| Mycophenolic acid (MPA) is a selective inhibitor of inosine monophosphate dehydrogenase (IMPDH). It possesses immunosuppressive, anticancer, antibacterial, and antiviral properties. An ester prodrug of MPA, mycophenolate mofetil (MMF, CellCept), has been developed as an immunosuppressive agent to prevent the rejection of organ allografts and GVHD after bone marrow transplantation.The main mechanism of MPA is that it inhibits IMPDH which is the rate-limiting enzyme in de novo synthesis of guanosine nucleotides. T-and B-lymphocytes are more dependent on this pathway than other cell types. Moreover, MPA is a fivefold more potent inhibitor of the type II isoform of IMPDH, which is expressed in activated lymphocytes, than of the type Iisoform of IMPDH, which is expressed in most cell types. MPA has therefore a more potent cytostatic effect on lymphocytes than on other cell types.Besides this, other mechanisms may also contribute to the efficacy of MPA. One of the most important is that MPA can induce cell apoptosis, and may be selective for lymphocytes and monocyte-macrophage lineage cells. Several reports show that MPA can induce apoptosis in activated T-lymphocytes, whereas opposite opinions also exit. Gu's group have examined the induction of apoptosis by MPA in interleukin-3 dependent murine hematopoietic cell lines, and found that MPA treatment, at clinically relevant doses, caused apoptosis in 32D myeloid cells and in FL5.12 and BaF3 pre-B cells in the ongoing presence of IL-3, suggesting their utility in chemotherapeutic regimens for hematological malignancies. We examined the induction of apoptosis by MPA in Molt-4 cells, which derive from a human leukemic T cell line, by morphological observation, DNA agarose gel eletrophoresis and flow cytometry assay. Raji cells, which derive from a human Burkitt's lymphoma cell line, was used as a cell control. Cyclosporine A (CsA) was used as a drug control.Apoptotic morphology was observed on Wright-Giemsa stain. Characteristic changes for apoptosis were found in Molt-4 cells after 72hours exposure to 10μmol/L MPA. The nuclear chromatin was condensed into crescents closely apposed to the nuclear membrane, and then fragmented into several dense spheres. The cells had condensed cytoplasm with increased electron density, and cytoplasmic blebs containing intact organelles and nuclear fragments. Transmission electron microscope confirmed these changes. No apoptotic morphological changes were observed in MPA treated Raji cells or CsA treated Molt-4 cells.Apoptotic ladder pattern of DNA fragmentation of Molt-4 cells was also observed in the DNA agarose gel electrophoresis in the presence of MPA. No ladders were found in MPA treated Raji cells or CsA treated Molt-4 cell.The positive cells of Annexin VFITC on the membrane were detected by flow cytometry. The results showed that treated with 0-10μmol/L MPA for 0A-48 hours, the percentage of Annexin V positive Molt-4 cells increased with dose (r=0.807, P0.001) and time (r=0.467, P=0.014), suggesting that MPA could induce apoptosis in Molt-4 cells in dose- and time-dependent manners. Exposed 48 hours to MPA ( 0, 0.1μmol/L, 1μmol/L, 10μmol/L), the percentage of Annexin V positive Raji cells were 0.92%±0.28%, 1.04% ±0.49%, 1.36% ±0.93% and 1.9% ±0.3% respectively, with no significant difference (F=1.79, P=0.227), suggestingthat MPA at these concentrations could not induce apoptosis in Raji cells. Exposed 48 hours to CsA ( 0, 0.1μmol/L, 1μmol/L), the percentage of Annexin V positive Molt-4 cells were 14.42%±1.46%, 15.08%±1.01% and 16.52% ±1.86% respectively, with no significant difference (F=1.576, P=0.282), suggesting that CsA at these concentrations could not induce apoptosis in Molt-4 cells.The cell cycle of Molt-4 cells was analyzed by flow cytometry, showing the apoptotic peak before G1 phase cells after treated with MPA at the concentrations from 1μmol/L to 10μmol/L. The percentage of apoptotic peak cells increased in a dose dependent manner (r=0.888, P=0.001). Furthermore, in...
|
PDF Full Text Request