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| Title: | 評估靈芝免疫調節蛋白減緩肺癌上皮間質型轉換及輔助化學療法與誘發細胞自體吞噬之機轉
Investigations of the Mechanisms of Alleviated Epithelial-Mesenchymal Transition, Chemotherapeutic Prevention and Autophagy Induction by Ganoderma Immunomodulatory Proteins |
| Authors: | 柯俊良 |
| Contributors: | 醫學研究所 |
| Keywords: | 細胞凋亡肺癌;細胞自體吞噬;腫瘤發生;上皮間質型轉換;靈芝免疫調節 蛋白;肺癌 |
| Date: | 2014 |
| Issue Date: | 2015-02-25T09:16:03Z (UTC)
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| Abstract: | 肺癌是全球癌症致死率前幾名的惡性腫瘤。本實驗室之前的研究發現靈芝免疫調節 蛋白(GMI)能抑制肺癌細胞轉移與誘發細胞自噬型死亡。本研究的目的為從肺癌防治到 治療肺癌，以不同層面探討 GMI的作用，分別為(1) GMI 抑制氯化鎳誘發之上皮間質型 轉換(EMT)，腫瘤發生與發炎反應；(2) GMI 促進 cisplatin 的抗癌效果；(3) GMI 降 低肺癌細胞與 docetaxel 導致之骨髓損傷與骨質疏鬆。國際癌症研究機構將鎳化合物 列為致癌物質。本實驗室之前的研究中發現，氯化鎳誘發 E-cadherin啟動子高度甲基 化抑制 E-cadherin表現，進而誘發肺癌細胞產生上皮間質型轉換。上皮間質型轉換已 知會導致組織纖維化，增加癌症轉移跟癌症幹細胞特性。第一年，我們將分析 GMI 抑 制氯化鎳誘發之上皮間質型轉換，癌幹細胞特性與發炎反應，探討 GMI 預防氯化鎳誘 發知腫瘤生成。我們將使用 MSP與bisulfite sequencing證實GMI透過反轉 E-cadherin 啟動子高度甲基化，而減緩氯化鎳誘發之上皮間質型轉換，並透過 Dot blot 與 ChIP 試驗，分析移除 DNA 甲基化的重要基因 TET 的功能以及 5hmC 的表現。以β-catenin 活性報導基因試驗，端粒酶活性試驗與 CD133/CD44流式細胞儀分析，評估 GMI抑制氯 化鎳誘發之癌幹細胞化。使用 NF-κB活性報導基因試驗，共軛焦顯微鏡與 ELISA，分析 GMI抑制氯化鎳刺激活化之發炎體表現與發炎反應。Cisplatin與 Docetaxel 是肺癌治 療常用的化療藥物，然而他們高劑量使用伴隨著副作用，影響病患的生活品質。第二 年將探討 GMI 如何促進 cisplatin 的抑癌效果。使用 VSV-G pseudotyped lentivirus-shRNA 系統抑制 Beclin-1、ATG5、LC3 表現建立細胞自噬能力缺陷細胞， 並使用他們釐清 GMI 是否透過細胞自噬增進 cisplatin 誘發細胞凋亡。分析 DNA 修補 基因 ERCC1 與抗凋亡基因 Bcl-2 受 GMI 抑制的情形，證實 GMI 透過抑制 DNA 修補與抗 凋亡機制促進 cisplatin 藥效。我們將使用動物腫瘤實驗分析活體中口服 GMI 與注射 cisplatin的協同作用。第三年，首先將使用動物模式印證 GMI抑制氯化鎳誘導上皮間 質型轉換，肺部纖維化以及肺腫瘤生成的能力。除此之外，我們將以動物模式分析 GMI 如何抑制肺癌細胞與 docetaxel 導致之骨髓脂肪生成與骨質疏鬆。使用人類骨髓幹細 胞與 Oil Red O 染色偵測脂肪生成。已知 IL-20 與 RANKL 會促使蝕骨細胞分化，因此 將使用 ELISA分析 GMI是否抑制肺癌細胞與 docetaxel所誘發之 IL-20與 RANKL表現。 並使用 TRAP與 ALP試驗分析前成骨細胞與前蝕骨細胞分化，證實 GMI是否抑制肺癌細 胞與 docetaxel 所導致之骨代謝失衡。本研究將證實 GMI 具有成為化學預防與化療佐 劑的潛力。GMI降低化療產生的副作用，將有助於改善癌症病患的生活品質。
Lung cancer is one of the most common malignancies in the world and a leading cause of cancer-related death in men and women. In our previous studies, GMI, a Ganoderma immunomodulatory protein, inhibited metastasis and induced autophagic cell death. The aim of this study is to investigate the effects of GMI on cancer prevention and cancer therapy from three aspects: (1) GMI inhibition of NiCl2-induced EMT, carcinogenesis, and inflammation; (2) GMI promotion of anti-cancer effects of cisplatin; and (3) GMI-induced decreases in lung cancer/docetaxel-mediated bone marrow damage and osteoporosis. The International Agency for Research on Cancer (IARC) classifies nickel compounds as carcinogenic to humans. In our previous study, NiCl2 induced epithelial-mesenchymal transition (EMT) through E-cadherin promoter hypermethylation. In the first year, we will analyze the effects of GMI on NiCl2-induced carcinogenesis. We will test the hypothesis that GMI alleviates NiCl2-induced EMT through reversal of E-cadherin promoter hypermethylation by MSP and bisulfite sequencing. Dot blot and ChIP assay will be used to investigate the function of TET, a DNA methylation removing gene, and expression of 5hmC. The inhibitory effects of GMI on NiCl2-induced cancer stemness will be observed by β-catenin activity reporter assay, telomerase TRAP assay, and CD133/CD44 flow cytometry. NiCl2-induced inflammasome expression and inflammation blocked by GMI will be analyzed by NF-κB activity reporter assay, confocal microscopy, and ELISA. Cisplatin and Docetaxel are chemotherapy drugs that are commonly used to treat lung cancer. However, they cause several side effects that affect patient quality of life. In the second year, the aim will be to study the role of GMI-induced effects of cisplatin. Autophagy-defective lung cancer cells will be established by VSV-G pseudotyped lentivirus-shRNA system for Beclin-1, ATG5 and LC3 silencing to clarify the role of GMI-induced autophagy on cisplatin-mediated apoptosis. The inhibitions of DNA repair gene ERCC1 and anti-apoptotic gene Bcl-2 will be used to demonstrate the mechanisms of GMI promotion of cisplatin-induced apoptosis. The synergistic effect of GMI on cisplatin will be investigated by xenograft tumor model. In the third year, animal models will be used to prove the inhibitory effect of GMI on NiCl2-induced EMT, lung fibrosis, and lung carcinogenesis in Beas-2B and A549 cells. The effects of GMI on lung cancer cell and docetaxel-induced bone marrow adipogenesis and osteoporosis will be investigated by animal model. Adipogenesis of bone marrow will be detected in human bone marrow mesenchymal stem cells by Oil Red O staining. IL-20 and RANKL have been documented to induce osteoclast differentiation. The expressions of IL-20 and RANKL in lung cancer cells after GMI and docetaxel treatment will be detected by ELISA. The differentiation and apoptosis of pre-osteoclast and pre-osteoblast will be investigated by TRAP and ALP assays to elucidate the role of GMI in lung cancer cell and docetaxel-induced imbalance in bone metabolism. This study may prove that GMI is a potential chemopreventive agent and chemotherapeutic adjuvant. As GMI decreases the side effects of chemotherapy this may lead to improved life quality for cancer patients. |
| URI: | https://ir.csmu.edu.tw:8080/ir/handle/310902500/10257 |
| Appears in Collections: | [醫學研究所] 研究計劃
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