Evaluation of the inhibitory effect of antisense oligodeoxynucleotides on the growth of hepatitis C-associated hepatocellular carcinoma cells in vitro.
OBJECTIVE
Hepatitis C virus (HCV) RNA is invariably detected in the serum and tumor tissue of anti-HCV-positive patients with hepatocellular carcinoma (HCC). The inflammation and cirrhosis caused by HCV could be the promoter for development of HCC or HCC could be the consequence of HCV infection independent of the effect of cirrhosis. The ability of the core protein of HCV to modulate gene transcription, cell proliferation and cell death by interacting with cellular genes that regulate cell growth and differentiation is involved in the pathogenesis of HCC. HCV NS3 protease is an attractive target for antiviral agent development because it is required for viral replication. Recent studies that constructed an in vitro model of HCC demonstrated that antisense oligodeoxynucleotides (AS-ODN) interfered with NS3 translation in a dose-dependent fashion and significantly inhibited protease activity. We studied the in vitro effect of AS-ODN on the rate of growth of the HCC cells grown in culture associated with HCV.


METHODS
Core biopsy was taken from 20 patients with HCC associated with HCV and each one was divided into two parts: group I to which antisense was added and group II which served as a control group. Comparison of cell viability between tubes with and without AS-ODN was done using MTT assay, LDH assay, cell cycle analysis, trypan blue exclusion test and colony formation in soft agar.


RESULTS
Colony formation in soft agar was inhibited in group I compared with the control group and the inhibition was highly significant (P < 0.01). The LDH concentration in culture supernatant and the trypan blue exclusion test, both reflecting cellular death, was higher in group I than group II and the difference was highly significant (P < 0.01). MTT assay showed a highly significant decrease in cell activation in group I than in group II (P < 0.01). The percentage of cells in the G(0)/G(1) phase was higher in group I than in group II and the difference was significant (P = 0.04). There was an insignificant difference between both groups in the percentage of cells in S phase (P = 0.378). The inhibitory effect of AS-ODNs on tumor cells in G(2)/M phase was highly significant compared with the control group (P < 0.01).


CONCLUSIONS
AS-ODN has a significant inhibitory effect on the growth of HCV-associated HCC cells grown in fluid culture, and there is potential for the use of AS-ODN as oncotherapy.
Amin
Mona A
MA
Department of Internal Medicine, Faculty of Medicine, Cairo University, Cairo, Egypt. monasleman@hotmail.com
Awadein
Mohamed R
MR
Gabr
Hala
H
D000368:Aged; D001483:Base Sequence; D006528:Carcinoma, Hepatocellular / Q000209:etiology / Q000473:pathology / Q000628:therapy* / Q000821:virology; D002455:Cell Division / Q000187:drug effects; D005260:Female; D016174:Hepacivirus / Q000235:genetics* / Q000472:pathogenicity; D019698:Hepatitis C, Chronic / Q000150:complications; D006801:Humans; D066298:In Vitro Techniques; D008113:Liver Neoplasms / Q000209:etiology / Q000473:pathology / Q000628:therapy* / Q000821:virology; D008297:Male; D008875:Middle Aged; D020319:Oligodeoxyribonucleotides, Antisense / Q000235:genetics / Q000494:pharmacology*; D014410:Tumor Stem Cell Assay; D017361:Viral Nonstructural Proteins / Q000235:genetics; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
D020319:Oligodeoxyribonucleotides, Antisense; D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases