매생이(Capsosiphon fulvescens) 당단백질에 의한 AGS 위암 세포주의 침윤 및 증식 저해 메커니즘

Abstract

Recently, cancer has been the leading cause of death in the world and has drawn much public health attention. Cancer death rates were reported to be accounted for 27.6% of all deaths in Korea during 2012. Gastric cancer, or stomach cancer, refers to cancer arising from any part of the stomach. Gastric cancer causes about 800,000 deaths worldwide per year. It is also one of the leading cancer type in Korea, accounting for about 16% of total cancer death rate. Gastric cancer can be caused by some foods, such as smoked foods, salted fish and meat, and pickled vegetables are associated with a higher risk. A recent case-control study reported that several food items and cooking methods are associated with increased or decreased risk of gastric cancer among Koreans. An increased risk of gastric cancer was noted among people who frequently consume broiled meats and fishes, salted side dishes (salted/fermented fish products) and salty stewed foods. In a recent cohort study in Seoul, green vegetables and soybean foods were associated with a decreased risk of gastric cancer. Seaweeds are suitable for human and animal feed applications. In Korea, seaweeds are harvested to be included in a great variety of dishes such as salads, soups, cookies, meals and condiments. The chemical composition of seaweeds is not well known as the terrestrial plants but it is known to be rich in carbohydrates, protein and minerals as well as bio-active compounds such as polyphenols, terpenoids, cartenoids and tocopherols. Seaweeds have been reported to produce a great variety of metabolic compounds which are not produced by terrerial plants. In addition to those mentioned above, seaweeds have been extensively studied because of their various biological activities including anticancer, anti-inflammatory, anticancer and immunomodulatory activities. Capsosiphon fulvescens has recently garnered attention because it also exhibits various bio-activities. C. fulvescens is a green seaweed traditionally eaten in the southwestern regions of Korea, showing its great potential to be used as an ingradient in formulated foods as well as in functional foods due to various biological functions of its constituents. Among the constituents, glycoprotein were major active compounds with various bio-activities such as anticancer, hangover effects, immune and anticoagulant activities. Anticancer effects have been reported in various cancer cells by using a C. fulvescens ingredient. In previous studies, the glycoproteins from C. fulvescens was confirmed the effects on the human gastric cancer AGS cells via the mechanism of cell death, and apoptosis by the Fas signal. In this study, we found that C. fulvescens glycoprotein (Cf-GP) could effectively inhibit AGS human gastric cancer cells invasion and proliferation from down-regulation integrin proteins via transforming growth factor-β1 (TGF-β1) and Wnt signaling pathway. TGF-β1 is a cytokine associated with various human cancers involving macrophages, brain cells, and keratinocytes. Previous studies have reported about TGF-β1 modulates cell migration, invasion and proliferation in gastric cancer cells. TGF-β1 induces the overexpression of growth factor focal adhesion kinase (FAK) protein in several cancers, and it activates phosphatidylinositol 3-kinase (PI3K)/AKT and small GTPase proteins, and upregulates integrin proteins. The small GTPases, which include Rho A, Rho B, Rac-1, and Cdc 42, are involved in the signaling pathways associated with diverse cellular functions, including cell proliferation and migration, in response to different growth factor receptors. The small GTPases also activate nuclear transcription factor-κB (NF-κB), which upregulates the expression of integrin receptor proteins, thereby contributing to cell migration. The Wnt ligand binds immediately to its receptor gene, Frizzled, and this binding site is divided into the canonical and noncanonical pathways. Among them, the canonical pathway is dependent on β-catenin and affects cancer cell adhesion. Wnt-1 proteins are secreted from cells. Thereafter, Wnt-1 protein combines with Frizzled proteins and lipoprotein-related protein 5/6 (LRP 5/6) of the two receptor molecules. The Wnt-1 signaling pathway is dependent on β-catenin. Wnt proteins bind the cysteine​​-rich glycoprotein, acting only in a limited range of ligands, and focal activation of the receptor-mediated signal transduction system acts as an important regulator of cell proliferation and differentiation factors. β-catenin is a factor related to cell adhesion. In normal cells, down-regulation of Wnt-1 signaling degrades β-catenin in the Axin-APC protein complex by GSK-3β. However, cancer cells show up-regulation of Wnt-1 signaling. Therefore, cancer cells continue to grow and metastasis occurs. The β-catenin degradation complex, an Axin degradation complex, inhibits the activity of GSK3-β when Wnt stimulation is external. Therefore, phosphorylation of β-catenin is inhibited. Additionally, normal cells operate E-cadherin in the nucleus, but cancer cells degrade E-cadherin. E-cadherin in the nucleus inhibits β-catenin and is involved in intercellular adhesion, which plays an important role in the maintenance of normal epithelial cells and epithelial structures; catenin in the cytoplasm and formation of the cadherin/catenin complex is involved, and intercellular adhesion fails to progress to invasive carcinoma. The change in cancer cell grade increases metastasis when the expression of E-cadherin is lost. Additionally, Snail, typically a zinc finger transcription factor, inhibits E-cadherin expression, which decreases the cytosolic level of β-catenin in the nucleus. Similar to Snail genes that are resistant to apoptosis, invasion and migration of cancer cells promotes the epithelial-mesenchymal transition (EMT) and induces increase of Snail levels in the nucleus through up-regulation of Wnt-1 signaling. Therefore, β-catenin is not degraded and cytoplasmic β-catenin levels increased, which stabilize β-catenin by moving to the nucleus, and the TCf/Lef transcription factor regulates the expression of various genes. Increases in cancer cell proliferation through the up-regulation of cell cycle-related proteins, c-myc and cyclin D, occur via activated TCf/Lef. In addition, representative β-catenin target genes increase cell proliferation of ICAM-1 and c-jun by increasing the expression of β-catenin and TCf/Lef. After activation of Wnt-1, TGF signal pathway, cancer cells has increased MMPs, integrins and tight junction proteins (TJs). Integrin receptors are located on the cell surface, where they are responsible for the adhesion of cells to extracellular matrix (ECM) proteins such as fibronectin and the transduction of extracellular signals to the cell. Integrins exist as heterodimers of two distinct transmembrane glycoprotein chains, called α and β subunits, that are noncovalently linked. The integrin family consists of 24 different α,β heterodimers. The binding of an integrin receptor to its extracellular ligand causes a signal to be relayed into the cell, resulting in the regulation of specific gene expression. The binding of integrin receptors to ECM molecules also produces cell adhesion, which is critical for cell migration, proliferation and differentiation. Tight junction proteins (TJs) and MMP proteins are known as overexpressed in many cancer cells as related to invasion of them. While MMP and TJs proteins are increased, the value of TEER is decreased. It facilitates material transfer between cells due to disorder of cell membrane permeability. In addition, adhesion, metastasis and invasion of cells are increased and cancer cells are proliferated. Therefore, recently, studies are progressed regarding not only apoptosis signal cancer cells but also invasion and metastasis of them. Additionally, they are closely related to cell membrane permeability. Although MMP and TJs levels are increased, the TEER value is lower. TEER facilitates material transfer between cells due to disrupted cell membrane permeability. Therefore, cancer cell membrane permeability is weakened due to overexpression of TJs, integrins and MMP proteins, resulting in enhanced metastasis and invasion. In this study, we focused on inhibited AGS gastric cancer cells invasion and proliferation by Cf-GP. First, we observed the effects of Cf-GP in AGS human gastric cancer cell viability by MTS assay. When it was treated on Cf-GP (5, 10, or 20 μg/ml) for 24 h. It was inhibited the AGS cells growth, and showed highest concentration 20 μg/ml in the apoptosis cells by approximately 50% reduction compared to control cell group and no toxicity to IEC-6 normal cell. Also, we conducted a wound-healing assay for confirmed cell migration and observed denuded zone through microscope. As the results, the gaps (denuded zone) distance between cell and cell were inhibited by dose dependent treated on Cf-GP. We evaluated its effect on invasion using hematoxylin staining. The Cf-GP treatment group had decreased cell invasion compared to control cells. Treatment with 20 μg/ml Cf-GP led to a 50% reduction compared to the control group. We thus evaluated the effect of Cf-GP on AGS cell invasion by measuring TEER. Change of cell membrane permeability by decreased TEER values. So, it was observed to be increased of cancer cells invasion and proliferation. In this study, we observed TEER values in AGS cells, it was increased upon Cf-GP treatment in a dose-dependent manner. In addition, we performed annexin V assay and cell cycle test for cell apoptosis rate by MuseTM annexin V and cell cycle. As the results, annexin V apoptosis rate has increased approximately 42.68% in final Cf-GP concentration (20 μg/ml) compared with the control group and cell cycle arrest at G0/G1 was confirmed by apoptosis. Here we found that TGF-β1 decreased FAK/AKT/PI3K/small GTPase expression by using western blot and RT-PCR. This results shows the inhibition of FAK by decreased TGF-β1. Compared to the control group, Cf-GP-treated cells exhibited significantly down-regulation of FAK/AKT/PI3K/small GTPase protein (Rac-1, Rho A, Cdc 42) and mRNA levels. But, Rho B was one of the Rho family proteins and activation of Rho B has a induced cancer cells apoptosis. In this study, Rho B was increased by Cf-GP. It was showed the results of the transcription factor NF-κB decreased due to inhibition of degradation of IκB and these growth factors. Also, it was showed the down regulation of Wnt-1 related proteins (APC, Axin, Frizzled, LRP-5, TCF-1, Lef-1) through inhibition of Wnt-1 by Cf-GP (5, 10, or 20 μg/ml) for 24 h. In addition, when it was treated on Cf-GP, we could observe that β-catenin was decreased due to activation of E-cadherin by inhibition of snail. And also, we observed that decrease of MMPs to the cell membrane degradation and increased of TIMP for inhibition MMPs by dose-dependent effect of Cf-GP (5, 10 or 20 μg/ml) for 24 h. Both at the level of proteins and mRNA, we confirmed the same results. Especially, MMP-2, 9 expression levels were observed to be associated with gelatin zymography, it was confirmed that inhibition at the level. In addition, interaction with MMPs in cell membrane permeability could allow to adjust in the expression levels of the TJs and integrin proteins indentified. As the result, it has significantly decreased of TJs (claudins, Zo-1 and occludin) and integrins levels of protein and mRNA level compared to the control group. This result showed the suppression of cancer cells invasion caused by inhibitory of overexpression TJs and integrin proteins. In general, cancer cells invasion is known to be involved up-regulation of angiogenesis proteins. Therefore, we could observe the inhibition of AGS cancer cells invasion and confirmed the expression of angiogenesis proteins. As the results, Cf-GP induced the decreases of angiogenesis proteins by inhibition of cells invasion. Therefore, we suggest that Cf-GP can reduce proliferation, migration and invasion of AGS cell, and its characteristics, may be of value in the development of a functional food.