Chemotherapy Resistance Study Based On The Combined Analysis Of Drug Resistance-related Proteins And Glucose-regulated Proteins In Human Lung Cancer

Background and Aim:As one of the most frequent malignancies, Lung cancer is the leading cause of cancer death in the world. Drug resistance remains a major problem for successful chemotherapy in lung cancer. Chemoresistance can be primary(intrinsic) where tumor cells do not respond to anticancer drugs from the beginning of the treatment or secondary (acquired) where tumor cells develop resistance during chemotherapy. As is well known, Drug resistance is a complex process mainly involving with pharmacokinetics, cancer-cell-specific issues and the tumor microenvironment. Various factors are mutually dependent and inter-acting in development of dug resistance。There are varieties of anticancer drugs for the treatment of lung cancer, such as VP-16, cisplatin. VP-16, one member of epiopodophyllotoxin, is the cell cycle specific drugs. Platium characterized by cisplatin can be used to treat all types of lung cancer. Through formation of cross-link, cisplatin affect the replication and synthesis of DNA and cause cell apoptosis.Cancer-cell-specific issues can be mediated by changes of a series of intracellular drug resistance-related protein. Among which, P-glycoprotein (P-gp), multidrug resistance-associated protein (MRP), lung resistance-related protein (LRP), Glutathione-s-transferase-π(GST-π) and Topoiso-meraseⅡα(TopoⅡα) has drew more attention. Many reports showed that P-gp, MRP, LRP, GST-π, and TopoⅡαplayed important roles in the intrinsic or acquired resistance in lung cancer. However, most of them only focused on one or two of these drug resistance related proteins, little information is known referring to all of these five mediators.In addition to cancer-cell-specific factors, mocroenviroment may also a crucial factor influencing chemoresistance in lung cancer. Because of poor vascularization, solid tumors usually contain hypoxic regions where glucolysis is the main means of ATP synthesis, and glucose deprivation often occurs, which trigger the glucose-regulated stress response of cancer cells, leading to the dramatically increased expression of glucose-regulated proteins(GRPs). GRP78 and GRP94 are the best studied GRPs. Our previous research showed that A23187 can induced the expression of GRP78 in multiple lung cancer cell lines, and its expression is related to the resistance to VP-16. Meanwhile, A23187 induced GRP94 expression is associated with the resistance to VP-16 as well in lung cancer. However, these results are all about the sigle cell line. In this work, we investigated the expression of GRP78 and GRP94 and evaluate whether their different expression can result in resistance discrepancy to VP-16 in mutiple lung cancer cell lines.Mutiple factors are suggested to be involved in drug resistance in lung cancer. Both drug resistance related proteins and GRPs can be important related contibutors. However, little is known about the correlationship between these factors.This work aimed at clarifying the effect of several drug resisitance related factors in lung cancer including P-gp, MRP, LRP, GST-πand TopoIIa on intrinsic and acquired drug resistance, the relationship between expression of GRPs and chemoresistance. Further more, correlation among them was well analyzed according to comprehensive factors mentioned above.Methods:1. The expression of P-gp, MRP, LRP, GST-πand TopoⅡwas examed by RT-PCR, Western blot and/or immunofluorescence in four histopa-thological subtypes of lung cancer cell lines (SK-MES-1, SPCA-1, NCI-H-460 and NCI-H-446) and the resistance to cisplatin, doxorubicin and VP-16 was detected by MTT assay. Analysis the relationship between the drug resistance-related protein and the resistance to cispaltin, doxorubicin and VP-16 in lung cancer.2. The expression of the drug resistance-related proteins in cisplatin- resistance lung adenocarcinoma cell line A549/DDP and the parental cell line A549 was detected by RT-PCR, Western blot or immunofluorescence3. The expression of P-gp with and without the pretreatment of verapamil was examed by RT-PCR and immunofluorescence in four histopa-thological subtypes of lung cancer cell lines. Cell survival to cisplatin and VP-16 was determined by MTT assay.4. The expression of GRP78 and GRP94 pretreated with A23187 was detected by RT-PCR, Western blot and immunofluorescence. MTT assay was used to determined cell survival to VP-16.5.Pearson correlation analysis was emplored to determine the relation-ship between drug resistance related proteins and GRPs so as to supply benefit indicator for individual treatment of lung cancer.Results:1. We found that the endogenous levels of P-gp, MRP, LRP, GST-πand TOPOⅡαin four cell lines vary. The level of GST-πin SK-MES-1was the highest, whereas the level of P-gp in SPCA-lwas the lowest. The chemosensitivity to cisplatin, doxorubicine and VP-16 in four cell lines were different. Both doxorubicin and Cisplatin were the most sensitive compounds to SPCA-1; VP-16 was the most sensitive compound to SK-MES-1. There was a positive correlation, between GST-πexpression and resistance to cisplatin, between TOPOⅡαexpression and resistance to VP-16; and a negative correlation between TOPOⅡαexpression and resistance to doxorubicin. Conclusively, different endogenous levels of P-gp, MRP, LRP, GST-πand TOPOⅡαin human lung cancer cell lines may suggest various chemosensitivity to cisplatin, doxorubicine and VP-16.2.Compared to parental cell line A549, the expression of P-gp and MRP enhanced and TOPO II a level decreased in cisplatin resistance cell line A549/DDP.3. The expression of P-gp at both mRNA and protein level can be obviously inhibited by verapamil in all the four cell lines. With the pretreat-ment of verapamil, NCI-H-446 was more sensitive to cisplatin; SPCA-1, NCI-H-460 and NCI-H-446 were more sensitive to VP-16 as well compared to the control group.4. We found that the endogenous levels of GRP78 in four cell lines vary, with NCI-H-460 was the highest. Compared to the control group, the expression of GRP78 and GRP94 at protein and GRP78 mRNA level with the A23187 pretreated was significantly increased in the four cell lines. The IC50 of VP-16 also showed significant elevation for the cells induced by A23187. There was a positive correlation, between endogenous levels of GRP78 and resistance to VP-16. With the pretreatment of A23187, the protein level of GRP78 and GRP94 was related to the resistance to VP-16 in four cell lines. And mRNA level of GRP78 was associated with the resistance to VP-16 in SK-MES-1, SPCA-1 and NCI-H-446 cell lines.5. Both drug resistance-related proteins and GRPs showed obviously basal expression in all four types of lung cancer cell lines. Except for P-gp,the other four drug resisitance-related protein exhibited similar expression tendency to GRP78 and GRP94 in all the fours cell lines.. There was a positive correlation between GRP78 and LRP(r=0.773, P<0.05).Conclusions:1. Co-expression of P-gp, MRP, LRP, GST-πand TOPOⅡa may play an important role on the intrinsic resistance in lung cancer, meanwhile the different expression in four histopathological subtypes of lung cancer cell lines may cause the various resistance to anticancer drugs. GST-πmay be significant for prediction of the intrinsic resistance to cisplatin, whereas TOPO II a may be a valuable protein for estimating the intrinsic resistance to doxorubicine and VP-16.2. P-gp, MRP and TOPOⅡαmay be important factors for the acquired resistance to cisplatin in lung cancer3. Down-regulation of P-gp is associated with the intrinsic resistance to cisplatin in NCI-H-446 cell line and to VP-16 in SPCA-1, NCI-H-460 and NCI-H-446 cell lines.4. Various chemosensitivity to VP-16 may be resulted from their different endogenous levels of GRP78 in human lung cancer cell lines. Expression of GRP78 and GRP94 at protein level is associated with the intrinsic resistance to VP-16 in all four cell lines, while mRNA expression of GRP78 is related to the resistance to VP16 in SK-MES-1, SPCA-1, NCI-H-446 cell lines.5. Both drug resistance-related proteins and GRPs are interacting important factors involving in lung cancer chemoresistance. Therefore, combined detection of two groups proteins can not only contibute to confir-ming chemoresistance mechanism and evaluating therapeutic effect, but also offer effective and rational guidelines in lung cancer chemo-treatment.