Downregulation of BHRF1 in chronic lymphocytic leukemia cells in order to enhance apoptotic effects of doxorubicin

Abstract

Resistance of leukemic cells to apoptosis is one of the most important problems in the treatment of various leukemias. Chemotherapy drugs such as doxorubicin have been routinely used in the treatment of leukemias such as chronic lymphocytic leukemia (CLL), which have unfortunately been associated with low efficacy. Therefore, one of the ways that can be used to treat leukemias is to inhibit the ways of resistance to apoptosis in leukemic cells. One of the important factors that plays an important role in resistance to apoptosis in malignant cells is BHRF1 factor, which is homologously Bcl-2 homologous and leads to resistance of leukemic cells to programmed cell death. Therefore, in this study, we decided to reduce their susceptibility to doxorubicin-induced cell death by inhibiting BHRF1 expression in CLL cells. For this purpose, we used chitosan-based nanoparticles to transfer BHRF1 siRNA and doxorubicin to leukemic cells. Materials and Methods: In this study, carboxymethyl dextran coated trimethyl chitosan nanoparticles were used for simultaneous delivery of BHRF1 siRNA and doxorubicin to leukemic and normal cells. H1NMR and FTIR tests were used to study the chemical structure of the synthesized nanocomplexes. The physicochemical properties of the nanoparticles were also evaluated by DLS. SEM microscope was also used to study the morphology of the produced nanoparticles. Also, the entry of nanoparticles into leukemic cells was examined by confocal microscopy and flow cytometry. Toxicity assessment of nanoparticles loaded with siRNA and doxorubicin molecules was also assessed by MTT assay. The expression of the desired genes was also examined using real-time PCR technique. Flow cytometry was also used to evaluate apoptosis in leukemic and normal cells. Results: Physicochemical analysis of the produced nanoparticles showed that they were about 105, zeta potential 18 and PDI <0.2 and could load siRNA and doxorubicin well and deliver them to malignant cells. Cytotoxicity showed that the nanoparticles did not have much toxicity on the cells, while nanoparticles loaded with siRNA and doxorubicin significantly caused cell death. In addition, co-administration of BHRF1 siRNA and doxorubicin to cells induced apoptosis in cells.