vitro:SHEC-4 cell volume than the other three slightly,four cell morphology little difference;CK and Ki67 showed positive in four ECCs.Growth curve showed the "S" curve,population doubling time was 28.79 h,34.98h,35.69h,35.8h,the three ECCs' population doubling time compared with SHEC-4 was significantly prolonged,and have statistical significance(p＜0.01).The number of colony formation rate of the three groups of cells increased than SHEC-4,but has no significance(p＞0.05);ECCs frozen cells after vaccination recovery rate of 70%to 81%.Cell cycle show SHEC-4—0.3 cDDP, SHEC-4-D-0.75,SHEC-4—1.0 cDDP proportional to the S phase,SHEC-4—0.3 cDDP, SHEC-4-D-0.75.SHEC-4—1.0 cDDP cell migration and cell invasion capacity increased significantly than SHEC-4.B The effect of BCRP in biological characteristics of esophageal squamous cell carcinoma cell line in vivo: Four weeks after Vaccination of 1×10~5 cells SHEC-4,SHEC-4-D-0.3, SHEC-4-D-0.75,SHEC-4-D-1.0 the tumor occurred 25%,62.5%,50%,75%respective. For 1×10~4 cells:SHEC-4 no tumor,and SHEC-4—0.3 cDDP,SHEC-4-D-0.75,cDDP SHEC-4-DDP-1.0 rate of 12.5%,1×10~3 inoculated tumor cells into the rate was 0%,0%, 25%and 12.5%,1×10~2 inoculation of tumor cells did not found,no significant difference between volume,no significant difference of the shape of the tumor cells and BCRP% between pro-generation content and filial generation(p＞0.05).Conclusion:Expression of BCRP in esophageal squamous cell carcinoma was significantly increased tumor cell proliferation,tumor,invasive,and may related with esophageal cancer metastasis and recurrence.Full summary:1.BCRP up-regulated expression in esophageal squamous cell carcinoma,correlated with differentiation,survival of patients with life,the recurrence rate.BCRP can be used for predicting the prognosis of patients.2.The use of cisplatin on ECCs screening can establish high expression in BCRP of esophageal squamous cell carcinoma.The possible mechanisms of the BCRP increased is the co-expression of LRP/BCRP expression in the same cell subsets and advantages cloning,therefore,clinical use of cisplatin also may lead high expression of BCRP.3.Up regulation of BCRP to esophageal cancer can lead resistant to some chemotherapeutic drugs(camptothecin,daunorubicin,etc.),these resistance can be reversed by the BCRP inhibitors.Clinical solution of esophageal cancer chemotherapy could use BCRP inhibitors too.4.Expression of BCRP in vivo and in vitro can be markedly increase esophageal squamous cell carcinoma's proliferation,invasiveness,tumorigenicity,and may be related to esophageal cancer metastasis and recurrence.