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.
Materials and Methods
Cell culture. The human breast epithelial cell line MCF10A was a kind gift from Dr. S. Maheswaran from the Massachusetts General Hospital Cancer Center (Charlestown, MA, USA). The human colorectal cancer cell line SW837, human pancreatic cancer cell line PANC-1, human stomach cancer cell line MKN45, human embryonic kidney cell line HEK293, and monkey kidney cell line COS-1 were obtained from ATCC (Rockville, MD, USA). The culture conditions used for the maintenance of these cell lines have been described previously.(27) Briefly, human pancreatic adenocarcinoma PANC-1, human kidney HEK293, and monkey kidney COS-1 cells were maintained in DMEM (Invitrogen, Carlsbad, CA, USA). Human mammary epithelial MCF10A cells were maintained in DMEM/F12, human gastric cancer MKN-45 cells were maintained in RPMI-1640, and human colorectal cancer SW837 cells were maintained in Leibovitz’s L-15 Medium (Invitrogen). All cell culture media were supplemented with 10% FBS (BioWest, Nuaillé, France) and 1% penicillin–streptomycin (Invitrogen). Cells were grown in a 5% CO2 atmosphere at 37°C.
Reagents. Protein-bound polysaccharide, which was supplied by Kureha (Tokyo, Japan), was dissolved with sterilized physiological saline and diluted to the indicated concentrations in the corresponding culture media. Human recombinant TGF-β1 was purchased from R&D Systems (Minneapolis, MN, USA).
Luciferase reporter assay. HEK293 or COS-1 cells (2 × 105) were seeded onto 24-well plates 24 h before transfection. Cells were transiently transfected with the p3TP-lux reporter plasmid (Addgene, Cambridge, MA, USA) and the phRL-TK vector (Promega, Madison, WI, USA) using FuGENE6 (Roche Diagnostics, Indianapolis, IN, USA). After 24 h, the cells were treated with or without 10 ng/mL TGF-β1 for 1 h then cultured in fresh medium in the presence of 0–500 μg/mL PSK. Cell lysates were prepared 24 h later and luciferase activity was assayed by the Dual-Luciferase Reporter System (Promega) and Lumat LB 9501 (Berthold Technologies, Bad Wildbad, Germany). Results were obtained from duplicate wells for each experimental condition and all experiments were carried out in triplicate. p3TP-lux luciferase activity was normalized to that of the control phRL-TK vector.
In order to assay the effects of the constitutively active TGF-β type I receptor, COS-1 cells were seeded at 10 000 cells/well in a 24-well plate and transfected with the p3TP-lux reporter plasmid, the phRL-TK vector, and the pRK5-mutated-TGF β type I receptor (T202D) (Addgene) using FuGENE6. Alternatively, cells were transfected with the p3TP-lux reporter plasmid, the phRL-TK vector, and pRK5 (Addgene) for the negative control. Culture medium was replaced 24 h after transfection and cells were treated with different concentrations of PSK overnight. Cells were harvested after treatment and luciferase assays were carried out. All assays were carried out in triplicate.
Western blot analysis. SW837, MCF10A, PANC-1, and MKN-45 cells were serum-starved for 24 h before treatment with 10 ng/mL TGF-β for 1 h. The cells were subsequently washed and treated with 50–100 μg/mL PSK. After 4 h, cells were lysed in RIPA buffer (Santa Cruz Biotechnology, Santa Cruz, CA, USA) supplemented with a protease inhibitor cocktail (Nacalai Tesque, Kyoto, Japan) and Halt Phosphatase Inhibitor Cocktail (ThermoFisher Scientific, Fremont, CA, USA).
Total protein extracts were analyzed by SDS-PAGE and Wblot analysis with anti-Smad2 (L16D3), anti-phospho-Smad2 (Ser465/467), anti c-Jun (60A8), anti-vimentin (V9) (Abcam, Cambridge, MA, USA), anti-E-cadherin (SHE78-7) (Takara Bio, Shiga, Japan), and anti-phospho-c-Jun (D47G9) antibodies (Cell Signaling Technology, Danvers, MA, USA). Actin, which was used as an internal control, was detected using an anti-actin antibody (C4) (BD Biosciences, San Jose, CA, USA). Immunoreactive proteins were visualized using the ECL Advanced Western Blotting Detection kit (GE Healthcare, Chalfont St Giles, UK).
Quantitative real-time PCR analysis. SW837, MCF10A, and PANC-1 cells were serum-starved for 24 h and subsequently treated with 10 ng/mL TGF-β for 1 h. The cells were washed and cultured in medium containing 50–100 μg/mL PSK for 24 h. Total RNAs were purified from cultured cells using Isogen (Nippon Gene, Tokyo, Japan). First-strand cDNAs were synthesized using the PrimeScript RT reagent kit (Takara Bio) for RT-PCR. Quantitative real-time PCR analysis was carried out using SYBR Premix Ex Taq and the Thermal Cycler Dice Real Time System (Takara Bio).
The following primer sequences were used: human serpin peptidase inhibitor, clade E, member 1 (SERPINE1), forward, 5′-CATTACTACGACATCCTGGAACTG-3′, reverse, 5′-AATGTTGGTGAGGGCAGAGAG-3′; human collagen, type1, alpha1 (COL1A1), forward, 5′-GTGCTAAAGGTGCCAATGGT-3′, reverse, 5′-ACCAGGTTCACCGCTGTTAC-3′; human transgelin (TAGLN), forward, 5′-TCCAGGTCTGGCTGAAGAAT-3′, reverse, 5′-TGCCTTCAAAGAGGTCAACA-3′; human fibronectin 1 (FN1), forward, 5′-ACGAGGAAATCTGCACAACC-3′ reverse, 5′-ACACACGTGCACCTCATCAT-3′; and human GAPDH, forward, 5′-GCACCGTCAAGGCTGAGAAC-3′, reverse, 5′-TGGTGAAGACGCCAGTGGA-3′. Human GAPDH was used for normalization. All experiments were carried out in duplicate.
Microscopy. MCF10A cells were cultured in the absence or presence of 2.5 ng/mL TGF-β1 and 500 μg/mL PSK for 11 days. Morphological changes were assessed by an inverted microscope (CKX41; Olympus, Tokyo, Japan) and the images were recorded at ×40 magnification.
Immunohistochemistry. MCF10A cells were cultured in the absence or presence of 2.5 ng/mL TGF-β1 and 50–500 μg/mL PSK for 7 days. Formalin-fixed cells were quenched with Dako REAL peroxidase-blocking solution (Dako, Glostrup, Denmark). Cells were then blocked with Dako Cytomation Protein Block Serum-Free (Dako) and stained with anti-vimentin (V9) (Abcam) or anti-E-cadherin (SHE78-7) (Takara Bio) antibodies, followed by incubation with HRP anti-mouse and anti-rabbit labeled polymers (Dako). Reaction sites were visualized with the EnVision+ kit/HRP (DAB) (Dako) and counterstained with hematoxylin. Images of stained cells were obtained using a digital microscope (VHX-600; Keyence, Osaka, Japan).
Wound healing assay. MCF10A cells were cultured in serum-free medium prior to wounding. Cells that had reached 80% confluency were scratched with a pipette tip, followed by treatment with 10 ng/mL TGF-β and 0–50 μg/mL PSK for 7 h. Phase contrast images were obtained using an inverted microscope (CKX41; Olympus) at a magnification of ×450.
Flow cytometry analysis. MCF10A cells were cultured in the presence or absence of 2.5 ng/mL TGF-β1 and 500 μg/mL PSK for 21 days. Cells were dissociated using PBS-based enzyme-free dissociation buffer (Invitrogen) and centrifuged. Cells were resuspended and stained with anti-CD44-allophycocyanin (APC) mouse monoclonal and anti-CD24-phycoerythrin (PE) (BD Biosciences) antibodies on ice for 30 min. Samples were then resuspended in PBS containing 2% FBS and examined using a FACSCalibur flow cytometer (BD Biosciences). APC-IgG and PE-IgG antibodies were used as controls. No-antibody and single-antibody controls were used to normalize the sample readings and to designate quadrants. The results were analyzed using CellQuest software (BD Biosciences).
Mammosphere culture. MCF10A cells were cultured in DMEM/F12 media with or without 10 ng/mL TGF-β1 or 500 μg/mL PSK for 12 days. Single-cell suspension cultures were prepared at a densities of 40 000, 20 000, 10 000, 5000, 2500, and 1250 cells per well in DMEM/F-12 supplemented with 2% B27 (Invitrogen), 20 ng/mL epidermal growth factor, and 20 ng/mL basic fibroblast growth factor (BD Biosciences) and seeded into six-well ultra low attachment plates (2.5 mL per plate). Culture medium was fed on day 4 and day 6, and the number of mammospheres was recorded 9 days after the start of the culture period.(28,29)
Statistical analysis. Statistical differences were determined for in vitro assays by Student’s t-test. Data were analyzed using spss software (SPSS, Chicago, IL, USA). We considered values of P < 0.05 statistically significant and values of P < 0.01 highly significant.
Inhibition of the TGF-β-responsive luciferase reporter by PSK treatment. Protein-bound polysaccharide-induced inhibition of the TGF-β pathway was investigated in HEK293 and COS-1 cells using the 3TP-lux luciferase reporter assay. 3TP-lux is a TGF-β1-responsive luciferase reporter gene that contains three consecutive tetradecanoylphorbol acetate response elements and a portion of the plasminogen activator inhibitor 1 (PAI-1/SERPINE1) promoter region.(30) The signal intensity was normalized by Renilla luciferase. After 24 h of serum starvation, the luciferase activities of 3TP-lux were increased in both cell lines after treatment with TGF-β1; however, 3TP-lux activity was suppressed in response to PSK treatment in a dose-dependent manner (Fig. 1A). Moreover, we examined the 3TP-lux activity of COS-1 cells that expressed the constitutively active mutant of TGF-β type I receptor (active TGF-βRI). As expected, 3TP-lux activity was increased by transfection of active TGF-βRI as well as by TGF-β1 treatment; however, 3TP-lux activity was suppressed after PSK treatment (Fig. 1B). It was previously reported that PSK selectively binds and reduces the active form of TGF-β1.(31) However, our results suggest that PSK inhibits signaling downstream of TGF-β receptors in addition to neutralizing TGF-β1.
Protein-bound polysaccharide inhibits Smad signaling and expression of TGF-β-signaling target genes. To determine whether PSK suppresses the Smad pathway,(32) SW837, MCF10A, PANC-1, and MKN45 cell lines with normal Smad2 expression and non-mutated TGF-β receptors(33,34) were treated with TGF-β1 after 24 h of serum starvation. Cells were subsequently treated with several concentrations of PSK for 4 h after TGF-β1 depletion and Smad2 phosphorylation was examined by immunoblotting. Western blot analysis showed a trend of PSK suppression of TGF-β1-induced Smad2 phosphorylation in a dose-dependent manner (Fig. 1C).
Transforming growth factor-β signaling activates target genes such as FN1, SERPINE1, TAGLN, and COL1A1. FN1 plays an important role in development and wound healing by promoting cell adhesion, migration, and cytoskeletal organization.(35)SERPINE1 regulates tumor cell invasion through the precise regulation of the peritumoral proteolytic microenvironment. TAGLN expression is required for epithelial cell proliferation and migration and is implicated in the regulation of fibrosis.(36)COL1A1 is the major fibrous collagen synthesized by wound fibroblasts during the repair process.(37) In the present study, the mRNA levels of FN1, SERPINE1, TAGLN, and COL1A1 were determined by quantitative RT-PCR. The mRNA levels of these downstream target genes of TGF-β1 signaling were elevated by TGF-β1 treatment after 24 h serum starvation in SW837, MCF10A, and PANC-1 cells. Treatment with PSK showed a dose-dependent tendency to decrease the mRNA levels of these target genes (Fig. 2, Fig. S1).
In addition to its effect on Smad signaling, TGF-β1 also activates the MAPK pathway, including ERK, JNK, and p38 kinase.(38,39) SW837 cells were serum-starved for 24 h, treated with TGF-β1, and treated with several concentrations of PSK for 4 h after TGF-β1 depletion. c-Jun phosphorylation was subsequently examined by immunoblotting. Unexpectedly, TGF-β1 treatment increased the levels of both c-Jun and phosphorylated c-Jun; however, PSK treatment decreased both c-Jun and phosphorylated c-Jun levels in a dose-dependent manner (Fig. S2).
Protein-bound polysaccharide inhibits EMT induced by activation of TGF-β pathway. The EMT process, which is a crucial step in tumor progression, can be induced by several cytokines and chemokines, including TGF-β.(40) After 7 days of treatment with TGF-β1, immortalized human mammary epithelial MCF10A cells showed a change in phenotype, becoming spindle-shaped with loose cell–cell contacts. However, MCF10A cells co-cultured with TGF-β1 and PSK did not show phenotypic changes and maintained epithelial characteristics (Fig. 3A).
Immunohistochemical analysis showed that E-cadherin expression was inhibited in TGF-β1-treated MCF10A cells after 7 days, but no changes in expression were detected when the cells were co-cultured with PSK. Moreover, the TGF-β1-induced upregulation of vimentin expression was diminished by PSK (Fig. 3B,C). Western blot analysis showed that PSK treatment inhibited both the TGF-β1 treatment-induced downregulation of E-cadherin expression and the upregulation of vimentin expression, suggesting that PSK suppresses TGF-β1-induced EMT (Fig. 3D).
The migratory ability of MCF10A cells stimulated with or without TGF-β or various concentrations of PSK was analyzed using a wound healing assay. Cultured cells reaching 80% confluency were pretreated with mitomycin C and scratched with a pipette tip, and the length of the wound was measured immediately after the incision and after 7 h of incubation with 10 ng/mL TGF-β and 0–50 μg/mL PSK (Fig. 3E). MCF10A cells did not show any migratory ability in the absence of EMT.(27) These results showed that MCF10A cells acquired a higher migratory ability in response to TGF-β1 treatment and this ability was abolished when these cells were treated with PSK.
Mani et al.(29) reported that the induction of EMT factors such as TGF-β1 in human mammary epithelial cells was associated with the acquisition of mesenchymal morphology and the expression of mesenchymal markers. This phenotypic EMT change increased the CD44+/CD24− cell subpopulation, which showed the properties of their mammary epithelial progenitors and an enhanced mammosphere-forming ability. In the present study, FACS analysis of the cell-surface markers CD44 and CD24 in the mammary epithelial cell line MCF10A showed that TGF-β1 treatment increased the CD44+/CD24− cell population from 1.98% to 18.6%. However, the CD44+/CD24− cell population did not change in MCF10A cells co-cultured with PSK (Fig. 4A), indicating that PSK inhibits the EMT-mediated generation of the CD44+/CD24− population. The mammosphere formation assay in MCF10A cells showed that TGF-β1 treatment triggered the formation of mammospheres, whereas mammosphere formation was decreased in cells that were not treated with TGF-β1 or co-treated with TGF-β1 and PSK (Fig. 4B).
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.
This research was supported by the Japanese Ministry of Education, Culture, Sports, Science and Technology Grant-in-Aid for Scientific Research (B) (#21791260 T.H.) and a Grant-in-Aid for the Global Center of Excellence (COE) Program entitled “Education and Research Center for Stem Cell Medicine” (Keio University, Tokyo, Japan).