Elsevier

Oral Oncology

Volume 48, Issue 8, August 2012, Pages 678-685
Oral Oncology

Growth inhibition and apoptosis by an active component of OK-432, a streptococcal agent, via Toll-like receptor 4 in human head and neck cancer cell lines

https://doi.org/10.1016/j.oraloncology.2012.02.005Get rights and content

Summary

Toll-like receptor 4 (TLR4) plays a significant role in cancer therapy as receptors of bacteria-derived immunotherapeutic agents such as OK-432, a streptococcal immunotherapeutic agent. In addition, recent reports demonstrated that TLRs, including TLR4, are also expressed in cancer cells as well as in immunocompetent cells. It is a problem in cancer therapy that the immunoadjuvant may activate survival signals such as nuclear factor (NF)-κB or mitogen-activated protein kinases (MAPKs) in cancer cells via TLRs. In the current study, we investigated responsiveness of human head and neck cancer cell lines against TLR4 ligands, OK-PSA, an active component of OK-432, and a lipopolysaccharide (LPS).

Stimulation with LPS or OK-PSA resulted in the activation of NF-κB in these cell lines expressing TLR4 and MD-2 that is a significant coreceptor for TLR4 signaling. Interestingly, OK-PSA induced cell-growth inhibition, while LPS enhanced the proliferation of the cancer cells. OK-PSA induced NF-κB activation more slowly than that induced by LPS. In addition, phosphorylation of p38 MAPK by OK-PSA was only slight compared with that by LPS. OK-PSA also induced apoptosis of the cancer cells mediated by the activation of caspase 1, 3 and 8 in a p53-independent manner.

These findings strongly suggest that active components of OK-432 may elicit anti-cancer effects via enhancing host immunity as well as via directly inducing the growth inhibition and apoptosis of head and neck cancer cells through TLR4 signal.

Introduction

OK-432, which is a penicillin-killed and lyophilized preparation of a low-virulence strain of Streptococcus pyogenes (group A),1 has been successfully used as an immunotherapeutic agent against many types of malignancies, including head and neck cancer (HNC).2, 3, 4, 5, 6, 7, 8 It has been reported that OK-432 elicits anti-tumor effects by stimulating immunocompetent cells such as macrophages, T cells, and natural killer (NK) cells, and by inducing helper T-cell 1 (Th1) type cytokines, including interleukin-2 (IL-2), IL-12, tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ).9, 10, 11, 12 We have isolated an active component [a lipoteichoic acid (LTA)-related molecule: OK-PSA] by affinity chromatography of a butanol extract of OK-432 on cyanogen bromide-activated Sepharose 4B bound by the mouse monoclonal antibody that recognizes an IFN-γ-inducing component of OK-432.13, 14 We have reported that OK-PSA is a far more potent inducer of Th1-type cytokines in human peripheral blood mononuclear cells (PBMCs) than the original OK-432, and that it engages in a marked antitumor activity in tumor-bearing mice.13, 15, 16, 17, 18, 19, 20 Furthermore, we showed that Toll-like receptor (TLR) 4 signaling is involved in regulating OK-432/OK-PSA-induced antitumor immunity in tumor-bearing mice21 as well as in oral cancer patients.22

TLRs are transmembrane proteins that belong to a newly recognized family of vertebrate pattern recognition receptors within the innate immune system.23, 24 Subsequent to ligand binding, TLRs initiate signaling through the sequential recruitment of myeloid differentiation protein 88 (MyD88), IL-1R-associated kinase, and TNFR-associated factor 6, which in turn activate downstream mediators such as mitogen-activated protein kinases (MAPKs) and nuclear factor-κB (NF-κB).25, 26 Among the family of ten identified human TLRs, TLR2 recognizes peptidoglycan (PGN),27 lipoprotein,28 etc., TLR4 recognizes lipopolysaccharide (LPS),29 and TLR9 recognizes bacterial unmethylated CpG-DNAs.30 MD-2 acts as a significant coreceptor in the TLR4 signaling.31

Recent report demonstrated that TLRs, including TLR4 and MD-2, are expressed in cancer cells32 as well as in immunocompetent cells, however, the role of the TLR ligands in cancer cells remains uncertain. In cancer therapy using OK-432, it is a problem that the immunopotentiator may activate survival signals such as NF-κB and MAP kinases in the carcinoma cells via the TLR4/MD-2 signal. In the current study, we conducted in vitro experiments to investigate the direct effects of TLR4/MD-2 ligands, OK-PSA and LPS, in human HNC cell lines.

Section snippets

Preparation of OK-PSA

OK-PSA was prepared from OK-432 (Chugai Pharmaceutical Co. Ltd., Tokyo, Japan) as described previously.13 The OK-PSA preparation was tested for LPS contamination using the Endospecy ES-50M set (Seikagaku Kogyo, Tokyo, Japan), according to the manufacturer’s recommended protocol.33

Cells and culture

As the human HNC cell lines, we used seven squamous cell carcinoma (SCC) cell lines, BHY, B88, HI, HNT, HSC-2, Ho-1-u-1, and KB, and five salivary gland carcinoma (SGC) cell lines, HSY, HSG, AZA1, AZA3, and TYS. BHY,

Expression of mRNAs for TLR4 and MD-2 in HNC cell lines

Of the 12 cell lines tested, expression of TLR4 mRNA was observed in all 12 cell lines by using the semiquantitative RT-PCR method, whereas MD-2, which is a coreceptor for TLR4 signaling, was expressed in only five cell lines (BHY, HI, HNT, HSC-2, and TYS). All TLRs tested were detected in human PBMCs (Fig. 1).

We also investigated the mRNA expression of the TLR4 and MD-2in the cell lines in detail using real-time quantitative PCR. The result of most correlations to the semiquantitative RT-PCR

Discussion

OK-432 is an immunotherapeutic agent in many types of malignancies, including HNCs.2, 3, 4, 5, 6 We have reported that OK-PSA, an active component of OK-432, augmented anti-cancer immunity via TLR4/MD2 signaling.15, 16, 17, 18, 19, 20, 21, 22 Recent reports demonstrated that TLRs including TLR4/MD-2 are expressed in cancer cells as well as in immunocompetent cells32; however, the role of the TLR ligands in cancer cells has been unclear. In cancer therapy using OK-432, it is a problem that the

Conflict of interest statement

None declared.

Acknowledgement

This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan.

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