Involvement of Toll-like receptor 4 signaling in interferon-gamma production and antitumor effect by streptococcal agent OK-432.
BACKGROUND
The streptococcal agent OK-432 has been used for immunotherapy of head and neck cancer, among other malignancies, but its mechanism of action is unknown. Because the Toll-like receptor 4 (TLR4)/MD-2 complex is important in enabling the mammalian immune system to recognize bacterial components, we investigated whether expression of the TLR4 and MD-2 genes is associated with OK-432-induced anticancer immunity.


METHODS
Peripheral blood mononuclear cells (PBMCs) from 28 patients with head and neck cancer were analyzed for TLR4 and MD-2 mRNA expression by reverse transcription-polymerase chain reaction (RT-PCR) analysis. PBMCs were treated in vitro with OK-432 or with OK-PSA (a lipoteichoic-acid-related molecule that is an active component of OK-432), and interferon-gamma (IFN-gamma) mRNA expression, an immune response measure, was analyzed by RT-PCR. Patient sera collected 24 hours after OK-432 administration were examined for IFN-gamma protein using an enzyme-linked immunosorbent assay. Lewis lung carcinoma-bearing wild-type C57BL/6 and TLR4-deficient mice (four mice per group) received intraperitoneal injections of OK-432, and tumor volumes and sera IFN-gamma levels were measured over time. All statistical tests were two-sided.


RESULTS
Twenty patients expressed both TLR4 and MD-2. Expression of TLR4 and MD-2 genes was associated with the in vivo IFN-gamma induction in 19 patients administered OK-432 (Fisher's exact test P<.001). Although both OK-432 and OK-PSA induced IFN-gamma expression from PBMCs in vitro, expression of TLR4 and MD-2 was associated only with IFN-gamma expression induced by OK-PSA (P<.001). In vivo intraperitoneal administration of OK-432 resulted in an increase of IFN-gamma in sera from wild-type mice but not in sera from TLR4-deficient mice. Tumors in wild-type mice treated with OK-432 were statistically significantly smaller than those in mice treated with saline (P =.007). By contrast, in TLR4-deficient mice, there was no difference in tumor volume between the two treatment groups.


CONCLUSIONS
TLR4 and MD-2 may mediate OK-432-induced anticancer immunity.
Okamoto
Masato
M
Second Department of Oral and Maxillofacial Surgery, Tokushima University School of Dentistry, 3-18-15 Kuramoto-cho, Tokushima 770 8504, Japan.
Oshikawa
Tetsuya
T
Tano
Tomoyuki
T
Ohe
Go
G
Furuichi
Sachiko
S
Nishikawa
Hidetomo
H
Ahmed
Sharif Uddin
SU
Akashi
Sachiko
S
Miyake
Kensuke
K
Takeuchi
Osamu
O
Akira
Shizuo
S
Moriya
Yoichiro
Y
Matsubara
Shuzo
S
Ryoma
Yoshiki
Y
Saito
Motoo
M
Sato
Mitsunobu
M
D000368:Aged; D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D000954:Antigens, Surface / Q000235:genetics / Q000378:metabolism*; D000970:Antineoplastic Agents / Q000494:pharmacology*; D029721:Drosophila Proteins; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006258:Head and Neck Neoplasms / Q000737:chemistry / Q000188:drug therapy* / Q000276:immunology*; D006801:Humans; D007274:Injections, Intraperitoneal; D007371:Interferon-gamma / Q000096:biosynthesis* / Q000097:blood / Q000187:drug effects; D008156:Luciferases / Q000032:analysis; D051218:Lymphocyte Antigen 96; D008297:Male; D008562:Membrane Glycoproteins / Q000172:deficiency / Q000187:drug effects / Q000276:immunology / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D008875:Middle Aged; D010844:Picibanil / Q000494:pharmacology*; D011112:Polymyxin B / Q000494:pharmacology; D012333:RNA, Messenger / Q000032:analysis; D012334:RNA, Neoplasm / Q000032:analysis; D011956:Receptors, Cell Surface / Q000172:deficiency / Q000187:drug effects / Q000276:immunology / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000187:drug effects; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors
D000900:Anti-Bacterial Agents; D000954:Antigens, Surface; D000970:Antineoplastic Agents; D029721:Drosophila Proteins; C495377:LY96 protein, human; D051218:Lymphocyte Antigen 96; D008562:Membrane Glycoproteins; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D011956:Receptors, Cell Surface; C495345:TLR4 protein, human; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors; D010844:Picibanil; D007371:Interferon-gamma; D008156:Luciferases; D011112:Polymyxin B