Effect of antisense oligomer in controlling c-raf.1 overexpression during diethylnitrosamine-induced hepatocarcinogenesis in rat.
OBJECTIVE
In ras-mediated signal transduction pathway, c-raf.1 is believed to have predominant oncogenic potential and has been found to be highly expressed in certain human and animal malignancies including hepatocellular carcinoma. In the present study, anticancer efficacy of antisense c-raf.1 oligomer on the inhibition of c-raf.1 mRNA overexpression during hepatocarcinogenesis was determined.


METHODS
Initially antiproliferating effect of the antisense oligomers was studied in vitro by measuring the rate of tritiated thymidine incorporation into DNA in rat hepatocellular carcinoma cells in culture medium. Based on the findings, the antisense treatment was carried out in rat hepatocarcinogenesis model-initiated with diethylnitrosamine and promoted using 2-acetylaminoflourene. Different drug-metabolizing enzymes, lipid peroxidation, liver morphology and histopathological studies along with c-raf.1 gene expression by in situ hybridization were performed.


RESULTS
c-raf.1 antisense oligomers exhibited an inhibitory effect (approximately 68%) on cancer cell proliferation in vitro. Gross and microscopic examination of liver showed fewer (29%) and smaller hyperplastic nodules and preneoplastic lesions (30%) in carcinogen and antisense oligomer-treated group as compared with carcinogen control group. Treatment of antisense c-raf.1 oligomers enhanced cytochrome P-450 content (81%) and reduced glutathione S-transferase activity (33%), UDP glucuronosyltransferase activity (74%) and MDA concentration (30%) in carcinogen and antisense oligomer-treated group as compared with carcinogen control animals. The oligomer treatment also resulted in less expression in terms of c-raf.1 expressed lesion count as compared to carcinogen control group.


CONCLUSIONS
The study demonstrates that the antisense oligomer targeted against c-raf.1 mRNA inhibits the overexpression of c-raf.1 gene during hepatocellular carcinoma in rats.
Das
Tanushree
T
Department of Pharmaceutical Technology, Jadavpur University, Kolkata, India.
Patra
Falguni
F
Mukherjee
Biswajit
B
D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D049109:Cell Proliferation / Q000187:drug effects; D002471:Cell Transformation, Neoplastic / Q000139:chemically induced / Q000378:metabolism* / Q000473:pathology; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D004052:Diethylnitrosamine; D006965:Hyperplasia; D015227:Lipid Peroxidation; D008113:Liver Neoplasms / Q000139:chemically induced / Q000378:metabolism* / Q000473:pathology; D008297:Male; D016376:Oligonucleotides, Antisense / Q000494:pharmacology*; D011230:Precancerous Conditions / Q000139:chemically induced / Q000378:metabolism / Q000473:pathology; D019908:Proto-Oncogene Proteins c-raf / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D017207:Rats, Sprague-Dawley
D000970:Antineoplastic Agents; D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; D004052:Diethylnitrosamine; D003577:Cytochrome P-450 Enzyme System; D019908:Proto-Oncogene Proteins c-raf