Severe impairment of anti-cancer effect of lipoteichoic acid-related molecule isolated from a penicillin-killed Streptococcus pyogenes in toll-like receptor 4-deficient mice

doi:10.1016/S1567-5769(01)00103-5

International Immunopharmacology

Volume 1, Issues 9–10, September 2001, Pages 1789-1795

https://doi.org/10.1016/S1567-5769(01)00103-5 Get rights and content

Abstract

A lipoteichoic acid-related molecule (OK-PSA) isolated from OK-432, a penicillin-killed Streptococcus pyogenes, is a potent inducer of Th1 cytokines, and elicits anti-cancer effect in tumor-bearing mice. Toll-like receptor (TLR) 4 is a member of the recently identified toll-like receptor family of proteins that has been implicated in lipopolysaccharide-induced cell signaling. In the present study, we have examined the role of TLR4 for OK-PSA-induced Th1-cytokine production and anti-tumor effect by using C3H/HeJ mice in which TLR4 function is impaired. Although OK-PSA strikingly induced Th1 cytokines [interferon (IFN)-γ, tumor necrosis factor (TNF)-α, interleukin (IL)-2, IL-12 and IL-18] in the splenocytes derived from control animals (C3H/HeN), OK-PSA did not induce the cytokines in the splenocytes from C3H/HeJ. Furthermore, C3H/HeJ-derived splenocytes acquired the responsiveness to OK-PSA stimulation by overexpression of TLR4 gene. Finally, OK-PSA administration significantly inhibited the tumor growth and lung metastasis of syngeneic squamous cell carcinoma cells in C3H/HeN; however, no effect of OK-PSA was observed in C3H/HeJ. These findings strongly suggest that TLR4 signaling is involved in regulating OK-PSA-induced anti-cancer immunity.

Introduction

OK-432, a penicillin-killed and lyophilized preparation of a low-virulence strain (Su) of Streptococcus pyogenes (group A) that was developed by Okamoto et al. [1], is commonly used as an immunotherapeutic agent in malignancies. It has been reported that OK-432 elicits anti-tumor effects by a stimulatory effect on immunocompetent cells and induces multiple cytokines [2], [3], [4], [5]. Since OK-432 is a whole bacterial preparation that contains many components, it is still unknown what the major effective component is for a cytokine induction as well as an anti-tumor effect of OK-432. We have generated an IgM mouse monoclonal antibody (mAb), TS-2, which recognizes an interferon (IFN)-γ-inducing molecule of OK-432 and have succeeded to isolate the IFN-γ-inducing molecule [lipoteichoic acid (LTA)-related molecule; OK-PSA] by an affinity chromatography of butanol extract of OK-432 on CNBr-activated Sepharose 4B bound TS-2 [6], [7]. OK-PSA is a potent inducer of Th1-type cytokines as well as killer cell activities on human PBMC and carries marked anti-tumor activity on tumor-bearing mice [6], [8], [9]. Toll-like receptors (TLRs) are mammalian homologues of the Drosophila toll receptor and have a role in innate recognition of bacteria [10] and TLR4 is recently identified as a signaling receptor for lipopolysaccharide (LPS) derived from gram negative bacteria [11], [12]. In the present study, we examined the role of TLR4 for the anti-tumor effect of OK-PSA by using C3H/HeJ mice in which TLR4 is mutated and its function is impared [12], [13].

Section snippets

Cells and media

A mouse squamous cell carcinoma cell line SCCVII, a mouse molony lymphoma cell line YAC-1 and a mouse fibrosarcoma cell line Meth-A were cultured in RPMI 1640 (Life Technologies, Gaithersburg, MD, USA) supplemented with 10% fetal calf serum (FCS), 100 μg/ml streptomycin, and 100 units/ml penicillin in a humidified atmosphere of 95% air and 5% CO₂ at 37 °C.

OK-PSA

OK-PSA, a lipoteichoic acid-related molecule, was isolated from OK-432 (Chugai Pharmaceutical, Tokyo, Japan) by an affinity chromatography on

Cytokine production by OK-PSA-stimulated spleen cells derived from C3H/HeN and C3H/HeJ mice

The spleen cells derived from C3H/HeN and C3H/HeJ mice were stimulated with OK-PSA for 48 h, then cytokines in the supernatants were measured by ELISA. Although Th1-type cytokines such as IFN-γ, TNF-α, IL-2, IL-12 and IL-18 were significantly induced by OK-PSA stimulation on the spleen cells derived from C3H/HeN mouse, these cytokines were not induced in the splenocytes from C3H/HeJ. Cytokine induction by LPS was also not observed in C3H/HeJ mice. The decrease of cytokine inducing activity of

Discussion

The results of the current study have clearly demonstrated that cytokine inducing and anti-tumor activity of OK-PSA is mediated by TLR4. TLR4 is one of the essential molecules for LPS signaling and plays a significant role for innate immunity. It can activate NFκB and MAP kinases mediated by MyD88 and induce cytokines and inflammatory mediators such as nitric oxide [15], [16]. Moreover, it was reported that TLR4 is also associated with the signaling of LTA derived from gram positive bacteria

Acknowledgements

This investigation was supported in part by Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan. A pEFBOS expression plasmid containing mouse TLR4 gene was kindly provided from Drs. Miyake and Akashi, Department of Immunology, Saga Medical School, Saga, Japan.

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Cited by (42)

Immunochemoradiotherapy for patients with oral squamous cell carcinoma: Augmentation of OK-432-induced helper T cell 1 response by 5-FU and X-rays irradiation
2013, Neoplasia (United States)
Eighty-one patients with oral squamous cell carcinoma (OSCC) received oral fluoropyrimidine UFT and radiotherapy (RT) with or without an immunotherapeutic agent OK-432. Both overall survival and progression-free survival of patients who received RT + UFT + OK-432 were significantly longer than those of patients who received RT + UFT (P = .0075 and P = .0175, respectively). Clinical response was also more favorable in RT + UFT + OK-432 group than in RT + UFT group (P = .0066). Next, in vitro experiments were conducted to examine the effect of 5-fluorouracil (5-FU) and X-ray irradiation in OK-432-induced immunity. Human peripheral blood mononuclear cells stimulated with OK-432 produced helper T cell 1 (Th1)-type cytokines as well as interleukin-10 (IL-10) and transforming growth factor-β (TGF-β), which are produced by Th2 and regulatory T cells (Tregs), respectively, and are inhibitory in antitumor immunity. OK-432-induced IL-10 and TGF-β but not Th1 cytokines were significantly inhibited by 5-FU and/or X-ray. 5-FU and X-ray also inhibited the expression of mRNAs for GATA-3 and Foxp3, which are transcription factors for Th2 and Tregs, respectively, but not for T-bet, a transcription factor for Th1. In addition, 5-FU and X-ray decreased the expression of mRNAs for suppressor of cytokine signaling 1 (SOCS1) and SOCS3. Antisense oligonucleotides for SOCS1 and SOCS3 markedly reduced OK-432-induced IL-10 and TGF-β. This is the first report clearly demonstrating that OK-432-based immunotherapy significantly enhanced the therapeutic effects of chemoradiotherapy in patients with OSCC as well as elucidating the mechanism of the synergistic effect of immunochemoradiotherapy in which 5-FU and radiation enhanced OK-432-induced Th1 response mediated by the inhibition of SOCS1 and SOCS3 gene expression.
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
2012, Oral Oncology
Citation Excerpt :
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.13,15–20 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
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.
Hypoxia-inducible factor-1 up-regulates the expression of toll-like receptor 4 in pancreatic cancer cells under hypoxic conditions
2012, Pancreatology
Citation Excerpt :
Kelly et a1 [15] found that activation of TLR4 signaling promotes growth and chemoresistance of epithelial ovarian cancer cells. Conversely, inhibition of TLR4 signaling delays tumor growth and prolongs the survival of animals [17,18]. Hypoxia is a common feature of solid tumors.
Hypoxia is a common characteristic of solid tumors. Recent studies confirmed that Toll-like receptor 4 (TLR4) plays a significant role in cancer invasion and progression. In this study, the correlation between the expression of TLR4 and the change of the protein level of Hypoxia-inducible factor-1 alpha (HIF-1α) was studied.
We examined 84 human pancreatic cancer tissues for expression of HIF-1α and TLR4 proteins. Panc-1 cells were exposed to normoxia (20% O₂) or hypoxia (<1% O₂) or treated with CoCl₂. TLR4 protein was analyzed by flow cytometry and immunostaining. Growth studies were conducted on cells with the HIF-1α inhibition isolated from stable transfected cell lines. Finally, TLR4 protein was detected by immunohistochemistry in vivo tumors.
There was a positive correlation between TLR4 and HIF-1α protein in pancreatic cancer tissues. Hypoxic stress induced TLR4 mRNA and protein expression in Panc-1 cells. Cells transfected with HIF-1α siRNA showed attenuation of hypoxia stress-induced TLR4 expression. In vivo growth decreased in response to TLR4 and HIF-1α inhibiton. Transient HIF-1α siRNA treatment could effectively curb tumor growth in vivo.
These results suggest that TLR4 expression in pancreatic cancer cells is up-regulated via HIF-1α in response to hypoxic stress and underscore the crucial role of HIF-1α-induced TLR4 in tumor growth.
Small interfering RNA-directed targeting of Toll-like receptor 4 inhibits human prostate cancer cell invasion, survival, and tumorigenicity
2009, Molecular Immunology
A major cause of tumor treatment failure is cancer cell metastasis. Toll-like receptor 4 (TLR4)-mediated signaling has been implicated in tumor cell invasion, survival, and metastasis in a variety of cancers. In this study, we investigated the biological roles of TLR4 in prostate metastatic cell invasion and survival, and the potential of gene silencing of TLR4 using small interfering RNA (siRNA) for treatment of cancer. In cultured human prostate cancer cell lines, TLR4 were higher PC3 and DU145 as compared with the poorly metastatic LNCaP indicating that up-regulation of TLR4 was positively correlated with metastasis of tumor cell. In the highly metastatic cancer cell PC3, gene silencing of TLR4 using siRNA significantly inhibited TLR4 mRNA expression and protein level. Knockdown of TLR4 in PC3 cells resulted in a dramatic reduction of tumor cell migration and invasion as indicated by a Matrigel invasion assay. Furthermore, TLR4 siRNA suppressed cell viability and ultimately caused the induction of apoptotic cell death. The effects were associated with abrogating TLR4-mediated signaling to downstream target molecules such as myeloid differentiation factor 88 (MyD88), adaptor-inducing IFN-beta (TRIF), and interferon regulatory factor-1 (IRF-1). In a mouse prostate cancer model, administration with the plasmid construct expressing siRNA for TLR4 obviously inhibited established tumor growth and survival. These studies revealed evidence of a multifaceted signaling network operating downstream of TLR4-mediated tumor cell invasion, proliferation, and survival. Thus, RNA interference-directed targeting of TLR4 may raise the potential of its application for cancer therapy.
A sensitive and reliable quantification method for mouse interleukin-12 p70 based on fluorometric sandwich ELISA (FS-ELISA)
2007, Cell Biology International
Citation Excerpt :
In agreement with the defect in LPS signaling in C3H/HeJ mice (Watson et al., 1978), the concentration of IL-12 p70 secreted by their PECs in response to LPS stimulation was very low but significant (see below) when compared with that from the PECs of C3H/HeN mice (Fig. 3a,b). Despite their low response to LPS, C3H/HeJ mice are known to respond well to OK-432 stimulation (Okamoto et al., 2001), and a high concentration of IL-12 p70 was secreted by the PECs of these mice (Fig. 3c). IL-12 plays a central role in promoting Th1 responses and thereby stimulates cell-mediated immunity.
Interleukin-12 (IL-12) p70 is an important cytokine secreted by antigen-presenting cells in response to antigenic stimulation; it is a heterodimer of p35 and p40 subunits. Here, we report a new, highly sensitive, and reliable method that employs fluorometric sandwich ELISA for quantification of the mouse IL-12 (moIL-12) p70 protein. Our method could quantify moIL-12 p70 in the range of approximately 0.5 to 500 pg/ml. In the assay, no signals were produced by the moIL-12 p40 monomer, homodimer [(p40)₂], or mouse IL-23 even up to a concentration of 500 pg/ml; this ensures that our assay has a high specificity for moIL-12 p70. To demonstrate that our method can determine natural moIL-12 in real physiological/pathological samples, we monitored the moIL-12 p70 secretion from peritoneal exudative cells after lipopolysaccharide (LPS) stimulation. IL-12 p70 secretion as early as 3 h after LPS stimulation was reliably detected due to the high sensitivity of the method.
Innate receptor activation patterns involving tlr and nlr synergisms in covid-19, ali/ards and sepsis cytokine storms: A review and model making novel predictions and therapeutic suggestions
2021, International Journal of Molecular Sciences

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International Immunopharmacology

Abstract

Introduction

Section snippets

Cells and media

OK-PSA

Cytokine production by OK-PSA-stimulated spleen cells derived from C3H/HeN and C3H/HeJ mice

Discussion

Acknowledgements

References (23)

Induction of interferon-γ in mouse spleen cells by OK-432, a preparation of Streptococcus pyogenes

Cell. Immunol.

Induction of Th1-type cytokines by lipoteichoic acid-related preparation isolated from OK-432, a penicillin-killed streptococcal agent

Immunopharmacology

Toll-like receptor-4 mediates lipopolysaccharide-induced signal transduction

J. Biol. Chem.

A new chromogenic endotoxin-specific assay using recombined limulus coagulation enzymes and its clinical applications

Clin. Chim. Acta

MyD88 is an adaptor protein in the hToll/IL-1 receptor family signaling pathways

Mol. Cell

Differential roles of TLR2 and TLR4 in recognition of gram-negative and gram-positive bacterial cell wall components

Immunity

Studies on the anticancer and streptolysin S-forming abilities of hemolytic streptococci

Jpn. J. Microbiol.

Augmentation of mouse natural killer cell activity by a streptococcal preparation, OK-432

J. Natl. Cancer Inst.

Streptococcal preparation OK-432 is a potent inducer of IL-12 and a T helper cell 1 dominant state

J. Immunol.

Local injectoin of OK-432 can augment the Th1-type T-cell response in tumor-draining lymph node cells and increase their immunotherapeutical potential

Int. J. Cancer

Purification and characterization of interferon-γ-inducing molecule of OK-432, a penicillin-killed streptococcal preparation, by monoclonal antibody neutralizing interferon-γ-inducing activity of OK-432

J. Immunother.

Cited by (42)

Immunochemoradiotherapy for patients with oral squamous cell carcinoma: Augmentation of OK-432-induced helper T cell 1 response by 5-FU and X-rays irradiation

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

Hypoxia-inducible factor-1 up-regulates the expression of toll-like receptor 4 in pancreatic cancer cells under hypoxic conditions

Small interfering RNA-directed targeting of Toll-like receptor 4 inhibits human prostate cancer cell invasion, survival, and tumorigenicity

A sensitive and reliable quantification method for mouse interleukin-12 p70 based on fluorometric sandwich ELISA (FS-ELISA)

Innate receptor activation patterns involving tlr and nlr synergisms in covid-19, ali/ards and sepsis cytokine storms: A review and model making novel predictions and therapeutic suggestions