Antitumor Effect And Pharmacokinetic Properties Of F951, A Novel Antisense Phosphorothioate Oligodeoxynucleotide To Bcl-2, On Human Lymphoma Cell In Vitro And In Mice

As an antiapoptotic oncogene, Bcl-2 is overexpressed in a variety of malignant tumors and has been an attractive target gene for antisense therapy due to its antiapoptotic and chemoresistant functions.F951 has been designed as a novel antisense phosphorothioate oligodeoxynucleotide to Bcl-2 with different sequence in our laboratory. To evaluate the potential therapeutic use of F951 in the treatment of cancer, we studied the antitumor efficacy of F951 alone or its combined efficacy with low-dose Ara-c on human Burkitt's lymphoma cell CA46 in vitro and xenografts in nude mice. Plasma pharmcokinetics, biodistribution, excretion and subcellular distribution of 3H-radiolabeled F951 were also investigated in mice and in CA46 cells respectively. The results indicate that F951 is a promising anticancer drug with favorable antitumor effect and pharmacokinetic properties.1. Efficacy on CA46 cells.Tow control groups, untreated group and FNS18(scramble sequence)-treated group, and tow experimental groups, F951 (15uM)-treated group and group treated with F951(15uM) in combination with Ara-c(2 u M) (F951+Ara-c), were designed in this study. It was found that F951 was able to specificallyinhibit the proliferation and growth of CA46 cells in vitro compared with control FNS18-treated and untreated cells. The combination F951 with low-dose Ara-c was able to suppress the proliferation and growth of CA46 cells beyond that obtained with the treatment given alone. 72h of treatment, a significant difference of cell growth was found by the MTT method among the four groups. The percent cell growth inhibition of F951+Ara~c-treated group, F951~treated group and FNS18 control group against the untreated group were 55. 10%, 38. 26% and 2. 06% respectively. After 24 hours treatment, significant reduction of Bcl-2 mRNA were found by real time RT-PCR method in F951~treated and F951+Ara-c~treated groups. Compared to the untreated group, the reduction excelled 50%. No same change was found in FNS18 control group. After 48 hours treatment, Bcl-2 protein markedly decreased in F951-treated and F951+Ara-c-treated groups examined by FACS. The Bcl-2 protein positive ratios of the four groups were 91.49%, 80. 14%, 67. 11% and 41.77% in order. Apoptotic cells in F951-treated and F951+Ara~c-treated groups were more popular than in untreated and FNS18-treated control groups detected by the TUNEL and MitoCapture?Mitochondrial Apoptosis Detection methods. In summary, F951 is able to specifically inhibit the growthand proliferation of CA46 cells and induce apoptosis in vitro. A synergistic antitumor effect is quite obvious when F951 combined with low-dose Ara~c.2. Antitumor effect of F951 on human Burkitt's lymphoma cell CA46 xenograft in nude mice.After receiveing intraperitoneal injection of CTX lOOmg/kg, Nude mice were administered a subcutaneouse injection of CA46 cells. When the tumor masses were visible, 20 tumor-bearing mice were randomly divided into four groups. Reagents of 0. 2ml of saline(NS), lOmg/kg of FNS18, lOmg/kg of F951 and lOmg/kg of F951 in combination with low-dose Ara-c(F951+Ara-c) were injected directly into the tumor masses of each groups respectively every day. The therapy lasted for 21 days.During the course of the therapy, tumor size in NS and FNS18 control groups increased continuously and in F951-treated group increased slightly in the first week and then reduced continuously. Continuous reduction of tumor size was found in F951+Ara-c~treated group as soon as the therapy beginning. According to tumor volume, there was no significant difference between NS control group and FNS18 control group (p>0.05), F951-treated group and F951+Ara-c-treated group (p>0.05). However, significant difference appeared between F951-treatedgroup and two control groups respectively after 21 days treatment (p<0.05). As early as the 7th day, significant difference had firstly appeared between F951+Ara-c-treated group and FNS18 control group (p<0.05). From the 14th day, significant difference appeared between F951+...