[국내논문]Hexane and Chloroform Fractions of Laetiporus sulphrueus var. miniatus Inhibit Thrombin-treated Matrix Metalloproteinase-2/9 Expression in Human Oral Squamous Carcinoma YD-10B Cells원문보기
Laetiporus sulphrueus var. miniatus is widely distributed worldwide, and has commonly been used as a medicinal mushroom. In the present study, we investigated the effects of water extract and solvent fractions from the Laetiporus miniatus as possible antioxidant, anti-thrombin and anti-invasive agen...
Laetiporus sulphrueus var. miniatus is widely distributed worldwide, and has commonly been used as a medicinal mushroom. In the present study, we investigated the effects of water extract and solvent fractions from the Laetiporus miniatus as possible antioxidant, anti-thrombin and anti-invasive agents against phorbol 12-myristate 13-acetate (PMA)- or thrombin-induced matrix metalloproteinase-2 (MMP-2) and MMP-9 activities. Samples were fractionated into n-hexane, $CHCl_3$, ethyl acetate, n-butanol, and water fractions, and individually analysed. The water fraction had the highest extraction yield at 34.90% (w/w), while the n-butanol fraction demonstrated the highest anti-oxidative activity at 81.44%. In the thrombin inhibitory activity test, the water fraction exhibited the highest activity at 94.64%. Even at the concentration of $40{\mu}g/mL$, evaluation of anti-proliferating activity in YD-10B cells did not reveal any cytotoxic effects. Although MMP-9 expression in YD-10B cells increased after the addition of PMA and thrombin, MMP-2 did not. Additionally, MMP-2/-9 levels in PMA-treated YD-10B cells (i.e., both mRNA expression and protein activation) were highly inhibited in the hexane and chloroform fractions. Compared with MMP-2 levels, MMP-9 mRNA expression and proteolytic activity were inhibited to a greater extent by the hexane and chloroform fractions in thrombin-treated YD-10B cells. Taken together, these results support that thrombin induces tumor invasion through MMP-2/9 and suggest that the L. miniatus may act as an effective functional food, conferring anti-oxidative, anti-thrombotic and anti-cancer activities.
Laetiporus sulphrueus var. miniatus is widely distributed worldwide, and has commonly been used as a medicinal mushroom. In the present study, we investigated the effects of water extract and solvent fractions from the Laetiporus miniatus as possible antioxidant, anti-thrombin and anti-invasive agents against phorbol 12-myristate 13-acetate (PMA)- or thrombin-induced matrix metalloproteinase-2 (MMP-2) and MMP-9 activities. Samples were fractionated into n-hexane, $CHCl_3$, ethyl acetate, n-butanol, and water fractions, and individually analysed. The water fraction had the highest extraction yield at 34.90% (w/w), while the n-butanol fraction demonstrated the highest anti-oxidative activity at 81.44%. In the thrombin inhibitory activity test, the water fraction exhibited the highest activity at 94.64%. Even at the concentration of $40{\mu}g/mL$, evaluation of anti-proliferating activity in YD-10B cells did not reveal any cytotoxic effects. Although MMP-9 expression in YD-10B cells increased after the addition of PMA and thrombin, MMP-2 did not. Additionally, MMP-2/-9 levels in PMA-treated YD-10B cells (i.e., both mRNA expression and protein activation) were highly inhibited in the hexane and chloroform fractions. Compared with MMP-2 levels, MMP-9 mRNA expression and proteolytic activity were inhibited to a greater extent by the hexane and chloroform fractions in thrombin-treated YD-10B cells. Taken together, these results support that thrombin induces tumor invasion through MMP-2/9 and suggest that the L. miniatus may act as an effective functional food, conferring anti-oxidative, anti-thrombotic and anti-cancer activities.
* AI 자동 식별 결과로 적합하지 않은 문장이 있을 수 있으니, 이용에 유의하시기 바랍니다.
문제 정의
miniatus in the context of oral cancer migration. Accordingly, the aim of this study was to assess the potential therapeutic and anti-invasive activities of the aqueous and organic solvent extracts from L. miniatus on human oral squamous carcinoma cells.
가설 설정
Recently, there has been greater progress made towards the development of new therapeutic drugs, derived from medicinal foods that present with little side effects, for the treatment of thrombotic disorders [31, 32]. In this study, we sought to investigate whether the water extract and solvent fractions of L. miniatus could function as potential preventive agents of vascular disease by measuring its antithrombotic activity. As shown in Fig.
However, the effect of thrombin on human oral squamous carcinoma cells is not fully understood. Thus, in this study we sought to evaluate whether the effects of the water extract and solvent fractions from L. miniatus are due to the regulation of thrombin-treated invasion. YD-10B cells were treated with different concentrations (0, 2.
제안 방법
The samples were processed through 30 cycles consisting of 1 minute at 94°C, 1 minute at 58°C for MMP-2, 58°C for MMP-9, 58°C for glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and 1 minute at 72°C.
To determine whether the cytotoxic effects of the water extract and solvent fractions from L. miniatus was attributable directly to cell death, the MTS cell proliferation assay was performed using the human oral squamous carcinoma cell line YD-10B (Fig. 4). Cells were treated with different concentrations (0, 20, 40, 80 and 100 µg/mL) of water extract and solvent fractions from L.
대상 데이터
1,1-diphenyl-2-picrylhydrazyl (DPPH), fibrinogen, thrombin, and phorbol 12-myristate 13-acetate (PMA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Cell Counting Kit-8 (CCK-8) was obtained from Dojindo Laboratories (Rockville, MD, USA).
Human oral squamous carcinoma cell line YD-10B was obtained from Korea Cell Line Bank (Seoul, Korea). Cells were grown in RPMI-1640 with 10% Fetal Bovine Serum (Life technologies, Gaithersburg, MD, USA) at 37°C in humidified atmosphere with 5% CO2 incubator.
데이터처리
Statistical analysis for anti-oxidant and anti-thrombin assay was performed with one-way ANOVA using Instat statistical software (GraphPad Software, Inc., La Jolla, CA, USA). For the statistical analysis of the cell viability assay, RT-PCR and gelatinase activity were obtained using Student’s t-test.
For the statistical analysis of the cell viability assay, RT-PCR and gelatinase activity were obtained using Student’s t-test.
성능/효과
miniatus could function as potential preventive agents of various diseases by measuring their anti-oxidative effects by examining DPPH radical scavenging. As shown in Fig. 2, the highest activity was observed in the butanol fraction (81.44%), followed by the ethyl acetate fraction (77.70%); those of water, chloroform and hexane fractions were 39.82%, 25.52%, and 9.86%, respectively. These results suggest that the butanol and ethyl acetate fractions of L.
miniatus could function as potential preventive agents of vascular disease by measuring its antithrombotic activity. As shown in Fig. 3, the highest inhibition (94.64%) was observed with the water extract, followed by the water fraction (at 90.10% of inhibition), with the fractions of butanol, chloroform, ethyl acetate and hexane fraction exhibiting 89.41%, 73.44%, 69.14%, and 64.97%, inhibition, respectively. Of note, these results corresponded to previous studies, where thrombin inhibition was significantly higher in the butanol fractions and organic solvent factions [33], suggesting that the fractions of L.
후속연구
miniatus demonstrated potent anti-cancer effects on HL-60 human myeloid leukemia cells, resulting from apoptosis activation. Therefore, we suggest that the hexane and chloroform fractions of L. miniatus might serve as potential antiinvasive agents in oral cancer, although further experiments will need to be carried out in order to identify the active compounds present within the hexane and chloroform fractions of L. miniatus.
참고문헌 (38)
Kakar P, Lip GY. Hypertension: endothelial dysfunction, the prothrombotic state and antithrombotic state and antithrombotic therapy. Expert Rev Cardiovasc Ther 2007;5:441-50.
Turpie AG. Oral, direct factor Xa inhibitors in development for the prevention and treatment of thromboembolic diseases. Arterioscler Thromb Vasc Biol 2007;27:1238-47.
Hart RG, Benavente O, McBride R, Pearce LA. Antithrombotic therapy to prevent stroke in patients with atrial fibrillation: a meta-analysis. Ann Intern Med 1999;131:492-501.
Kim J, Lee JW, Kim SI, Choi YJ, Lee WK, Jeong MJ, Cha SH, Lee HJ, Chun W, Kim SS. Thrombin-induced migration and matrix metalloproteinase-9 expression are regulated by MAPK and PI3K Pathways in C6 glioma cells. Korean J Physiol Pharmacol 2011;15:211-6.
Choi MS, Kim YE, Lee WJ, Choi JW, Park GH, Kim SD, Jeon SJ, Go HS, Shin SM, Kim WK, et al. Activation of protease-activated receptor 1 mediates induction of matrix metalloproteinase-9 by thrombin in rat primary astrocytes. Brain Res Bull 2008;76:368-75.
Radjabi AR, Sawada K, Jagadeeswaran S, Eichbichler A, Kenny HA, Montag A, Bruno K, Lengyel E. Thrombin induces tumor invasion through the induction and association of matrix metalloproteinase-9 and beta-1 integrin on the cell surface. J Biol Chem 2008;283:2822-34.
Juarez J, Clayman G, Nakajima M, Tanabe KK, Saya H, Nicolson GL, Boyd D. Role and regulation of expression of 92-kDa type-IV collagenase (MMP-9) in 2 invasive squamous cell carcinoma cell lines of the oral cavity. Int J Cancer 1993;55:10-8.
Gweon EJ, Kim SJ. Resveratrol induces MMP-9 and cell migration via the p38 kinase and PI-3K pathways in HT1080 human fibrosarcoma cells. Oncol Rep 2013;29:826-34.
Overeem C van. Laetiporus miniatus (Jungh.) Icon Fung Malay 1925;12:1.
Imazeki R, Hongo T. Colored llustrations of mushrooms of Japan. Vol. II. Osaka: Hoikusha;1989.
Hong MR, Kim YS, Joo AR, Lee JK, Kim YS, Oh DK. Purification and characterization of a thermostable beta-1,3-1,4-glucanase from Laetiporus sulphureus var. miniatus. J Microbiol Biotechnol 2009;19:818-22.
Hwang HS, Lee SH, Baek YM, Kim SW, Jeong YK, Yun JW. Production of extracellular polysaccharides by submerged mycelial culture of Laetiporus sulphureus var. miniatus and their insulinotropic properties. Appl Microbiol Biotechnol 2008;78:419-29.
Yoshikawa K, Bando S, Arihara S, Matsumura E, Katayama S. A benzofuran glycoside and an acetylenic acid from the fungus Laetiporus sulphureus var. minatus. Chem Pharm Bull (Tokyo) 2001;49:327-9.
Leon F, Quintana J, Rivera A, Estevez F, Bermejo J. Lanostanoid tripenes from Laetiporus sulphureus and apoptosis induction on HL-60 human myeloid leukemia cells. J Nat Prod 2004;67:2008-11.
He JB, Tao J, Miao XS, Bu W, Zhang S, Dong ZJ, Li ZH, Feng T, Liu JK. Seven new drimane-type sesquiterpenoids from cultures of fungus Laetiporus sulphureus. Fitoterapia 2015;102:1-6.
Zhang JW, Wen GL, Zhang L, Duan DM, Ren ZH. Sulphureuine B, a drimane type sesquiterpenoid isolated from Laetiporus sulphureus induces apoptosis in glioma cells. Bangladesh J Pharmacol 2015;10:844-53.
Saba E, Son Y, Jeon BR, Kim SE, Lee IK, Yun BS, Rhee MH. Acetyl eburicoic acid from Laetiporus sulphureus var. minatus suppresses inflammation in murine macrophage RAW 264.7 cells. Mycology 2015;43:131-6.
Oh SH, Choi SY, Lee NR, Lee JN, Kim DS, Lee SH, Park SM. Cell migration and anti-inflammatory effect of Red Ginseng extracts fermented with Laetiporus sulphureus. J Soc Cosmet Sci Korea 2014;40:297-305.
Sinanoglou VJ, Zoumpoulakis P, Heropoulos G, Proestos C, Ciric A, Petrovic J, Glamoclija J, Sokovic M. Lipid and fatty acid profile of the edible fugus Laetiporus sulphurous.Antifungal and antibacterial properties. J Food Sci Technol 2015;52:3264-72.
Klaus A, Kozarski M, Niksic M, Jakovljevic D, Todorovic N, Stefanoska I, Van Griensven LJ. The edible mushroom Laetiporus sulphureus as potential source of natural antioxidants. Int J Food Sci Nutr 2013;64:599-610.
Kim YJ, Kim CK, Kwon YJ. Isolation of antioxidative components of Perillae semen. Korean J Food Sci Technol 1997;29:38-43.
Doljak B, Stegnar M, Urleb U, Kreft S, Umek A, Ciglaric M, Strukelj B, Popovic T. Screening for selective thrombin inhibitor in mushrooms. Blood Coagul Fibrinolysis 2001;12:123-8.
Kozarski M, Klaus A, Jakovljevic D, Todorovic N, Vunduk J, Petrovic P, Niksic M, Vrvic MM, Griensven LV. Antioxidants of edible mushrooms. Molecules 2015;20:19489-525.
Yun YP, Do JH, Ko SR, Ryu SY, Kim JH, Song HC, Park YD, Ahn KS, Kim SH. Effects of Korean red ginseng and its mixed prescription on the high molecular weight dextran-induced blood stasis in rats and human platelet aggregation. J Ethnopharmacol 2001;77:259-64.
Kim KH, Lee HJ, Lee JH, Jang YS, Kim DK, Shim BS, Cho KH, Ko SG, Ahn KS, Kim SH. Blockade of glycoprotein IIb/IIIa mediates the antithrombotic activity of butanol fraction of Actinostemma lobatum Maxim. J Ethnopharmacol 2008;116:431-8.
Kim EJ, Che ZM, Park YJ, Hwang YS, Kim KY, Jung DW, Jeon NK, Choi YW, Lee EJ, Kim J. Morphogenesis and biological significance of spindle cell transformation in a spindle cell carcinoma. Cancer Lett 2009;275:61-71.
Koo BH, Park MY, Jeon OH, Kim DS. Regulatory mechanism of matrix metalloproteinase-2 enzymatic activity by factor Xa and thrombin. J Biol Chem 2009;284:23375-85.
Hwang YS, Park KK,Chung WY. Kalopanaxsaponin a inhibits the invasion of human oral squamous cell carcinoma by reducing metalloproteinase-9 mRNA stability and protein trafficking. Biol Pharm Bull 2012;35:289-300.
Weng CJ, Chau CF, Hsieh YS, Yang SF, Yen GC. Lucidenic acid inhibits PMA-induced invasion of human hepatoma cells through inactivating MAPK/ERK signal transduction pathway and reducing binding activities of NF-kappaB and AP-1. Carcinogenesis 2008;29:147-56.
Park SK, Hwang YS, Park KK, Park HJ, Seo JY, Chung WY. Kalopanaxsaponin a inhibits PMA-induced invasion by reducing metalloproteinase-9 via PI3K/Akt- and PKCdelta-mediated signaling in MCF-7 human breast cancer cells. Carcinogenesis 2009;30:1225-33.
※ AI-Helper는 부적절한 답변을 할 수 있습니다.