靈芝免疫調節蛋白經由調控整合素相關訊息傳遞路徑與影響腫瘤微環境來降低黑色素瘤之轉移作用

Ganoderma immunomodulatory protein reduced the metastasis of melanoma via regulating integrin-related signaling pathway and modulated tumor microenvironment

呂俊德(Chun-Te Lu)

指導教授 ： 柯俊良

中山醫學大學/醫學院/醫學研究所/博士(2019年)

https://doi.org/10.6834/CSMU201900023

摘要

背景：
腫瘤細胞的轉移，是個多重步驟而且腫瘤微環境複雜作用的過程。就晚期黑色素瘤治療而言，標靶治療和免疫檢查點阻斷劑是目前最有效的治療，但仍然有易產生抗藥性和反應率低的缺點。靈芝免疫調節蛋白(Ganoderma immunomodulatory protein) GMI，除了可以增強人體免疫反應，對肺癌細胞，具有誘導細胞自噬，加強化療藥物作用細胞凋亡效果。然而GMI對黑色素瘤的作用機制目前仍尚未清楚。
研究目的：
針對於黑色素瘤，探討靈芝免疫調節蛋白GMI是否透過影響細胞存活，細胞水平移行作用，垂直侵襲，細胞毒殺，以及血管新生，來限制黑色素瘤細胞的轉移。進ㄧ步分析GMI合併標靶藥物chidamide，或合併ketoconazole抗黴菌藥物，可能的協同抑制作用以及相關訊息傳遞路徑。並且利用動物試驗，證實合併標靶藥物chidamide和免疫治療藥物Pembrolizumab的效果。
研究方法及資料：
我們分析單獨使用GMI，合併GMI和chidamide，以及合併GMI和ketoconazole，對於黑色素瘤的細胞型態、細胞存活率和移行能力之影響。細胞移行及存活率使用CCK-8和MTT來測定；細胞凋亡和細胞移行相關蛋白表現量使用西方墨點分析法來偵測；細胞週期分布使用流式細胞儀來測定。利用小鼠尾靜脈注射腫瘤，觀察肺轉移腫瘤轉移分析，評估合併GMI和chidamide，以及合併GMI和Pembrolizumab抑制轉移的效果。
研究結果：
在細胞癒合實驗中，GMI能顯著的抑制黑色素瘤A375.S2細胞的生長與移行，而合併處理GMI和chidamide協同性地誘發細胞凋亡的發生。GMI降低Integrin α5、αV、 β1和β3的表現量，而合併處理GMI和chidamide能抑制p-FAK、FAK、phospho-Rb、phosphor-Chk1和Survivin的表現量，並增加cleaved caspase-7 和LC3 II/I的表現量。當A375.S2細胞過度表現Integrin-αV時，便會活化下游p-FAK的路徑。
合併處理GMI和ketoconazole，具有抑制細胞遷移和侵襲的能力，可能是透過活化p-AMPK pathway，並且抑制下游pACC 表現，來影響細胞的轉移。同時，LC3-II 的表現增加，Survivin下降，MCP-1 表現降低，可能有細胞自噬 autophagy參與，影響細胞的存活。在動物實驗中，本研究發現GMI單獨，或合併chidamide，Pembrolizumab都能有效的抑制腫瘤的轉移。
結論與建議：
GMI合併處理chidamide或ketoconazole，都有抑制細胞移行能力。GMI和chidamide合併，透過調控整合素相關的訊息傳遞路徑來抑制黑色素瘤細胞的移行能力和生長速度。GMI和ketoconazole也可經由AMPK-ACC 路徑的活化，影響細胞移動和生長。在活體動物實驗中，本研究發現GMI協同chidamide和免疫藥物Pembrolizumab降低遠端轉移的能力。本研究證實GMI對於轉移性黑色素瘤，是一個具有潛力的治療選擇。

關鍵字

靈芝免疫調解蛋白 ； 黑色素瘤 ； 西達苯胺 ； 酮康唑 ； 遠端轉移 ； 整合素

並列摘要

Background:
Metastasis is a multistep process and complex interactions with tumor microenvironment. For treatment of advanced melanoma, recent advances have been made in target therapies and immune checkpoint inhibitors, but the major disadvantages of treatment are the drug-resistance and low response rate. In lung cancer, Ganoderma immunomodulatory proteins (GMI) could enhance human immune reaction and potentiate Cisplatin induced apoptosis via autophagy. However, the role of GMI in melanoma is not clear.
Objective：
For metastasis of melanoma, we investigated the inhibiting effects of GMI through cell viability, cell migration, cell invasion, cytotoxicity and angiogenesis. We also combined GMI and target therapy drug, chidamide or GMI and antifungal drug, ketoconazole, to study the synergic effect and related signal pathway in melanoma inhibition. In animal model, we proved the anti-metastasis effect of GMI combination treatment with chidamide and Pembrolizumab.
Methods and Materials：
We analyzed the morphology, viability, and migration of melanoma cells under single use of GMI and combination GMI with chidamide or ketoconazole. Cell migration and viability were measured by CCK-8 and MTT. The activities of apoptosis- and migration-related proteins were detected on Western blot. Flow cytometry was used to analyze cell cycle distribution and sub-G1 fraction in treated melanoma cells. An in vivo rat tail vein injection animal model was used to evaluated the anti-metastasis effect of GMI and combination with chidamide or Pembrolizumab.
Results：
GMI significantly inhibited cell growth and migration of A375.S2 cells on Wound healing assay. GMI combined with chidamide synergistically induced apoptosis. GMI inhibited the expressions of Integrin α5, αV, β1, and β3. The levels of p-FAK, FAK, phospho-Rb, phosphor-Chk1 and Survivin decreased and the levels of cleaved caspase-7 and LC3 II/I increased with combination treatment of GMI and chidamide. Integrin-αV overexpression activated p-FAK pathways in A375.S2 cells. In vivo, GMI combined with chidamide suppressed melanoma distal metastasis.
GMI combined with ketoconazole inhibited cell migration and invasion through activating AMPK -ACC pathway and pACC depression. The level of LC3-II increased, surviving and MCP-1 decreased in combination treatment revealed autophagy may involve in cell survival.
Conclusion and Suggestion：
GMI has synergic effect on cells migration inhibition when combination with either chidamide or ketoconazole. GMI combined chidamide inhibited the migration and growth of melanoma cells via integrin-related signaling pathway. GMI and ketoconazole also effected the migration and growth of melanoma cells through activating AMPK-ACC pathway. In vivo study, GMI reduces distal metastases when used with chidamide and Pembrolizumab. GMI is a potential treatment choice for metastatic melanoma.

並列關鍵字

Ganoderma immunomodulatory proteins ； melanoma ； chidamide ； ketoconazole ； distal metastasis ； integrin

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