Antitumor Effect Of F951, A Novel Antisense Phosphorothioate Oligodeoxynucleotide To Bcl-2, On Human Small Cell Lung Cancer In Vitro And In Mice

As an oncogene, Bcl-2 is overexpressed in a variety of malignant tumors andhas been an attractive target gene for antisense therapy due to its antiapoptoticfuntion and chemoprotective effects. F951 is a novel antisense phosphorothioate oligodeoxynucleotide to Bcl-2 ,which was designed in our laboratory and has different sequence to others. Toevaluate the potential therapeutic use of F951 in the treatment of cancer and fund abase for the clinical experience, we studied the antitumor efficacy of F951on humansmall cell lung cancer cell line (NCI-H446, which has been demonstrated high Bcl-2expression.) in vitro and its xenografts in nude mice. The first part of this experience is to establish a successful xenotransplantationmodel of a human small cell lung cancer cell line (NCI-H446) in nude mice.NCI-H446 cells were transplanted subcutaneously into nude mice. The growthfeatures, histology, ultramicroscope structure and karyology of the successfullytransplanted tumors were observed. After the first generation, the xenografts weretransplanted subcutaneously into nude mice with serial passages for another threegenerations. After another three generations transplantation the latent period wasshorten, and the spontaneous regression wasn't observed. The tumor grows well,liver metastasis can be observed in several cases. The morphological features ofNCI-H446 cell were preserved. The karyotype of tumor cells belongs to human. The second part is to treat the xenografts of NCI-H446 cell in nude mice with 5F951 alone or chemotherapy drug (etopside, VP16) combined. Mice bearinggrowing tumors with a volume of 40-150 mm3 were individually identified andrandomly assigned to the control or treatment groups (6 animals/group), and thetreatment started at day 1. The therapy lasted for 14 days. Reagents wereadministered via injection intraperitoneal (ip) or injection directly into the mass oftumor (it) respectively everyday, expect VP16 (injected at day 4-7, 11-14).According to the injection way, mice were divided into five groups respectively,there were saline (NS) control group, no sense (FNS18) control group, F951(10mg/kg it, 15mg/kg ip) group, VP16 (8mg/kg it, 12mg/kg ip) group, F951 incombination with VP16 (F951+VP16) group. Tumor growth were evaluated bytumor volume and tumor weight; the survival of mice bearing tumor was describedby survival time; levels of Bcl-2 mRNA and protein in tumor cells were assayed byreal time RT-PCR and FACS respectively; histological and microstructural changeswere detected by light microscope and transmission electron microscoperespectively; tumor cell apoptosis was tested by TUNEL assay. The results showed that there was no significant difference between NS controlgroup and FNS18 control group in each examination item. The results of treatmentwith F951 alone indicated that whether F951 it or F951 ip, the relative tumorvolume (RTV) of F951 group was obviously low than NS control group; the percentof tumor growth inhibition reached to meaningful effect (over 50%); 100% of thetumors in F951-it group complete regressions, 80% of the tumors in F951-ip groupcomplete regressions; the median growth delay days were obviously long than NScontrol group, the growth delay index of F951-it group was meaningful effect; therewas significant difference of the tumor weight (weighed after 14 days treatment)between F951 group and NS control group, the percent of tumor inhibition was82.02%, 74.17% respectively in F951-it and F951-ip group; the survival time wasrelatively longer than NS control group, the percent of 120 days survival wasmarkedly high. The results of treatment combined with F951 and VP16 indicated that InF951-it+VP16-it group, there were four mice had no tumor regrowth up to 3 months 6after complete regression (...