Study On The Antitumor Effect And Toxicity Of F951, A Novel Antisense Phosphorothioate Oligodeoxynucleotide To Bcl-2, On AML, Both In Vitro And In Mice

As one of the antisense phosphorothioate oligodeoxynucleotide to Bcl-2 with different sequence, F951 developed in our laboratory has been demonstrated to dramatically inhibit the growth of Burkitt's lymphoma both in vitro and in mice. Its valuable pharmacokinetic parameters have been investigated in previous studies in vivo. The present study is to investigate the antitumor effect of F951 on Acute Myeloid Leukemia (AML) with overexpression of Bcl-2 protein, and the toxicity of F951 as well, both in vitro and in mice, so as to provide valuable data for its further clinical application.In this study, antitumor effects of either F951 or F951 combined with low-dose of Ara-C to AML were determined in HL-60 cells in vitro and in nude mice with HL60 cells xenograft. In addition, CHL cells and BALB/c mice were used to assess the general, hereditary and acute toxicities of F951 to normal cells and to mice.The efficacy of F951 against HL-60 cellsHL-60 cells were cultured with F951 in variant doses alone or with F951 combined with low-dose Ara-C. The proliferations of HL-60 cells were assayed by MTT and Typan Blue dye exclusion test.Expressions of Bcl-2 protein and its mRNA were measured by FACS and semi-quantitative RT-PCR respectively. The apoptotic cells were detected by transmission electron microscope, DNA ladder,MitoCapture?Mitochondrial Apoptosis Detection method the and the elevation of caspase activity in plasma. The results show that F951 inhibit the HL-60 cells proliferation significantly in the range of dosages studied, which indicated dose-dependent effects and synergistic effects while in combination with low-dose Ara-C. These may be due to its capacities to inhibiting Bcl-2 expression specifically, down-regulating Bcl-2 mRNA and protein in HL-60 cells, and then triggering cell apoptosis.The efficacy of F951 against AML xenograft in nude mice F951 alone and in combination with low-dose Ara-C were administered via intraperitoneal injection or injection directly into the mass of tumor respectively to evaluate their effects on growth of tumor and survival of mice with HL60 cells xenograft. In the experiment, levels of Bcl-2 mRNA and protein in tumor cells were assayed by real time RT-PCR and FACS respectively; histological and microstructural changes were detected by light microscope and transmission electron microscope respectively; tumor cell apoptosis detected by mitochondrial apoptosis, activity of caspase, TUNEL assay, microstructure in electron microscope, after the treatment with either F951 alone or in combination of Ara-C. The results showed that regimens of F951 both inhibited growth of AML xenograft significantly and prolonged the duration of survival of mice with AML xenograft. In comparison, the efficacy of F951 administered by injection into mass looked better than intraperitoneal, and presented synergistic effect when combined with Ara-C. Thus, it has been concluded that F951 could specifically inhibit or downregulate Bcl-2 expression in nude mice with AML xenograft so that the apoptosis of tumor cell may be triggered, and antitumor effects of chemotherapy agents could be promoted bythis way.General pharmacological effects and acute toxicity of F951 in vitro in miceAccording to 'Instructional principle for preclinical study on novel medicine', BALB/c mice were used to investigate the toxic reactions of different dosages of F951 administered intravenously via tail-vein in mice.General pharmacological investigation: four groups of dosages (2, 6, 18, 54 mg.kg-1 respectively ) intended to clinical use of F951 were set to test its general pharmacological effects. Behaviors of mice were examined systematically, including hepatic function, renal function, blood cell counts, state of coagulation, as well as pathological and microstructural profiles of major organs. As a result, no adverse effects except for its therapeutical effects of F951 were found in the genral pharmacological test. The hepatic function, renal function, blood cell counts, state of coagulation, as well as...