Supplementary file 1_Lentinan inhibits colorectal cancer stemness by binding CD133 and suppressing the CD133/p85/p-AKT signaling axis.docx

BackgroundColorectal cancer (CRC) ranks third in the global cancer incidence rate worldwide. Cancer stem cells (CSCs) are key drivers of CRC recurrence, metastasis, and therapy resistance, while therapies against them remain limited. Lentinan is widely recognized for its strong immune-enhancing and broad-spectrum directly antitumor activities, however, whether it has potential for cancer stemness remains unknown.
MethodsThe CRC cell lines HCT116 and SW620 were selected for pharmacodynamic investigation. Furthermore, tumor sphere formation and limiting dilution assays were used to assess the impact of SLNT on stemness of CRC. Using cell sorting alongside cluster of differentiation 133 (CD133) knockdown and overexpression experiments, the key molecular targets of SLNT in exerting anti-CRC effects were identified. To further elucidate the underlying molecular mechanism, we performed localized surface plasmon resonance (LSPR), cellular thermal shift assay (CETSA), and molecular docking simulations. Additionally, western blot analysis was conducted to validate the key signaling molecules involved.
ResultsOur results demonstrated that lentinan significantly suppressed the proliferation and stemness of CRC cell lines HCT116 and SW620. We identified CD133 as a critical functional target and confirmed a direct binding interaction between lentinan and CD133. Finally, we revealed that lentinan suppresses stemness by inhibiting the CD133/ phosphatidylinositol 3-kinase 85 kDa regulatory subunit (p85)/ phosphorylated AKT serine/threonine kinase (p-AKT) signaling axis in CRC.
ConclusionOur findings not only shed new light on the anti-tumor mechanism of lentinan, highlighting the scientific potential of natural polysaccharides in cancer treatment, but also offers new options for the clinical drug therapy of CRC.

研究背景：结直肠癌（Colorectal cancer, CRC）是全球发病率排名第三的恶性肿瘤。癌症干细胞（Cancer stem cells, CSCs）是结直肠癌复发、转移及治疗耐药的关键驱动因素，但当前针对癌症干细胞的治疗手段仍十分有限。香菇多糖（Lentinan）因兼具强大的免疫增强活性与广谱直接抗肿瘤作用已得到广泛认可，但其是否具备调控癌症干细胞干性的潜力尚不明晰。
研究方法：本研究选取结直肠癌细胞系HCT116与SW620开展药效学探究。进一步采用肿瘤球形成实验与极限稀释实验，评估香菇多糖（后文简称SLNT）对结直肠癌干性的影响。通过细胞分选结合分化簇133（Cluster of differentiation 133, CD133）敲低与过表达实验，明确香菇多糖发挥抗结直肠癌作用的关键分子靶点。为进一步阐明其潜在分子机制，本研究开展了局部表面等离子体共振（Localized surface plasmon resonance, LSPR）、细胞热位移实验（Cellular thermal shift assay, CETSA）以及分子对接模拟。此外，通过蛋白质免疫印迹（Western blot）分析验证所涉及的关键信号分子。
研究结果：本研究结果表明，香菇多糖可显著抑制结直肠癌细胞系HCT116与SW620的增殖及癌症干性。研究鉴定出分化簇133（CD133）为关键功能靶点，并证实香菇多糖与CD133之间存在直接结合相互作用。最终揭示，香菇多糖可通过抑制结直肠癌中CD133/磷脂酰肌醇3-激酶85kDa调节亚基（p85）/磷酸化丝氨酸/苏氨酸激酶AKT（p-AKT）信号轴，从而阻断癌症干性。
研究结论：本研究结果不仅为香菇多糖的抗肿瘤机制提供了新的科学见解，凸显了天然多糖在癌症治疗中的应用潜力，同时也为结直肠癌的临床药物治疗提供了全新选择。