GENETIC POLYMORPHISM AND RELATIVE EXPRESSION OF MDR1 IN ACUTE LYMPHOBLASTIC LEUKEMIA

Abstract

Chemoresistance against multiple anti-cancer drug regimens being reported in acute lymphoblastic leukemia (ALL) is one of the major therapeutic concerns against the refractory childhood leukemia in the region. The current research project was aimed to study the genetic basis of individualized drug responsiveness and multidrug resistance among the local population suffering from ALL, based on a most important SNP rs1045642 (C3435T) on exon 26 of mdr1 gene. The SNP rs1045642 is associated with functional polymorphism of human permeability glycoprotein (P-gp). One hundred total human subjects were enrolled (n=100) both male and female ALL patients from local public hospitals including a healthy group (n=20). Automated complete blood counts (CBC) and microscopic examination of peripheral blood film were done. A phenotypic study of P-gp in the normal group was performed through HPLC-based measurement of plasma digoxin level followed by its oral administration. Protein expression of Mdr1 among the study population was evaluated by plasma P-Glycoprotein activity by highly specific Enzyme-Linked Immunosorbent assay (ELISA) based on the human P-gp monoclonal antibody. The mdr1 gene region of genomic DNA extracted from human lymphocytes was amplified by PCR using specific primers and the PCR product was run on agarose gel electrophoresis. The obtained amplified PCR product was restricted with the restriction endonuclease enzyme MboI specific for the mdr1 gene (for rs1045642) by restriction fragment length polymorphism (RFLP) to study the genotypes. The genotype frequency of MDR1 C3435T polymorphism for CC, CT, and TT variants were 35%, 43.75%, and 21.25% respectively. The MDR1 C3435T polymorphism has found significantly associated (p-value = 0.013) with plasma P-gp expression (ng/mL). The wild-type homozygous 3435CC individuals demonstrate the hyperexpression of P-gp as compared to 3435TT genotypes. The higher P-gp activity in 3435CC genotypes was found to be associated with relatively lower plasma digoxin concentration at predefined time intervals after oral digoxin intake (0.25mg), owing to the P-gp mediated rapid drug efflux resulting in relatively poor absorption from the intestine. The mutant homozygous 3435TT genotype presented lower P-gp activity allowing the higher digoxin uptake resulting in increased digoxin plasma level as compared to individuals with CC genotype. The findings for heterozygous CT genotypes were found inconclusive as variable P-gp phenotypes were observed among the study population. The B-Cell Lymphoma protein-2 (BCL-2) confers chemoresistance as well, through influencing the cancer pathophysiology. Plasma BCL-2 was also quantified by double antibody ELISA method. Plasma total oxidant status and total antioxidant status of leukemia and control samples were evaluated. Serum level of Lactate Dehydrogenase (LDH) predicts the increased anaerobic glycolysis and associated with metabolic modulation in the cancer cells. A strong Warburg effect explained by exaggerated anaerobic glycolysis in cancer cells was depicted by marked high plasma LDH concentration in leukemia population as compared to control group (p<0.05). In the present research, the interplay of BCL-2, total oxidative status (TOS) and LDH has been investigated in ALL patients. The student’s t-test and one-way ANOVA was applied to find out the statistical significance of study groups and genotypic association with phenotypes was evaluated by applying the Pearson’s chisquare test of association. The phenotypic behavior of study population associated with genotypes has been presented by component bat charts, frequency distribution histograms, and probability plots.