Mechanisms Of Tumor Resistance To-Tinib Agents And No-Donating Compound Against Multidrug Resistant Cancer Cells

Background: Multidrug resistance (MDR) is a major obstacle to successful cancer chemotherapy, sothe mechanisms of drug resistance and investigation of novel strategy are urgently required to improve theefficacy of chemotherapeutic agents against MDR. The epidermal growth factor receptor (EGFR) is overexpressed in many human cancers, including hepatocellular carcinoma prevalence in our country. RecentlyEGFR-TKI targeted drugs have been tested in advanced liver cancer, and some made breakthroughprogress in clinic. But the potential problem of drug resistance deserves our attention. It has been reportedthat NO was able to overcome MDR-ABC mediated multidrug resistance. In the present study, weinvestigated the mechanisms of tumor resistance to EGFR-TKI, including erlotinib、gefitinib、vandatinib inHepG2resistant cells and evaluated NO-donating compound AB-19against classic ABC transportermediated multidrug resistance in cancer cells.1. The mechanisms of tumor resistance to erlotinib、gefitinib、vandatinib.Objective: To establish three human hepatocellular carcinoma cell lines which its resistance toErlotinib、Gefitinib、Vandatinib and investigate their possible resistant mechanisms. Methods: The cell lineHepG2was cultured by gradually increasing dose of Erlotinib、Gefitinib、Vandatinib and high-dosestimulation in vitro to generate their resistance cell lines HepG2/Erlotinib、 HepG2/Gefitinib andHepG2/Vandatinib. To study their possible mechanisms of the resistance, SRB was used to test theirresistant indexes; cell growth curve, doubling time and cell cell cycle phase distribution were measured;RT-PCR, Western blotting, and Flow cytometry were used to examine drug-resistant bcrp mRNA and theirprotein level, cDNA microarray and2-DE technique were used to detected the different genes and proteinsbetween HepG2and HepG2/Gefitinib. Results:The resistant cell lines HepG2/Erlotinib、HepG2/Gefitiniband HepG2/Vandatinib were established with resistant index of4to10. The resistance cell line also hadcross resistance to cisplatin、doxorubicin、mitoxantrone、topotecan and paclitaxel while HepG2/Erlotinib excepting for paclitaxel. The doubling time of resistant cell lines were longer than their parental cells. TheS and G2/M phases of resistant cell lines were increased while G0/G1phase was decreased. The resistantcell lines showed higher levels of bcrp mRNA and BCRP protein compared with parental cells. Theup-regulation of genes and proteins by cDNA microarray and2-DE technique may lead to find itsresistance factors to Gefitinib. Conclusion: Three resistant human hepatocellular carcinoma cell lineHepG2/Erlotinib、HepG2/Gefitinib and HepG2/Vandatinib were established. They showd the typicalresistant phenotype and biological characteristics. The high BCRP expression may contribute to itsresistance to the three drugs and the up-regulation of some genes and proteins detected by cDNAmicroarray and2-DE technique may lead to its resistance to Gefitinib, these possibilities need to beevaluated in the future studies.2. NO-donating compound against multidrug resistance in cancer cells.Objective: To evaluate the antiproliferative activities of NO-donating compound AB-19comparedwith core structure AB against classic MDR in prental and resistant cells, incluing HEK293/vector,HEK293/BCRP, HEK293/MRP1, K562, K562/A02. To test the effect of AB-19of on MDR-related P-gp、BCRP、MRP1and other signal protein. Methods: MTS and SRB were used to test the antiproliferativeactivities of compounds AB-19and AB against HEK293/vector, HEK293/BCRP, HEK293/MRP1, K562,K562/A02, HepG2and LO2cells; The levels of nitrate/nitrite produced by AB-19or AB in the cells weredetermined by the nitrate/nitrite colorimetric assay kit; To further determine the contribution of NOreleased by AB-19to its antiproliferative activity against tumor cells, the NO-eliminating experiment wascarried out by adding NO scavenger hemoglobin. The effects of AB-19on the intracellular accumulation offluorescent substrates were testd by flow cytometry. Western blotting were used to test the effect of AB-19on the MDR-related P-gp、BCRP、MRP1and other signal protein. Conclusion: Compound AB-19showedsignificantly potent growth inhibitory effects on MDR cell lines, and exhibited no difference in potencyagainst the parental sensitive and drug-resistant cell lines which overexpress three MDR-ABC transporters.AB-19exhibited selective antiproliferative activities against tumor cells as compared with nontumor cells,probably due to produce much higher levels of NO in tumor cells. Moreover, AB-19inhibited a number ofkey signaling pathways implicated in cancer drug resistance and tumor proliferation, including p-AKT、p-ERK、p-NF-κB and HIF-1α in a dose-and time-dependent manner. Given that acquired MDR to NO donors was difficult to achieve and genetically unstable, compounds like AB-19may be promisingcandidates for further intensive study for the intervention of human multidrug resistant cancer cells.