本土產硫磺菌含硫多醣體之物化性質、化學結構及抗乳癌活性研究

Abstract

藥用菇類應用於保健食品及民俗用藥的歷史悠久。多醣體 (polysaccharides, PS) 為菇類主要的活性成分之一，其多元的生物活性深受物化性質及結構影響。含硫多醣體 (sulfated polysaccharides, SPS) 為一種單醣上帶有硫酸根的PS，由於其特殊的物化性質而具有優異的生物活性，故廣泛應用於醫療及保健食品。天然SPS的來源主要為海藻，迄今尚未發現其存在於菇類子實體中，直到本研究團隊於臺灣南投豐丘採集到一株食藥用菇類「硫磺菌 (Laetiporus sulphureus)」，並經初步研究後發現其子實體富含SPS，然而目前有關硫磺菌PS和SPS之物化性質及其生物活性研究仍十分缺乏。本研究目的擬探討本土產硫磺菌之PS及SPS的物化性質差異，並選用三陰性乳癌細胞株MDA-MB-231作為抗癌活性模式細胞，分析PS及SPS之抗乳癌活性，並進一步解析活性多醣體的化學結構與分子作用機轉，以及評估活性多醣體與乳癌化療藥物合併施用的潛力。結果顯示，硫磺菌PS及SPS的硫酸根 (0.16%及16.17%)、蛋白質 (0.43%及4.33%) 和糖醛酸 (10.98%及3.43%) 含量，以及單醣組成、分子量和構形皆具有顯著差異。SPS對MDA-MB-231細胞增生的抑制活性優於PS，且對乳癌細胞具有選擇性細胞毒性。SPS主要透過阻滯細胞週期於G0/G1期進而抑制乳癌細胞增生。此外，其亦能阻止乳癌細胞遷徙，並且具有活化巨噬細胞並增強毒殺乳癌細胞的活性。根據SPS的分子量分布特徵，可將其分為三個不同分子量的分餾物，其中以中分子量群含硫多醣體F2對乳癌細胞增生的抑制效果最佳，且能選擇性毒殺乳癌細胞。F2主要透過抑制表皮生長因子受體 (EGFR) 的磷酸化與蛋白質表現，進而減弱ERK1/2和Akt磷酸化，阻礙NF-κB與GSK-3β/β-catenin途徑的活化，導致下游CDK4與cyclin D1的蛋白質表現量下降，同時促使p21的蛋白質表現量提高，最後引發乳癌細胞週期停滯於G0/G1期。此外，F2還能透過EGFR介導之途徑下調MMP-9及MMP-2的蛋白質表現量，進而抑制乳癌細胞的遷徙及侵襲。F2主要由半乳糖、甘露糖、岩藻糖及葡萄糖組成，莫耳比例約為4：3：3：2，分子量為23.0 kDa，其主鏈的重複單元由24個α-(1→6)半乳糖殘基與6個β-(1→6)葡萄糖殘基構成，部分半乳糖殘基的2-O處連接α-甘露糖、α-岩藻糖或α-岩藻糖-3-O-α-甘露糖，而部分葡萄糖殘基在3-O處與β-葡萄糖-3-O-β-葡萄糖連接，硫酸根基團主要位於岩藻糖上。F2能增強小紅莓 (doxorubicin) 對乳癌細胞的毒殺效果，同時減少其對正常乳房表皮細胞的毒害，其中以序列性處理模式的效果優於同步處理模式。F2主要透過增強小紅莓誘導的cleaved PARP蛋白質表現量，進一步促進細胞凋亡，進而達到兩者在序列性處理下的協同抗乳癌效果。總結來說，硫磺菌含硫多醣體F2具有獨特的化學結構，並會透過調控EGFR介導的訊息傳遞途徑，對乳癌細胞產生抗增生與抗轉移效果；此外，F2與小紅莓的協同抗癌作用顯示其在乳癌合併治療中的應用潛力。

Medicinal mushrooms have a long history of being widely used as functional foods and folk medicines. Polysaccharides (PS) are one of the major bioactive components in mushrooms, and their diverse bioactivities are mainly affected by their physicochemical properties and structures. Sulfated polysaccharides (SPS) are a group of polysaccharides characterized by the presence of sulfate groups on monosaccharides; they possess remarkable bioactivities due to their unique physicochemical properties. Currently, natural SPS are mainly derived from marine algae, it has not been reported in mushroom fruiting bodies. It was until our research team first collected an edible and medicinal mushroom, Laetiporus sulphureus, from Fengqiu, Nantou, Taiwan. Preliminary studies discovered that its fruiting bodies contained a high amount of SPS. However, to-date, studies on the physicochemical properties and anti-breast cancer activities of PS and SPS from L. sulphureus remain limited. This study aimed to investigate the differences of physicochemical properties between PS and SPS from local L. sulphureus, followed by using the triple-negative breast cancer cell line MDA-MB-231 as a cancer model organism to examine their anti-breast cancer activities. Furthermore, the chemical structures and mechanism(s) of action of the most bioactive polysaccharides, and their potential of combining with chemotherapy drugs for breast cancer therapy were also analyzed. Results showed that the contents of sulfate (0.16% vs. 16.17%), protein (0.43% vs. 4.33%), and uronic acid (10.98% vs. 3.43%), as well as the monosaccharide composition, the molecular weight, and the conformation of PS were significantly different from SPS. SPS exhibited more potent inhibitory effect on the MDA-MB-231 cell proliferation than PS, and it also possessed selective cytotoxicity against breast cancer cells. The inhibitory effect of SPS on breast cancer cells was mainly mediated by arresting cell cycle at G0/G1 phase. Furthermore, it also inhibited cancer cell migration and was shown to activate macrophages, consequently led to enhance a cytotoxicity against breast cancer cells. Based on the molecular weight distribution of SPS, it was separated into three different fractions. Among the three SPS fractions, the medium-molecular-weight sulfated polysaccharide fraction, F2, exhibited the most potent inhibitory effect on the breast cancer cell proliferation, and it showed selective cytotoxicity against breast cancer cells. F2 inhibited the phosphorylation and protein expression of epidermal growth factor receptor (EGFR), thereby attenuating the phosphorylation of ERK1/2 and Akt, blocking the activation of NF-κB and GSK-3β/β-catenin pathways, and resulted in the down-regulation of downstream targets, CDK4 and cyclin D1, protein expressions, as well as the up-regulation of p21 protein expression, to induce the breast cancer cell cycle arrest at G0/G1 phase. Furthermore, F2 also inhibited breast cancer cell migration and invasion by down-regulating MMP-9 and MMP-2 protein expressions through the EGFR-mediated pathway. F2 was composed of galactose, mannose, fucose, and glucose in a ratio of approximately 4:3:3:2, with a molecular weight of 23.0 kDa. The repeat unit of the F2 backbone was composed of twenty-four α-(1→6)-galactosyl residues and six β-(1→6)-glucosyl residues. Parts of the galactosyl residues were substituted at 2-O by α-mannosyl, α-fucosyl, or α-fucosyl-3-O-α-mannosyl residues, and the parts of glucosyl residues were substituted at 3-O with β-glucosyl-3-O-β-glucosyl residues. Sulfate groups were primarily located on the fucose residues. F2 enhanced the cytotoxic effect of doxorubicin on breast cancer cells while reducing its toxicity to normal mammary epithelial cells, with sequential treatment showing greater efficacy than simultaneous treatment. F2 enhanced doxorubicin-induced cleaved PARP protein expression, promoting cell apoptosis and synergistically enhancing anti-breast cancer effects under the sequential treatment. In conclusion, the sulfated polysaccharide F2 from L. sulphureus possessed a unique chemical structure and exhibited anti-proliferative and anti-metastatic effects on breast cancer cells by regulating EGFR-mediated signaling pathways. Additionally, the synergistic anti-cancer activity of F2 combined with doxorubicin highlighted its potential application in breast cancer combination therapy.