Role of the Fas/FasL pathway in combination therapy with interferon-alpha and fluorouracil against hepatocellular carcinoma in vitro.
OBJECTIVE
Several studies have reported the efficacy of combination therapy of interferon (IFN) alpha and 5-fluorouracil (5-FU) for hepatocellular carcinoma (HCC). However, the mechanism underlying the clinical anti-tumor effects of this treatment is not well understood. The aim of this study was to determine the role of Fas/FasL signaling in the anti-tumor effect of this combination therapy.


RESULTS
We used six human hepatoma cell lines, three of which are known Fas-expressing cells. Growth of Fas-positive hepatoma cell lines was inhibited by an agonistic anti-Fas antibody in a dose-dependent manner, and these effects were enhanced by IFNalpha or 5-FU alone, but even more so by combination therapy using both agents. Annexin-V assay implicated apoptosis as the main mechanism underlying these growth inhibitory effects, although changes in Fas expression regulated by IFNalpha and/or 5-FU did not correlate with increased apoptosis. Caspase-3 activation was exclusively increased by IFNalpha/5-FU combination treatment, which was compatible with enhancement of the synergistic apoptotic effect, and other caspases and apoptotic factors (FLIP, BCL-xl, and Bax) were also regulated by IFNalpha/5-FU. 51Cr-release assay revealed that pretreatment with IFN activated cytotoxicity of peripheral blood mononuclear cells (PBMCs) against HCC cells. The largest interaction was observed when both PBMC and HCC cells were pretreated with the combination of IFNalpha/5-FU. These cytotoxicities were markedly inhibited by a neutralizing anti-Fas antibody.


CONCLUSIONS
Our results indicated that IFNalpha/5-FU combination treatment enhances the induction of apoptosis and the cytotoxic effect of PBMCs via the Fas/FasL pathway. The Fas/FasL pathway seems, at least in part, to contribute to the anti-tumor effects of IFNalpha/5-FU against HCCs.
Nakamura
Masato
M
Department of Surgery, Graduate School of Medicine, Osaka University, Osaka, Japan.
Nagano
Hiroaki
H
Sakon
Masato
M
Yamamoto
Tameyoshi
T
Ota
Hideo
H
Wada
Hiroshi
H
Damdinsuren
Bazarragchaa
B
Noda
Takehiro
T
Marubashi
Shigeru
S
Miyamoto
Atsushi
A
Takeda
Yutaka
Y
Umeshita
Koji
K
Nakamori
Shoji
S
Dono
Keizo
K
Monden
Morito
M
D000906:Antibodies / Q000008:administration & dosage / Q000627:therapeutic use; D000970:Antineoplastic Agents / Q000008:administration & dosage / Q000627:therapeutic use; D017209:Apoptosis / Q000187:drug effects; D001483:Base Sequence; D053446:CASP8 and FADD-Like Apoptosis Regulating Protein / Q000235:genetics; D006528:Carcinoma, Hepatocellular / Q000188:drug therapy* / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D020169:Caspases / Q000378:metabolism; D045744:Cell Line, Tumor; D004273:DNA, Neoplasm / Q000235:genetics; D004789:Enzyme Activation / Q000187:drug effects; D053222:Fas Ligand Protein / Q000378:metabolism*; D005472:Fluorouracil / Q000008:administration & dosage / Q000627:therapeutic use*; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D066298:In Vitro Techniques; D007370:Interferon Type I / Q000008:administration & dosage / Q000627:therapeutic use*; D008113:Liver Neoplasms / Q000188:drug therapy* / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012334:RNA, Neoplasm / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins; D019014:fas Receptor / Q000037:antagonists & inhibitors / Q000378:metabolism*
D000906:Antibodies; D000970:Antineoplastic Agents; D053446:CASP8 and FADD-Like Apoptosis Regulating Protein; C481004:CH-11 anti-fas antibody, human; D004273:DNA, Neoplasm; C491901:FAS protein, human; D053222:Fas Ligand Protein; D007370:Interferon Type I; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D011994:Recombinant Proteins; D019014:fas Receptor; D020169:Caspases; D005472:Fluorouracil