Screening Of Drug Resistance Related Protein By Proteome And Mechanisms Of APL Cells Resistant To ATRA

Introduction: Acute promyelocytic leukemia (APL) is a special subtype of the acute myelocytic leukemia whose morbidity account for approximate 3.3 to 17.4 percentages. The ATRA-induced differentiation methods, the pioneer treatment of APL established by domestic scientist in 1986, has been proven to elevating the complete remission (CR) rate of APL up to 85% and been widely recognized as a success model. However, the recurrence of APL is frequent within few months after treatment with ATRA alone, and this will results in ATRA resistance. Some research had elucidated that plasma concentration of ATRA did not change with increased administration, and this suggested that ATRA resistance is related to changes in pharmacokinetics. In Vitro, the insensitivity to ATRA in primary recurrent APL cell and the formation of ATRA resistant cell lines suggest that more complicated mechanisms must be involved in ATRA resistance.Former researches indicate that the mechanism of drug resistance of APL cells to ATRA changes not only on the level of gene, but also relates to the change of proteome arrangements, especially the change of protein signal molecules. Proteins are the direct carriers of life functions. When the abundance of mRNA is not positively correlated with the content of proteins or when modulation, such as protein modification, is involved, the study of this field is out of the reach of the genome. With the development of proteome research, it is feasible to study the rules of life activities from the proteins level of cell. Proteome can help find new markers for early diagnosis of tumor through large scale and high-flux analyzing all proteins expressed in some cell or some kind of tissue at the very time in the very place. It can also break through the limitation of conventional study on the very protein or gene, and provide new methods to study the mechanism of tumor drug resistance. Therefore, proteome is widely applied to the study of the pathogenesis, the drug resistance, and the diagnosis of malignant tumors.This study analyzed the expression differences of protein in retinoic acid sensitive and resistant cell strains with proteomic methods, screened and identified the possible proteins related to retinoic acid resistance, and discussed the mechanism of its drug resistance.In recently years, great effects have been achieved on the treatment of tumors with retinoic acid combined with interferons. As a member of GRIM family, the overexpression of GRIM19 can increase the death rate of IFN/RA sensitive cells. IFN-βcombined with RA can also induce the expression of GRIM19 in other tumor cells. Stat3 signaling pathway is related to cell proliferation, differentiation and apoptosis, the activation of which may result in abnormal proliferation and malignant transformation of cells. Cell death caused by GRIM19 may relate to the transcription inhibition of Stat3 and its downstream genes; it may also relate to the production of reactive oxygen species.Objective: to screen the resistance related protein in APL cell by proteomics; and to survey the inhibitory effect by combination of IFN-αand ATRA on NB4 and NB4R1, to analyze GRIM19, STAT3, Bcl-2, Bax, M2-PK, Eno, NF-κB/p65 expression after IFN-αand ATRA treatment; then to discuss the mechanism of APL cells to ATRA. Method: To perform the 2-dimensional gel electrophoresis analysis of ATRA sensitive (NB4) and resistant cell line (NB4-R1); to identify of the differential expression protein by MALDI-TOF-MS; to observe the proliferation rate by MTT and evaluate the optimized inhibitory concentration. And to observe the mRNA expression of IκBαby RT-PCR method and detect the expression of GRIM19, STAT3, Bcl-2, Bax, M2-PK, Eno, NF-κB/p65 by Western blotting methods.Result: There were 36 differential expression proteins, among them 15 were up-regulated and 17 were down- regulated in NB4-R1 cell line, subsequently it was identified as pyruvate kinase, enolase, chain A, Triosephosphate Isomerase, glutamate dehydrogenase, and 3 unnamed proteins was also found. Compare to control group and the group of ATRA, IFN-αtreatment alone, 40uM ATRA and 1000u/ml IFN-αadministration significantly suppressed the proliferation of NB4 and NB4-R1 cell; Enhanced GRIM19, Bax expression and decreased STAT3, Bcl-2, M2-PK, Eno, IκBαexpression were also observed in NB4 cell line by RT-PCR and Western blotting methods. No GRIM19 expression was detect in NB4-R1 control group, the expression of GRIM19, Bax were significantly low as compare to NB4 cell line treated with the same dose of IFN-α/ATRA. In NB4-R1cell line, STAT3, Bcl-2, Bax, IκBαexpression were relative high as compare to NB4 cell line, and there is no obviously change in the group treated with IFN-α, ATRA alone or combination.Conclusion:1. Our result demonstrated that differential expression of proteins may be involved in ATRA resistance by participating the process of glycometabolism, stress reaction and signal transduction.2. the abnormal expression of GRIM19/STAT3 induced by IFN-α/RA treatment is related to PK expression, and this indicates the relationships between GRIM19/STAT3 signal transduction pathway and ATRA resistance of tumor cells.3. Bcl-2 expression was related with GRIM19, GRIM19 and Bcl-2 expression is a sensitive index of mitochondrial function monitor. GRIM19 has a close relationship with glycometabolism.4. IFNαand ATRA showed a synergistic suppression effect on ATRA resistance cell line, this suggests that IFNαcan increase sensitivity of NB4-R1 cell line to ATRA, and this may be from the participating of GRIM19, STAT3 and/or Bcl-2/Bax pathway.5. HSP60, G protein, vitamin D binding protein and tropomyosin 3 expression also changes in RTRA resistance cell line, the results of NF-kB activation induced by IFN-α/RA indicates that other resistance mechanisms can not be excluded.