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Identification of Properties of Fungal Metabolites and Gene expression in response to anti-tumour intervention by polysaccharide-K (PSK)
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운지버섯을 이용한 건강기능성식품 개발
<내 용 요 약>
구름버섯은 면역증강작용, 항암작용 등이 잘 알려진 버섯이다.
구름버섯(Coriolus versicolor)은 전국적으로 분포되어 있고 그 기능성이 잘 알려져 있음에도 불구하고 기타의 버섯에 비해 산업화되어 있는 종이 아니다.
일반 소규모 건강원 등에서는 자연산 구름버섯을 채취하여 사용하고 있고 일부의 중소기업에서는 원료를 수입하여 사용하고 있는 실정이다.
이러한 현상을 극복하고 새로운 건강기능성식품의 개발이 절실히 요구되는 실정이다.
버섯원료의 대량배양방법에는 고체배양과 액체배양방법이 있는데 식품원료의 경우 대규모 자본이 투입되는 액체배양방법보다는 고체배양방법이 일반적이다. 그러나 고체배양의 경우 배지의 성분까지 같이 추출하여
사용함으로써 지표물질인 β-(1→4)-D-glucan의 순도가 떨어지고 배양기간 오래 걸린다는 단점을 가지고 있다. 따라서 일정한 기능성을 갖는 산업용 단백다당류를 생산하기 위해서는 액체배양의 최적화실험을 통하여 수율향상, 생산기간 단축 등 생산성을 향상시킴과 동시에 산업용배지선발, 단백다당류 추출조건, 성분분석, 유전자칩을 이용한 생리활성검색 등의 일련의 기술개발이 요구되고 있는 상황이다.
구름버섯에는 항암, 항염증, 면역증강 및 간염의 치료에 뛰어난 효과를 나타내고 있는 것으로 보고되었고, 이러한 효과는 β-(1→4)-D-glucan이 주쇄로 이루어진 Krestin(PSK)라는 단백다당체 화합물로부터 유래되는 것으로 알려지고 있으며, 이외에 ergosterol 및 β-sitosterol과 같은 생리활성을 갖는 테르펜류의 화합물이 함유되어 있는 것으로 알려지고 있다. 이러한 생리활성을 갖는 물질을 분리 정제하여 식품에 적용함으로써 기능성 제품화 할 수 있는 기술개발이 절실히 요구되고 있다.
따라서 유효성분을 다량 함유한 구름버섯 개체를 선발하고 대량증식하여 PSK 등의 유효성분을 분리하고 정제하여 기능성식품에 적용하는 기술을 개발하고 유전자 칩을 이용하여 구름버섯 유래의 단백다당류의 생리활성을 검색하여 면역증강, 항암작용 외에 새로운 생리활성을 탐색하였다
운지버섯 균사체 생산량과 세포 외 다당체 생산량을 비교하여 YD001를 선발하였다.
운지버섯의 액체배양 시 균체량과 다당체의 생성량에 있어서 배양 온도, 배양 pH, 접종량, 최적배지를 결정하였으며 각 배양조건에 따른 다당체의 함량 및 유용성분의 분리 및 정제 공정을 확립하였다.
본 연구결과에 의해 운지버섯이 metastasis에 효능이 있는 것으로 나타났으며 PSK가 전이를 막고 colorectal
cancer mouse의 생존률을 10-20%를 증가시켰다. cDNA microarray를 이용하여 인간의 colorectal adenocarcinoma cell line 인 HCT116를 대상으로 하여 __EXPRESSION__ prfile을 분석하였다.
Cell line에 대해 250 ug/ml PSK를 120 h동안 노출 시킨 결과 112개의 유전자 up-regulate하게 되었고 233개의 유전자는 down-regulate한 결과를 나타내었다.
이들 중 cell cylce을 조절하는 lymphotactin（Lptn)，multi drug resistance protein 3 (MRP3), transgelin (TAGLN)을 포함하는 유전자들과 연관된 연구를 통해 암치료의 효과를 획득할 수 있을 것으로 사료된다.
이러한 결과를 통하여 현재 운지버섯 균사체 기능성 식품이 제조, 판매되고 있으며 추후에 관계된 유전자와
연관된 임상실험을 통하여 신의약개발의 기초자료로 사용하고자 한다.
<원 문>
Identification of Properties of Fungal Metabolites and Gene __EXPRESSION__ in response to anti-tumour intervention by polysaccharide-K (PSK)
Jae Gun Park*† , Kim Sung Soo*, Jung Dae Lim**, Yang Ho Park***
*Young Dong Healthy Supplements Co. LTD,
***College of Agriculture and Life Science, Kangwon National University, Chunchon 200-701, Korea
** Biological Modifier Response International Health Town Cooperation
INSTRUDUCTION
The number of different mushroom species on earth is estimated at 140 000, of which may be only 10% are known. Meanwhile, of those approximately 14 000 species that we know today, about 50% are considered to possess varying degrees of edibility, more than 2000 are safe, and about 700 species are known to possess significant pharmacological properties (Chang, 1996; Wasser and Weis, 1999; Reshetnikov et al., 2001; Wasser, 2002). Mushrooms have long been attracting a great deal of interest in many areas of foods and biopharmaceuticals. They are well known for their nutritional and medicinal values (Chang, 1996; Reshetnikov et al., 2001; Mattila et al., 2000; Smith et al., 2002; Gao et al., 2004)
]. Many species of mushrooms are cultivated worldwide. Mushroom extracts have been increasingly sold as dietary supplements. The market value of mushroom dietary supplement products worldwide is about US$5–6 billion per year (Wasser et al., 2000).
Medicinal mushrooms have an established history of use in traditional oriental therapies. Historically, hotwater-soluble fractions (decoctions and essences) from medicinal mushrooms were used as medicine in the Far East, where knowledge and practice of mushroom use primarily originated (Wasser, 2002; Hobbs, 1995; 2000). Mushrooms such as Ganoderma lucidum (Reishi), Lentinus edodes (Shiitake), Inonotus obliquus (Chaga), Coriolus versicolor (Yun Zhi )and many others have been collected and used for hundreds of years in Korea, China, Japan, and eastern Russia (Wasser, 2002)
Coriolus versicolor (formerly Trametes versicolor, Polyporus versicolor) is a mushroom which grows on tree trunks and belongs to the more-advanced Basidiomycetes class of fungi. This mushroom has long been treasured in the East; in Asia it is known as mushroom of Yun Zhi, and in Korea it is called Goorum or “cloud fungus.” In Japan around 1965 a chemical engineer investigated Coriolus versicolor for its anticancer constituents after observing his neighbor’s life-threatening cancer was cured after taking Yun Zhi. This led to the discovery of PSK (Polysaccharide-K).21 The closely-related PSP (Polysaccharide-Peptide) was first isolated in China some time later, around 1983.
PSK, a protein-bound polysaccharide obtained from cultured mycelia of Coriolus versicolor in basidiomycetes, is a biological response modifier, diverse operations of which include an antitumor action. PSK has a preventive effect on chemical carcinogen-induced, radiation-induced, and spontaneously developed carcinogenesis. According to a Japanese newspaper, about one-third of cancer patients in Japan also take supplements, including traditional ingredients for general well-being (e.g., mushrooms), as biological response modifiers (BRMs) without telling their physicians. The benefits for cancer of non-specific immunopotentiation with BRMs, i.e., OK-432, BCG, polysaccharides, and endogenous cytokines, are thought to be due to 1) immunomodulatory effects, 2) direct anti-neoplastic effects, 3) chemotherapeutic protection of normal tissue, and 4) restorative effects in patients who have been immunosuppressed by both recent surgery and subsequent chemotherapy (Agha-Mohammadi and Lotze, 2000).
However, despite their popularity, as yet there has been no scientific evaluation of the effectiveness of these BRMs in cancer. Polysaccharide-K (PSK), or Krestin, a protein-bound polysaccharide, is a BRM prepared from the mushroom Coriolus versicolor, and has been used in traditional Chinese medicine for centuries (Ng, 1998). PSK (Sankyo Co., Tokyo, Japan) is widely used in adjuvant therapy after surgery or radiotherapy in Japan and other Asian countries, and the Japanese National Health Insurance scheme covers the use of PSK for gastric, colorectal, and lung cancers. Surprisingly, randomised controlled clinical studies have revealed that PSK in adjuvant therapy for gastric, colorectal, esophageal, and lung cancers significantly extends the 5-year survival rates of patients by 10-20% (Nakazato et al., 1994; Mitomi et al., 1992; Ogoshi et al., 1995 ; Hayakawa et al., 1997). Compared with colorectal cancer patients who did not receive PSK, patients receiving PSK showed a lower local recurrence rate (9.5% in the PSK group versus 11.9% in non-PSK group), systemic recurrence rate (16.3% in PSK group versus 19.8% in non-PSK group), lymph node recurrence rate (1.8% in PSK group versus 5.3% in non-PSK group), and peritoneal dissemination rate (1.8% in PSK group versus 3.5% in non-PSK group), and a higher 3-year survival rate (77.2% in PSK group versus 67.7% in non-PSK group) (Mitomi et al., 1992). PSK produces very few serious adverse side effects, and its characteristics allow long-term oral administration. The antineoplastic effects of PSK have also been reported in animal models, and shown to involve radical trapping and modulation of cytokine production and effector cell functions (Ehrke et al., 1983; Hosokawa et al., 1986.). In vitro studies confirmed that PSK induces gene __EXPRESSION__ of several cytokines including TNF-•, IL-1, IL-2, IL-4, IL-6, IL-7, and IL-8, amongst others (Hirose et al., 1990; Nio et al., 1991; Noguchi et al., 1995). The anti-tumour actions of PSK are considered to be not only host-mediated but also to involve a direct regulatory action on the growth factor production and enzyme activities of tumours (Mickey et al., 1989; Saji et al., 1991). To unravel the mechanism that induces anti-neoplastic immunity, we investigated alterations in gene __EXPRESSION__ that are potentially required for direct action of PSK, using a cDNA microarray containing 891 human cDNA fragments in a human lymph node (metastasis) adenocarcinoma cell line, NCI-H2087 with a wild-type p53 gene. Mutations of the p53 gene have been found in approximately 50% of colorectal carcinomas and are associated with lymphatic dissemination and a poorer prognosis (Goh et al., 1994; Hamelin et al., 1994). We therefore examined alterations in __EXPRESSION__ of candidate genes in NCI-H2087 cells, and in SW480 cells which have a mutant p53 gene, before and after exposure to PSK.
.
TEXT
Cell culture and PSK treatment.
A human lymph node (metastasis) adenocarcinoma cell line, NCI-H2087, with a wild-type p53 gene was purchased from the LGC promochem (TEDDINGTON U.K.) , and a cell line with a mutant p53 gene, SW480, was obtained from the Human Science Research Resource Bank (Tokyo, Japan). The cells were grown in RPMI-1640 medium (Gibco, Grand Island, NY, USA) supplemented with fetal bovine serum (10% (v/v), HyClone, Logan, UT, USA), glutamine (2 mM), penicillin (100,000 units/l), streptomycin (100 μg/l), and gentamycin (40 mg/l). Cell cultures were maintained at 37°C, in a humidified atmosphere of 5% CO2/95% air. For the cell growth study, 106 cells were plated per 60 mm dish and treated with PSK (Kureha Chemical Co.,Tokyo, Japan) at the indicated concentrations. Cells were counted using a haemocytometer on the days indicated. Cells were then prepared for flow cytometry, isolation of total cell RNA and cDNA analysis using the SpotBotTM Complete Desktop Manufacturing System (Telechem).
Flow cytometry.
Floating and trypsinized adherent cells were collected, suspended in PBS (-), fixed with 70% (v/v) ethanol, and stained with propidium iodide (50 g/ml). DNA content was analysed using a FACScan (Becton-Dickinson, Lincoln Park, NJ, USA) with CellQuest and Mod Fit LT 1.0 software (Verity Software House, Inc., Topsham, ME, USA). Cell debris and fixation artifacts were gated out.
Isolation of RNA.
After treatment, cell lines grown to log phase as monolayers were washed twice with PBS and total RNA was extracted using Isogen (Nippon Gene, Tokyo, Japan), an acid guanidine thiocyanate-phenol-chloroform method. Isolated RNA was electrophoresed through 1.0% agarose-formaldehyde gels to verify the quality of the RNA, and RNA concentrations were determined from absorbance measurements at 260 and 280 nm.
Identification of gene __EXPRESSION__ profiles by cDNA microarray.
cDNA synthesis, microarray hybridisation, scanning, gridassisted spot identification, and analysis were performed according to the manufacturer's instructions (Clontech laboratories, Inc.). Briefly, 100 g of total cellular RNA was primed with oligo (dT) and reverse-transcribed in the presence of Cy3-labeled or Cy5-labeled dATP, using 1 l of MMLV reverse transcriptase. The resulting Cy3- and Cy5-labeled cDNAs were treated with RNase I for 10 min at 37°C, combined, purified using a NucleoSpin‚ Extraction Spin Column, and assessed for radioactivity by scintillation counting. Sample and control-labelled probes were mixed
together and hybridised to cDNA microarray slides containing 8581 human genes in the Atlas Plastic Human 8K (#7905-1) and Human Cytokine/Receptor (#7744-1) Arrays. Names of these genes are available at http://www.clontech. com/atlas/genelists/index.shtml. The hybridisation solution, ExpressHyb, was placed on a pre-treated microarray slide, and then incubated in a hybridisation chamber overnight at 50°C. After hybridisation, the slide was washed at room temperature, first with 0.2X SSC, 0.1% SDS for 20 min with gentle shaking, then twice with 0.2X SSC (20 min each time). Hybridised slides were scanned, the scanner output images were localised by overlaying a grid on the fluorescent images, and these were then analysed using the AtlasImagesoftware. Each slide contained 9 housekeeping genes to normalise the signal intensities of the fluorescent dyes. The intensities of Cy5 and Cy3 were adjusted so that the mean Cy5 and Cy3 intensities of probe cDNA binding to the housekeeping genes were equivalent. Both final reported intensities (green and red) were filtered, and the spots with intensity <1.5 were eliminated. The ratios of red to green and green to red intensities for all targets were determined. The cDNA microarray results comparing cells treated with or without PSK are based on two completely independent experiments involving separate cell treatments, separate RNA isolations, and separate microarray assays.
CONCLUSIUON
Suppression of cell growth by PSK.
We examined the effects of PSK at various concentrations (0-1,000 μg/ml) on the growth of lymph node (metastasis) adenocarcinoma cell line, NCI-H2087. Exposure to 500 and 1000 μg/ml of PSK suppressed the growth of NCI-H2087 cells by 54.1 and 43.4% at 96 h, respectively (Fig. 1), and of SW480 cells by 69.5 and 60.8% at 96 h, respectively (data not shown).
Figure 1. The growth curve of NCI-H2087 following continuous exposure to 0-1000㎍/㎖ PSK. ◆, control; ■, 10㎍/㎖; ▲, 100㎍/㎖; ×, 500㎍/㎖; ○, 1000㎍/㎖. Each point represents the mean of three independent experiments
Flow cytometry.
Neither NCI-H2087 nor SW480 cells showed any marked difference in cell-cycle distribution pattern before or after treatment with 500 g/ml PSK for 96 h, while the proportion of apoptotic cells, which appeared in a region to the left of the G1 peak, increased from 4.3 to 14.7% in NCI-H2087 cells after treatment (Fig. 2)
Cell cycle distribution in Apoptotic cell Without PSK
(%) With PSK
(%)
Sub-G1 4.3 14.7
G0/G1 58.6 56.1
S 10.4 9.9
G2/M 26.7 19.3
Figure 2. Flow cytometry of NCI-H2087 cell without (A) and with esposure to 500㎍/㎖ PSK for 96h (B). The four values in each represent the percentage of sub-G1, G0/G1, S and G2/M phase. The arrowhead indicated the percentage of sub-G1.
Analysis of __EXPRESSION__ profiles.
Gene __EXPRESSION__ was analysed in NCI-H2087 cells following 96 h of treatment with 500 g/ml PSK (Atlas Plastic Human 8K Arrays #7905-1). Under basic selection conditions, a total of 345 genes were selected from 8,581 human genes as elements associated with exposure to PSK in NCI-H2087 cells. These genes were identified on the basis of altered __EXPRESSION__ with two-fold or higher ratios, and included 112 up-regulated and 233 down-regulated genes. Further analyses of gene __EXPRESSION__ patterns allowed us to identify 19 genes showing a four-fold or more change in __EXPRESSION__, including 7 up-regulated and 12 down-regulated genes (Table I). Genes showing up-regulated __EXPRESSION__ included nm23, vascular endothelial growth factor, drug resistance protein 3 (MRP3), lymphotactin (Lptn) in order of increasing ratio. Genes showing down-regulation included Kinesin-like 4, transgelin(TAGLN) and Pirin in order of decreasing ratio. Following exposure to 500 g/ml PSK for 24 or 96 h, no significant change could be identified in any of the cytokines we examined (Atlas Human Cytokine/Receptor Arrays #7744-1).
Table 1. Representative differnetial gene __EXPRESSION__ defined by a 4-fold or greater change in signal intensity, induced by 500㎍/㎖PSK treatment for 96h in the NCI-H2087 cell line.
Genebank Acession Definition Spot intensity
Control PSK treatement
Up-regulated NM_000269 non-metastatic cells 1, protein (NM23A) 5 54
NM_003786 ATP-binding cassette, sub-family C (CFTR/MRP), member 3 10 46
M32977 Vascular endothelial growth factor 12 43
NM_002995 Small inducible cytokine-subfamily C, member 1 (lymphotactin, Lptn) 7 42
Down-regulated NM_007317 Kinesin-like 4 32 7
NM_003186 transgelin (TAGLN) 31 1
NM_003662 Pirin 19 1
Figure 3. Image of rpresentative differnetial gene __EXPRESSION__ profile by DNA chip in 500㎍/㎖PSK treatment for 96h in the NCI-H2087 cell line.
The nm23 gene encodes a nucleoside diphosphate kinase (p19/nm23 ; EC.2.7.4.6; NDP kinase ). This enzyme is a hexamer of two different subunits (A, B , 152 amino acids each, displaying 88 percent homology with each other). These subunits form all possible isoenzymes (A6, A5B. AB5, B6), which differ in their isoelectrical points. NDP kinase catalyses the phosphorylation of nucleoside diphosphates into triphosphates required for the biosynthesis of nucleic acids. NDP kinase can also phosphorylate GDP in GTP-binding proteins and therefore acts as an activator for such proteins. N-terminal sequence determination of a nucleoside diphosphate kinase isolated from dark-grown oat (Avena) tissue and composed of six 18 kDa subunits shows that 87 percent of the 23 amino acids sequenced are identical with human nm23 protein.
The A-chain of the enzyme is identical with human nm23 which is encoded by a gene mapping to chromosome 17q22. nm23 is called also Metastasis inhibition factor . The B-chain of the enzyme (gene on chromosome 17q21.3) is callednm23-H1. nm23-H1 has been implicated in cell death by apoptosis , following its activation by Granzyme A
nm23 RNA levels are highest in cell lines with low metastatic potential but do not correlate with cell sensitivity to host immune responses. A markedly reduced __EXPRESSION__ of nm23 caused, among other things, by deletion mutations, correlates with a high metastasizing potential of some tumors including mammary and colorectal carcinomas. This is however not a general phenomenon and is not observed in all tumors. The __EXPRESSION__ of nm23 is enhanced in many solid tumors. In human neuroblastomas the enhanced __EXPRESSION__ of nm23 correlates well with the progressive development of an aggressively growing tumor. Low nm23 RNA levels are associated with histopathologic indications of high metastatic potential of human breast tumors. Somatic allelic deletion of the nm23 gene has been observed in DNA from human breast, renal, colorectal, and lung carcinomas. Mutations have been observed also in aggressive childhood neuroblastomas. Resistance to initial chemotherapy in acute monocytic leukemia has been found to be associated with elevated nm23-H1 mRNA levels and these correlated with poor prognosis.
We now focus on the effects of PSK once the progression of carcinogenesis fm begun, and review what is now known of the preventive action of PSK on cancer metastasis. Recent research reports that PSK suppresses pulmonary rnetastasis of methylcholantbrene-induced sarcomas, human prostate cancer DU 145M, and lymphatic metastasis of mouse leukemia P388, and that it has prolonged the survival period in spontaneous metastasis models. PSK also suppresses the metastasis of rat hepatoma AH60C, mouse colon cancer colon 26, and mouse leukemia RL male I in artificial metastasis models. PSK influences the steps of cancer metastasis in a number of ways: (a) by suppression of intravasation through the inhibition of tumor invasion, adhesion and production of cell matrix-degrading enzymes; (b) by suppression of tumor cell attachment to endothelial cells through the inhibition of tumor cell-induced platelet aggregation; (c) by suppression of tumor cell migration after extravasation through the inhibition of tumor cell motility; and (d) by suppression of tumor growth after extravasation through the inhibition of angiogenesis, the modulation of cytokine production, and the argumentation of effector cell functions. In addition, PSK has suppressed the malignant progression of mouse tumor cells through superoxide trapping.
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