Journal of Cancer Research and Therapeutics

ORIGINAL ARTICLE
Year
: 2014  |  Volume : 10  |  Issue : 1  |  Page : 43--49

Anti-cancer Effects of CME-1, a Novel Polysaccharide, Purified from the Mycelia of Cordyceps sinensis against B16-F10 Melanoma Cells


Thanasekaran Jayakumar1, Chong-Chi Chiu2, Shwu-Huey Wang3, Duen-Suey Chou1, Yung-Kai Huang4, Joen-Rong Sheu1,  
1 Department of Pharmacology and Graduate Institute of Medical Sciences, Taipei Medical University, Taipei, Taiwan
2 Department of Surgery, Chi-Mei Medical Center, Tainan, Taiwan
3 Core Facility Center, Office of Research and Development, Taipei, Taiwan
4 School of Oral Hygiene, College of Oral Medicine, Taipei Medical University, Taipei, Taiwan

Correspondence Address:
Joen-Rong Sheu
Department of Pharmacology, Graduate Institute of Medical Sciences, Taipei Medical University, 250 Wu-Hsing St., Taipei 110
Taiwan
Yung-Kai Huang
School of Oral Hygiene, College of Oral Medicine, Taipei Medical University, Taipei 110
Taiwan

Abstract

Background: Matrix metalloproteinases (MMPs) play important roles in the invasion and migration of cancer cells. In melanoma, several signaling pathways are constitutively activated. Among these, the mitogen-activated protein kinase (MAPKs) signaling pathways are activated through multiple signal transduction molecules and appear to play major roles in melanoma progression. Therefore, the inhibition of MAPK signaling might be a crucial role for the treatment of melanoma cancer. Aims: We examined the anticancer effect of CME-1, a novel water-soluble polysaccharide fraction, isolated from Cordyceps sinensis mycelia on B16-F10 melanoma cells. Materials and Methods: B16-F10 cells were exposed to different concentrations of CME-1 (250, 500 and 800 μg/ml) for 24 h in 5% CO 2 incubator at 37°C. Western blot analysis was performed to detect the expression of MMP-1, p-p38 MAPK, p-ERK1/2, and IkB-α in B16-F10 cells. Cell migration test was performed by wound healing migration assay. Results: CME-1 suppresses cell migration in a concentration-dependent manner. Western blotting analysis revealed that CME-1 led to the reduction on the expression levels of MMP-1 and down regulated the expression of phosphorylated extracellular signal-regulated kinase (ERK1/2 and p38 mitogen-activated protein kinase (p38 MAPK). CME-1 restored the IkB-degradation in B16F10 cells. Conclusions: These results indicate that CME-1 inhibited MMP-1 expressions in B16F10 melanoma cells through either NF-kB or ERK/p38 MAPK down regulation thereby inhibiting B16F10 cell migration. Therefore, we proposed that CME-1 might be developed as a therapeutic potential candidate for the treatment of cancer metastasis.



How to cite this article:
Jayakumar T, Chiu CC, Wang SH, Chou DS, Huang YK, Sheu JR. Anti-cancer Effects of CME-1, a Novel Polysaccharide, Purified from the Mycelia of Cordyceps sinensis against B16-F10 Melanoma Cells.J Can Res Ther 2014;10:43-49


How to cite this URL:
Jayakumar T, Chiu CC, Wang SH, Chou DS, Huang YK, Sheu JR. Anti-cancer Effects of CME-1, a Novel Polysaccharide, Purified from the Mycelia of Cordyceps sinensis against B16-F10 Melanoma Cells. J Can Res Ther [serial online] 2014 [cited 2019 Oct 20 ];10:43-49
Available from: http://www.cancerjournal.net/text.asp?2014/10/1/43/131365


Full Text

 Introduction



Malignant melanoma of the skin is the most frequent cause of mortality from skin cancer and approximately 20-25% of patients with malignant melanoma die of metastatic disease. Metastasis is one of the major causes of mortality in cancer patients. Tumor metastasis is the end result of a complex series of steps involving multiple tumor - host interactions. [1] One important step in this process is invasion into tissues, which requires proteolytic degradation of extracellular matrix (ECM) components. Matrix metalloproteinases (MMPs) are a family of zinc-binding enzymes that cleave ECM components and the expression levels of MMPs are correlated with tumor invasiveness. [2] MMP expression is low in most normal cells under physiologic conditions; however, MMP expression is dramatically increased in a variety of cancer types, where it is indicative of invasive disease with a poor clinical prognosis. [3] One MMP family member capable of degrading the most abundant proteins of the ECM (collagen types I and III) is the interstitial collagenase, MMP-1. MMP-1 is over expressed in invasive melanoma [4] and is required for melanoma cell invasion through a synthetic ECM in vitro. [5]

In normal cells, MMP-1 expression can be induced by a variety of growth factors, including bFGF, epidermal growth factor, interleukin-1, and tumor necrosis factor α. [6] This induction requires the activation of the mitogen-activated protein kinase (MAPK) pathways, which in turn act in part through activation of AP-1 transcription factor. [7],[8] There are at least three major human MAPK families: The extracellular response kinases (ERKs), p38, and Jun N-terminal kinases (JNKs). Specific pharmacologic inhibitors of these MAPK pathways, such as U0126, which blocks the phosphorylation and activation of MEK1/2 and SB203580, which inhibits the kinase activity of p38, have been useful in analyzing the role of MAPK pathways in transcriptional activation of MMP-1 in normal cells. [6] In animal models, MMP inhibitors prevent tumor dissemination and formation of metastases. [9] However, these compounds have failed to live up to expectations, mostly due to their systemic toxicities. [10]

Medicinal mushrooms have been widely used as tonic foods and herb remedies since ancient times, and their medicinal properties have been increasingly recognized through modern scientific research. In the search of alternative medicines and natural therapeutics for cancer therapy, medicinal mushrooms are among the most promising targets because of their notable immunomodulatory activities. [11] Cordyceps, is a special type of mushroom which is formed on an insect larva infected by Cordyceps sinensis. The anti-tumor activities of cultivated Cordyceps fungal mycelia have been described in previous studies. [12] A number of bioactive constituents from Cordyceps species have been reported, including cordycepin, antibacterial and antitumor adenosine derivatives, sterols, polyphenolics and polysaccharides. [13] Polysaccharides isolated from Cordyceps species are major antioxidant phytochemicals, have been shown to have anti-inflammatory, antitumor and immunomodulatory activities. [14]

CME-1, a novel water-soluble polysaccharide, isolated from the mycelia Cordyceps sinensis, containing mannose and galactose in a respective ratio of 4:6. A recent study has been demonstrated that CME-1 protects RAW264.7 cells against oxidative stress through inhibition of sphingomyelinases (SMase) activity and reducing C16- and C18-ceramide levels. [15] However, there is no information available about the anticancer effect of CME-1 against melanoma cells. Malignant melanoma originates from melanocytes in the basal layer of the epidermis and its incidence is rapidly increasing. Well known as a chemotherapy-resistant cancer, melanoma might be a suitable target for therapy with natural compounds. Hence, for the first time this study is aimed to elucidate the anticancer effect of CME-1 against B16-F10 murine melanoma cells via investigating the possible mechanism of inhibiting ERK/p38MAPKs signaling pathways.

 Materials and Methods



(3-(4, 5-Dimethylthiazol-2-yl)-2), 5-diphenyl tetrazolium bromide (MTT) was purchased from Sigma (St. Louis, MO). Anti-mouse and anti-rabbit immunoglobulin G-conjugated horseradish peroxidase (HRP) was purchased from Amersham Biosciences (Sunnyvale, CA, USA) and/or Jackson-Immuno Research (West Grove, PA, USA). A rabbit polyclonal antibody specific for IkB-α was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The anti-p38 MAPK and anti-phospho-p42/p44 ERK (Thr202/Tyr204) were from Cell Signaling (Beverly, MA, USA). The anti-mouse monoclonal antibody for MMP-1was purchased from Millipore (USA). The Hybond-P polyvinylidene difluoride (PVDF) membrane and enhanced chemiluminescence (ECL) Western blotting detection reagent and analysis system were obtained from Amersham (Buckinghamshire, UK). All other chemicals used in this study were of reagent grade.

CME-1 [Figure 1]a was extracted according to the method described by Wang et al., [15] Briefly, dried powder (200 gm) of cultured Cordyceps mycelia were extracted by using double-distilled H 2 O (200 ml) three times for 3 h at room temperature (25°C). Extracts were combined and concentrated to give 65 gm (33%) of crude residue (water-soluble fraction of Cordyceps sinensis mycelia [CME]). Two grams of CME were fractionated by gel filtration column chromatography (Sephacryl G-15 column; 2.5 × 45 cm) with the double-distilled H 2 O eluent, which yielded the polysaccharide CME-1 (12%). The CME-1 fraction was collected using the phenol-sulfuric acid method and the absorption was read at 490 nm. [16] For the polysaccharide composition analysis, CME-1 fractions were hydrolyzed in 4 M trifluoroacetic acid at 112°C for 8 h, and the hydrolysate was measured with high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD), using an ICS-3000 ion chromatography system (Dionex, Sunnyvale, CA). The hydrolysate was eluted with a mixture of water and 200mM NaOH in a volume ratio of 92:8, through a CarboPac PA-10 column (2 × 250 mm; Sunnyvale, CA). Molecular masses of the fractions were predicted using diffusion ordered spectroscopy (DOSY) experiment (AV600 NMR spectrometer; Bruker, Rheinstetten, Germany). [17] The chemical structure of CME-1 was determined according to a previous report [18] and by using a GC-MS profile. CME-1 with 99% or higher purity was dissolved in phosphate-buffered saline PBS and diluted with culture medium. Vehicle-treated cells served as a control.

Highly metastatic B16F10 murine melanoma cells were obtained from the National Institute of Preventive Medicine, Department of Health, Executive Yuan (Taipei, Taiwan) and were cultured in RPMI1640 medium (Sigma) supplemented with 10% fetal bovine serum (Gibco, Carlsbad, CA, USA), 100 U/ml penicillin (Gibco), 100 U/ml streptomycin (Gibco), HEPES (18 mM) and NaHCO 3 (23.57 mM) (pH 7.4) in an atmosphere containing 5% CO 2 .

Cells were plated at a density of 1 × 10 4 per well into 12-well plates in RPMI1640 medium with 10% fetal bovine serum. After the required confluence reached, cells were exposed to different concentrations of CME-1 (250, 500, 800 and 1000 μg/ml) for 24 h in 5% CO 2 incubator at 37°C. At 22 h, the MTT solution was added to each well at a final concentration of 0.5 mg/ml. After 2 h of incubation, the supernatant was discarded and replaced with dimethyl sulfoxide DMSO to dissolve the formazan product, which was measured at 550 nm in a spectrophotometric plate reader. The following formula was used to calculate the percent cell viability: Percentage cell viability = (absorbance of the experiment samples/absorbance of the control) 100%.

The wound healing assay is one of the earliest developed methods to study directional cell migration in vitro. B16-F10 cells were seeded in a six-well plate and allowed to attach overnight to 90-95% confluence. Subsequently, cell monolayers were wounded by sterile plastic pipette tips and washed with PBS twice to remove floating cells. Cells were then incubated in RPMI1640 medium with 250, 500 and 800 μg/ml CME-1 for up to 24 h. Cells migrated into the wound surface and the number of migrating cells was determined under an inverted microscopy at 24 h. The percentage of inhibition was expressed using untreated wells at 100%.

Western blot analyses were performed as previously described. [19] Lysates from each sample were mixed with 6 × sample buffer (0.35M Tris, 10% w/v SDS, 30% v/v glycerol, 0.6 M DTT, and 0.012% w/v bromophenol blue; pH 6.8) and heated to 95°C for 5 min. Proteins were separated by electrophoresis and transferred onto PVDF membranes for MMP-1, p-p38 MAPK, p-ERK1/2 and IkB-α. The membranes were blocked with 5% non-fat milk in tris-buffered saline TBS-0.1% Tween 20 and sequentially incubated with primary antibodies and HRP-conjugated secondary antibodies, followed by ECL detection (Amersham Biosciences). The BIO-PROFIL Bio-1D light analytical software (Vilber Lourmat, Marue La Vallee, France) was used for the quantitative densitometric analysis. Data of specific protein levels are presented as multiples relative to the control.

Experimental results are expressed as the mean ± S.E.M. and are accompanied by the number of observations. The experiments were assessed by the method of analysis of variance (ANOVA). If this analysis indicated significant differences among the group means, then each group was compared using the Newman-Keuls method. P < 0.05 was considered statistically significant.

 Results



[Table 1] shows that CME-1, with a molecular mass of 27.6 kDa, contained 95% carbohydrates and had a mannose/galactose/glucose composition ratio of 39.1:59.2:1.7. The structure was suggested to consist of a backbone possessing (1 - 4)-linked mannose with galactose branches attached to the O- 6 of mannose [Figure 1]a.{Table 1}{Figure 1}

In order to estimate the toxicity of CME-1 treatment on B16F10, melanoma cells were incubated in the presence of CME-1 at concentrations of 250-1000 μg/ml and tumor cell viability was determined after 24 h by MTT test. Results of MTT assay showed that a 24 h treatment of CME-1 exhibited no cytotoxicity in B16F10 cells [Figure 1]b except that the studied concentration of 1000 μg/ml. This concentration range was then omitted to all subsequent experiments.

Cell migration was investigated by a wound healing in vitro assay, in which cells migrate bi-directionally from the edges of a scratched wound. Migration ability was reduced by 51.95 and 40.69%, with respect to control cells, in 500 and 800 μg/ml CME-1-treated B16F10 cells [Figure 2] a and b, respectively. The results indicated that CME-1 inhibited the migration of B16-F10 cells in a concentration dependent manner.{Figure 2}

Our finding that CME-1 had an inhibitory effect on cell migration encouraged us to examine its effects on the expressions of MMP-1. As shown in [Figure 3], analysis of the immunoblot image revealed that levels of MMP-1 in untreated B16F10 cells increased compared to levels detected in CME-1 treated cells. CME-1, at concentrations of 250, 500, and 800 μg/ml significantly inhibited the expressions of MMP-1 approximated at 0.853 ± 0.032, 0.641 ± 0.018 and 0.537 ± 0.023, respectively as compared to the untreated control cells (1.00 ± 0.00){Figure 3}

It is well known that the MAPK pathway participates in MMP-1 activation and that ERK and p38 are critically involved in MMP-1 induction. [20] Therefore, we investigated whether CME-1 suppresses ERK and p38 in B16F10 cells. Western blot analyses showed that the phosphorylation of ERK1/2 and p38MAPK were markedly increased in normal B16 melanoma cells. However, treatment of CME-1 at concentrations of 250-800 μg/ml was significantly reduced the expression of phosphorylated ERK1/2 and p38 in a concentration dependent manner, compared with those in the normal melanoma cells [Figure 4] a and b.{Figure 4}

Degradation of IkB-α lead to the nuclear translocation of NF-kB, which exists as a complex of NF-kB and IkB-α in the cytoplasm. It has also been demonstrated that the MAPK pathways regulated metastasis could be mediated through the nuclear NF-kB-regulated MMP expression and secretion. To elucidate whether the inhibitory action of CME-1 on matrix metalloproteinases (MMP)-1expression was due to an effect on degradation of IkB-α, the cytoplasmic levels of protein were examined by Western blot analysis. As shown in [Figure 5], it has been demonstrated that CME-1 treatment significantly reversed IkB-α degradation in melanoma cells in a concentration (250, 500, and 800 μg/ml) dependent manner and the respective degradation rates were about 1.098 ± 0.02, 1.346 ± 0031 and 1.652 ± 0.035, as compared with normal melanoma cells (1.000 ± 0.00).{Figure 5}

 Discussion



Matrix metalloproteinases (MMPs), are critical facilitators of tumor invasion, their expression patterns have been studied during different stages of melanoma progression and found to be temporally and spatially regulated. [4] Particular MMPs contribute to specific stages in melanoma progression, with the interstitial collagenase MMP-1 being expressed in later, more invasive tumors. [4] Increasing evidence suggested that MMP-1 is an important player in tumor progression and that invasive potential of melanoma cells are to be decreased by inhibiting MMP-1 production. [5] In the present study, we found for the first time that CME-1, a novel water-soluble polysaccharide, isolated from the extract of Cordyceps sinensis mycelia, inhibited the protein expression of MMP-1 in B16 melanoma cells via down regulating the expressions of phosphorylated ERKs and p38.

It has been demonstrated that the drug's and natural substances can inhibit carcinogenesis and development of tumors through the induction of cellular terminal differentiation, [21] avoiding the typical cytotoxicity of chemotherapeutic agents. Our attention was then focused on the wound healing migration assay to verify the anti-metastatic effect of CME-1 in B16 melanoma cells and the results discovered that treatments with CME-1 decreased B16 melanoma cell migration in a concentration-dependent manner, with negligible cell toxicity [Figure 1]b, [Figure 2]a and b. These results imply that the potential ability of CME-1 in reducing melanoma cell migration may be through the induction of cell differentiation. These findings are in accordance with the results of Man et al., [22] as they found that saponin Paris H suppressed cancer cells migration through down-regulation of MMP-1, -3 and -14 in B16F10 melanoma cells without causing cell toxicity.

MAPKs play a key role in oncogenic and tumor-suppressing activities; they are present in most cell types and can activate MMP gene expressions. [23] It was considered that ERKs, p38 s and JNKs have distinct physiological properties and it was generally accepted that ERKs are pro-oncogenic, while p38 s and JNKs inhibit proliferation. Researchers have challenged these concepts, as several MAP kinases may phosphorylate the same substrates and affect each other via cross-talk reactions and feedback mechanisms. In human melanoma it has been demonstrated that ERK is constitutively active [24] and its activity is essential for striking the malignant phenotype characterized by cell growth, invasion and metastasis. ERK over expression or constitutive activation of this pathway has been shown to play an important role in the pathogenesis and progression of breast and other cancer types, making the components of this signaling cascade potentially important as therapeutic targets. [25] MMP expressions can be induced by various growth factors and cytokines, including epidermal growth factor, interleukin-1 and tumor necrosis factor- α. [26] These inductions require the activation of MAPKs pathway. [27] Studies have also shown that the ERK1/2 pathway mediates the activation of the MMP-1 promoter via an AP-1 element by Ras, serum, phorbol ester, insulin and oncostatin M. A study also described that SB203580, a p38 inhibitor decreases MMP-1 production in melanoma cells, and they suggested that this inhibition is an indirect effect mediated by inhibition of the ERK pathway. [28] Besides, Mtsukova et al., [29] reported that tamoxifen [member of the selective estrogen receptor modulator (SERM) family, widely used in the treatment and prevention of breast cancer] inhibits MMP-1 activation through the suppression of p-ERK1/2 in melanoma cells. Our results also evidently demonstrate that CME-1 inhibits the phosphorylation of ERK1/2 in B16 melanoma cells, which indicates that the inhibition of MMP-1 may be at least in part due to suppression of the ERK signaling pathway. The MAP kinase family including p38 MAPKs is thought to play an important role in melanogenesis. [30] The activity of p38 MAPK is required for induction of MMP-1 gene expression. [31] Therefore, it was suggested that the inhibition of p38 MAPK signaling or the decreased levels of its activity might be crucial role for the treatment of melanoma cancer. A study reported that some Chinese herbal formulas inhibit melanogenesis through the suppression of p38 MAPK via inhibiting the melanogenic enzymes in B16F10 melanoma cells. [32] In the present study, CME-1 was significantly inhibited p38 MAPK expression in melanoma cells, these finding supported that the inhibitory effect of CME-1 on MMP-1 expression may be due to the effects on p38 MAPKs activation, as it has been demonstrated that ERK and p38 MAPK suppression can inhibit MMP-1 in response to various stimuli. [6]

NF-kB activation could be an event that promotes melanoma progression and metastasis. [33] The NF-kB family consists of several different proteins, such as p50, p52, p65 (RelA), RelB, and c-Rel, all of which share a conserved Rel homology domain responsible for the dimerization, nuclear localization and DNA binding of the NF-kB protein. The biological activity of this protein depends on its concentration, nucleocytoplasmic distribution, and DNA binding activity. These are controlled by a family of inhibitory proteins called inhibitor of kB (IkB) proteins, to which the NFkB proteins are bound under unstimulated conditions. The best characterized protein of the family is IkBα, which binds to the p65/c-Rel and p65/p50 heterodimers, the latter being most ubiquitous and biologically active NF-kB dimer. IkBα shifts the subcellular distribution of NF-kB predominantly toward the cytoplasm and prevents DNA-NF-kB binding by occupying the Rel domain. [34] On stimulation by a growth factor such as the tumor necrosis factor (TNF), IkBα undergoes rapid phosphorylation, ubiquitinylation, and subsequent degradation by the 26 S proteasome, [35] resulting in the release, nuclear entry and DNA binding of NF-kB. [36] Dissociation and nuclear translocation of NF-κB facilitate cell proliferation, angiogenesis and metastasis, leading to aggressiveness of tumors. [37] It was reported that MAPK pathways play a critical role in regulating the expression of MMPs by activating NF-κB. [38] Therefore, targeting NF-κB may be beneficial for suppressing metastasis. [39] A recent study established that holothurian glycosaminoglycan inhibited metastasis and thrombosis via targeting of NF-κB pathway in melanoma B16F10 cells. [40] So also, in the present study a significant recovery of the degradation of IkBα by CME-1, may provide a molecular basis underlying the CME-1-mediated inhibition of MMP-1 via inhibition of IkBα degradation in B16 melanoma cells.

 Conclusion



The overall data suggest that CME-1 possesses a significant anticancer effect against melanoma cells via inhibiting MMP-1 expression with consequence suppression of cell migration. This effect may be due to suppressing the ERK/p38 MAPK pathways and by recovering IkBα degradation in melanoma cells. These results may provide a new possible opportunity for the development of potential therapeutic agent to the treatment of tumors.

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