| Background and purpose: Treatment of the acute promyelocytic leukemia with arsenic trioxide obtained prominent therapeutic effect in clinical work, especially the patients of drug resistance to retinoic acid and refractory acute promyelocytic leukemia. The mechanisms of treating tumor with arsenic trioxide included: (1)the degradation of PML-RARαprotein; (2)to regulate the expression of interrelated genes of apoptosis; (3) to induce cell apoptosis by the mitochondria-dependent pathway; (4) to induce tumor cells differentiation; (5)to resist tumors by protoplasmic poison and peroxylradicals; (6)to damage DNA directly; (7)to inhibit angiogenesis; (8) to downregulate the activity of the telomerase. The main mechanisms of treating the acute promyelocytic leukemia with arsenic trioxide included that it induced APL cells differentiation and aopotosis, as well as controlled cell cycle progress and interrelated signal transduction pathway. Rapamycin was a macrolide antibiotic with antifungal, immunosuppressive, and antitumor properties, it formed a complex with FKBP-12, a specific immunophilin intracellular, and inhibited the catalytic activity of mTOR protein kinase by binding to the FKB domain of mTOR. Rapamycin weakened or blocked p70S6K and 4EBP1/eIF4E proteins phsphorylation and activation, which were downstream effectors of mTOR, to intercept the conduction of nutrition factors and growth factors transduction signal, and to inhibit the initiation of translation. Rapamycin arrested cell cycle in G1 phase to antitumor by degrading the CDK-cyclin complexes kinase activity. In recent years, the tumor is known as a kind of "cell cycle disease", so the cell cycle phases became targets as anticancer drugs, the effect is weaker that single target drug induced apoptosis of the tumor cells, but the effect of multitude target drugs maybe surpass single drug, and the toxic reaction relieve possibly. According to the cell cycle different targets or phases, this experiment applied rapamycin in combination with arsenic trioxide to treat the APL cell line NB4 cells, and expect that arsenic trioxide and rapamycin have synergetic effect in apoptosis. The purpose of this experiment is to investigate the synergistic action of arsenic trioxide and rapamycin in inducing NB4 cell apoptosis, by observing the effect of apoptosis, cell cycle and the expression of related proteins, so as to provide an experiment evidence to treatment of APL more effectivly. Methods: NB4 cell treated with different concentrations (0.5~4.0μmol/L) of arsenic trioxide or in combination with rapamycin (20nmol/L) 16h or 20h, the cell proliferation was detected by trypan blue dye-exclusion; the morphologic variance of apoptotic cells was observed by Giemsa dye; apoptosis and cell cycle were investigated by flow cytometry analysis; agarose gel electrophoresis detected apoptotic DNA ladder; western blot analysis detected the expression and modify of apoptosis related pretein caspase-3, ERK1/2 signaling pathway protein, mTOR downstream signaling pathway protein 4EBP1/eIF4E and p70S6K, as well as cell cycle protein p34cdc2 and cyclin B1. Results: Treatment with arsenic trioxide for 16h, the total cell number and viable count of NB4 cells reduced by trypan blue exclusion. After Giemsa staining, NB4 cells changes in morphous were observed under the light microscope, these changes included cell volume became smaller,cellular membrane was integrated, karyopycnosis became the irregular shape or the round and formed typical apoptotic bodies. Flow cytometry results showed that the fraction of NB4 cells in G2/M phase progressively increased with an increment of arsenic trioxide from 0.5~4.0μmol/L and a prolongation of treatment time, accompanying a reduction of the cells in G1 phase. DNA content analysis indicated that 0.5μmol/L arsenic trioxide treated NB4 cells in 16h, the hypodiploid apoptotic peak (AP) preceded to appear in G1 phase, and with an increment of arsenic trioxide, the fraction of apoptotic cell increased. Agarose gel electrophoresis of genomic DNA from NB...
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