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Transforming growth factor-β (TGF-β) is involved in the regulation of cell proliferation, differentiation, and apoptosis and is associated with epithelial–mesenchymal transition (EMT). Inhibition of the TGF-β pathway is an attractive strategy for the treatment of cancer. We recently screened for novel TGF-β inhibitors among commercially available drugs and identified protein-bound polysaccharide (PSK) as a strong inhibitor of the TGF-β-induced reporter activity of 3TP-lux, a TGF-β1-responsive luciferase reporter. Protein-bound polysaccharide is used as a non-specific immunostimulant for the treatment of gastric and colorectal cancers in Japan. The anticancer activity of this agent may involve direct regulation of growth factor production and enzyme activity in tumors in addition to its immunomodulatory effect. Although several clinical studies have shown the beneficial therapeutic effects of PSK on various types of tumors, its mechanism of action is not clear. In the present study, Western blot analysis showed that PSK suppressed the phosphorylation and nuclear localization of the Smad2 protein, thereby suggesting that PSK inhibits the Smad and MAPK pathways. Quantitative PCR analysis showed that PSK decreased the expression of several TGF-β pathway target genes. E-cadherin and vimentin immunohistochemistry showed that PSK suppressed TGF-β1-induced EMT, and FACS analysis showed that PSK inhibited the EMT-mediated generation of CD44+/CD24− cells. These data provide new insights into the mechanisms mediating the TGF-β-inhibiting activity of PSK and suggest that PSK can effectively treat diseases associated with TGF-β signaling. (Cancer Sci 2012; 103: 317–324)
Transforming growth factor-β (TGF-β) is involved in various biological activities, such as cell proliferation, differentiation, and apoptosis(1–3) and is also considered a major inducer of epithelial–mesenchymal transition (EMT) during development.(4,5) Inactivation of the TGF-β pathway during the early stages of carcinoma may contribute to carcinogenesis because TGF-β signaling is implicated in the negative regulation of cell proliferation.(2,6) Paradoxically, TGF-β is often overexpressed in malignant cells and alters tumor-specific cell fates and facilitates immunosuppression, deposition of ECM proteins, and angiogenesis.(7,8) Cancer cells overexpressing active TGF-β1 showed increased metastatic ability,(9) and targeting of TGF-β signaling prevented metastasis in several neoplastic tumors including breast, prostate, and colorectal cancers.(10,11) Furthermore, recent studies have suggested new roles for TGF-β signaling in the tumor microenvironment associated with the regulation of cancer stem cells and their niches.(12,13) Clinical studies have shown a positive correlation between TGF-β1 expression and metastasis and poor prognosis in gastric, breast, and colorectal carcinomas.(14–18) Thus, the inhibition of invasion and metastasis through inhibition of the TGF-β pathway could be a promising treatment strategy. However, the application of inhibitors in standard cancer therapy requires both careful evaluation of the clinical benefits and the development of effective strategies to overcome the side-effects associated with the toxicity of these agents.
A previous study suggested that protein-bound polysaccharide (PSK) modulates the biological activity of TGF-β1 and β2 by binding to their active forms.(19) Protein-bound polysaccharide obtained from Basidiomycetes has been used as an agent in the treatment of cancer in Asia for over 30 years.(20,21) The anticancer activity of PSK, which is derived from the fungus Coriolus versicolor, has been documented in experimental models in vitro(22) and in human clinical trials. Several randomized clinical trials have shown that PSK has anticancer potential in adjuvant cancer therapy, with positive results in the treatment of gastric, esophageal, colorectal, breast, and lung cancers.(23–26) These studies suggest that the efficacy of PSK is due to its ability to act as an immunomodulator of biological responses, but the mechanism of action of PSK has not been fully elucidated.
We recently screened for TGF-β inhibitors among commercially available drugs and identified PSK as a strong inhibitor of 3TP-lux, a TGF-β-responsive luciferase reporter. The present study investigated the inhibitory effect of PSK on the TGF-β pathway and TGF-β-induced EMT as possible mechanisms that mediate the anticancer activity of PSK.
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Protein-bound polysaccharide has been used as a non-specific immunostimulant for the treatment of cancer patients in Japan for more than 30 years without the occurrence of adverse side-effects. The antitumor activity of PSK has been documented in various experimental models and beneficial therapeutic effects were shown in several types of tumors in clinical studies. The addition of PSK to adjuvant chemotherapy significantly prolonged survival after curative surgery in a large prospective trial of patients with gastric and colorectal cancer.(23,25) Protein-bound polysaccharide is a non-specific immunopotentiator that exerts immunomodulatory action by inducing the production of interleukin-2 and γ-interferon, thereby stimulating lymphokine-activated killer cells and enhancing natural killer cells.(41) Proetin-bound polysaccharide also has a favorable effect on the activation of leukocyte chemotactic locomotion and phagocytic activity.(42,43) Moreover, it was recently revealed that PSK is a specific Toll-like receptor 2 agonist and has potent antitumor effects through stimulation of both CD8+ T cells and natural killer cells.(44)
The anticancer effect of PSK might involve a direct regulatory action on growth factor production and enzyme activity in tumors in addition to the immunomodulatory activities mentioned above. Zhang et al.(31) reported that PSK selectively bound and reduced the active form of TGF-β1, thereby inhibiting tumor invasiveness through direct inhibition of TGF-β1 production, MMPs, and the urokinase-type plasminogen activator system. In this study, we showed the inhibitory effect of PSK on the TGF-β pathway and TGF-β-induced EMT.
In the present study, PSK inhibited the Smad pathway, the major regulator of TGF-β signaling,(38) by suppressing Smad2 protein phosphorylation. Protein-bound polysaccharide also decreased the levels of both c-Jun and phosphorylated c-Jun, which were increased by TGF-β1 treatment, suggesting that the effect of PSK involves the MAPK pathway. Treatment with PSK inhibited the expression of several TGF-β pathway target genes and prevented TGF-β1-induced EMT. These findings indicate that PSK inhibits TGF-β-associated pathways such as Smad and MAPK signaling even if these pathways have been activated, and the ability of PSK to inhibit these pathways suggests that it acts upstream of Smad and MAPK signaling. Although the interaction between PSK and TGF-β receptors was evaluated by immunoprecipitation, the results were inconclusive because the PSK extracted from the fungus C. versicolor exists as a glycoprotein complex rather than a single molecule. Thus, an additional study should be undertaken to clarify the biological mechanism of PSK action.
Various TGF-β signaling inhibitors, including antisense oligonucleotides against TGF-β2,(45–47) mAb against TGF-β,(48–50) and small molecule inhibitors,(51) have recently been developed. Among them, a soluble antisense oligonucleotide that is specific for human TGF-β2 mRNA, AP12009, has been used to target the TGF-β pathway in vivo and is currently in clinical trials for malignant gliomas.(45,46) A phase II study of Belagenpumatucel-L, a TGF-β2 antisense gene-modified allogeneic tumor cell vaccine for non-small-cell lung cancer, suggested that this compound provided a survival advantage and was well tolerated; therefore, this agent should be investigated further. In addition, mAbs to TGF-β are currently in clinical trials including CAT-152, which is used to prevent the progression of fibrosis after trabeculectomy for primary open-angle or chronic angle-closure glaucoma,(48) and CAT-192, which is used to treat early-stage diffuse cutaneous systemic sclerosis.(49)
Although the results obtained with TGF-β inhibitors in clinical trials are promising, TGF-β-based therapeutic strategies must be carefully considered in each case. Because a large number of cellular context-dependent factors contribute to the dynamic regulatory roles of TGF-β signaling, an alteration of this balance could have a significant effect on the characteristics of certain cells and induce oncogenic transformation. The potentially deleterious effects of these strategies in normal tissues must be considered.
In the 30-year history of its clinical use, PSK has not shown severe side-effects, and it has prolonged the survival of cancer patients and reduced the recurrence of tumors.(23,25) The anticancer activities of PSK are reportedly derived from its immunomodulatory effects and its inhibitory effect on TGF-β. The present data, which confirm the inhibition of the TGF-β pathway by PSK, suggest that this agent is not only effective as an anticancer drug but could also be applied as a TGF-β inhibitor in diseases caused by the aberrant activation of the TGF-β pathway such as primary open-angle glaucoma, diffuse cutaneous systemic sclerosis, and pulmonary fibrosis.
In conclusion, the present results show the effect of PSK on TGF-β pathway inhibition and indicate that PSK could be a promising new agent for the treatment of diseases associated with alterations in TGF-β signaling.