The Establishment Of Bortezomib Resistant Myeloma Cell Line And Study Of Its Drug Resistance Mechanism

Background and objectivesMultiple myeloma (MM) is an incurable B-cell malignancy resulting in significant morbidity and mortality. MM accounts for 1% of all cancers and slightly more than 10% of hematological malignancies. Although many therapeutic advances such as combined chemotherapy and hematopoietic stem cell transplantation have been made to improve the survival rate of patients with MM, a higher proportion of the patients cannot expect the long-term remission due to drug-resistant disease, and minimal residual disease, leaving limited therapeutic option.Proteasome inhibitors (PIs), such as bortezomib, have excellent clinical activity in patients with multiple myeloma. Although they exert broad effects on cancer cells, their ability to (1) stabilize pro-apoptotic members of the BCL-2 family, (2) inhibit the two major pathways leading to NF-κB activation, and (3) cause the build-up of misfolded proteins appear to be particularly important. In addition, PIs may disrupt tumor–stromal interactions that drive NF-κB activation and angiogenesis and in such a way sensitize cancer cells to other agents. Still, drug resistance ultimately emerges in all patients that initially respond to PIs.In clinical trials with bortezomib in relapsed or refractory multiple myeloma (MM), where the drug displayed significant single-agent anti-tumor activity, and it received FDA approval in 2003. Although bortezomib has produced unsurpassed antitumor activity in MM, drug resistance has already emerged as a major challenge. Some patients with multiple myeloma failed to respond to the bortezomib therapy, and no one has been cured of disease with the drug. Thus, laboratory efforts are underway to identify the mechanisms underlying resistance and develop strategies to reverse it.In this article we try to establish a bortezomib resistant myeloma cell line and study of its drug resistance mechanism.Part I. The establishment of poteasome inhibitor bortezomib resistant myeloma ce11 lines and study of its gene expression profileThe bortezomib-resistant NCI-H929 cell line (NCI-H929B)was derived from the parental NCI-H929 cell line in our laboratory,by stepwise increasing extracellular concentrations of bortezomib over a period of 8 months, starting at a concentration of 10 nM (IC50 concentration: 20.7 nM) up to a concentration of 200 nM bortezomib.To investigate the stability of the resistance phenotype, an aliquot of NCI-H929B cells was cultured in the absence of bortezomib for a period of up to 2 weeks.Bortezomib-resistant NCI-H929 myeloma cells were selected by continuously exposing cells to gradually increasing concentrations of bortezomib. Over a period of 8 months,H929 cells grown in the presence of 200 nM (NCI-H929B )bortezomib were used for further characterization. This cell line (NCI-H929B) exhibits a 23.5-fold level of resistance to bortezomib as compared to the parental cell line.Gene chip was used to detect gene expression of the NCI-H929 cell and NCI-H929B cell. 317 genes were up-regulated in NCI-H929B cell, while 356 genes were down-regulated in NCI-H929B cell. Based on previous gene expression profiling analysis, We used GO- analysis and Pathway- analysis to decide whether or not to follow up a category that appears enriched in different genes,then we would know the statistical reliability of the apparent enrichment. The source of analysis comes from pathway database, such as KEGG and Genmapp.By the Pathway analysis we found that Apoptosis pathway maybe very important in the resistance of Bortezomib. Quantative PCR analysis of bortezomib resistance associated gene Casp7,TRAF2,FADD,Casp8,MAP3K5and Hsp86 were examined. The mRNA expression levels of HSP 86 gene were increased significantly in H929B compared with parental NCI-H929 cell, but FADD,Casp8,MAP3K5 gene were decreased significantly in NCI-H929B cell compared with parental NCI-H929 cell.Conclusion:Bortezomib resistant myeloma cell line could be established by stepwise increasing extracellular concentration induction. There are no significant differences in cell cycle distribution and growth curve between NCI-H929 cell and NCI-H929B cell. Gene expression profiling and Pathway-analysis demonstrated that Apoptosis pathway maybe very important in the resistance of Bortezomib.Quantative PCR analysis showed that the mRNA expression levels of Caspase 8 gene were decreased significantly in NCI-H929B compared with parental NCI-H929 cell. Inhibition of Caspase 8 may lead to resistance of bortezomib in myeloma.Part II. Differential Proteomics Study of Bortezomib sensitive and drug-resistant myeloma cells. We separated the whole proteins of NCI-H929 and NCI-H929B myeloma cell lines by two-dimensional gel electrophoresis. Gel-image analysis revealed that there were 17 differential protein spots. A total of 14 differential protein spots were successfully identified by MALDI-TOF-MS,of which HSP 75,Alpha-enolase, Annexin A1, Stress-induced-phosphoprotein 1, Protein disulfide-isomerase A3, Putative deoxyribonuclease TATDN1,Heat shock protein beta-1,Tubulin beta-6 chain, Keratin and F-actin-capping protein subunit beta were up-regulated compared with NCI-H929 cell, and Adenosylhomocysteinase,ATP synthase subunit beta, mitochondrial and Arylsulfatase J precursor were down-regulated compared with NCI-H929 cell. Western blot was used to further verify differential proteins in order to ensure the reliability of the proteome results. The results showed that expression of protein DJ-1 was significantly higher in NCI-H929B cell line and primary myeloma cells of bortezomib resistant myeloma patientsConclusion:Fourteen differential proteins are characterized .11 differential proteins were up-regulated in NCI-H929B cell line, and 3 were significantly reduced. Western blot verified that expression of protein DJ-1 was significantly higher in NCI-H929B cell line and primary myeloma cells of bortezomib resistant myeloma patients. This result suggested protein DJ-1 maybe important in bortezomib resistance.