植物化合物 cumingianoside A和 deoxyelephantopin 及其衍生物DETD-35用於抑制抗藥性或轉移性黑色素瘤功效之研究

Abstract

突變的原癌基因BRAF為治療黑色素瘤藥物之標的。可惜的是，黑色素瘤對BRAF抑制劑藥物如vemurafenib (PLX4032) 所產生的抗藥性使得此一標靶治療策略受到質疑，因而目前帶有BRAF突變基因的轉移性黑色素瘤的治療目前仍是一大挑戰，且對於能夠耐久的藥物反應或是減緩標靶藥物副作用的新穎治療策略仍有迫切需求。本論文旨在探討皂素cumingianoside A (CUMA)、倍半萜內酯DET及其半有機合成衍生物DETD-35針對抗PLX4032之BRAF突變黑色素瘤或轉移性黑色素瘤的抑制效果，並了解背後的分子機制。首先，我們建立從楝科(Meliaceae) 蘭嶼樫木 (Dysoxylum cumingianum) 葉子及枝條萃取三萜類皂素CUMA的方法，並探討CUMA在活體內及細胞實驗中抑制抗PLX4032之BRAF突變黑色素瘤細胞(A375-R)的活性。 我們的結果顯示CUMA處理能夠抑制A375-R生長，且此活性隨CUMA濃度與處理時間增加。CUMA亦能抑制A375-R細胞中CDK1/cyclin B1的表現而造成細胞生長週期G2/M期的停滯。再者，CUMA能夠透過增進caspase 3及caspase 7的活性和PARP的水解促進細胞凋亡。我們也觀察到CUMA會在A375-R誘發類細胞自噬，其中包含自噬相關的基因表現上升以及自噬小體的生成。我們更發現CUMA會誘發細胞內質網壓力，且與內質網壓力抑制劑(4-PBA)共同處理A375-R細胞時能夠延緩細胞的凋亡。更重要的是，在異種移植的抗PLX4032之BRAF突變黑色素瘤小鼠模式中，不管是以口服方式給予單一處理CUMA或是共同給予CUMA與PLX4032皆能在不影響正常器官的狀況下有效抑制腫瘤生長。本論文是第一篇探討CUMA在活體內及體外試驗中抗黑色素瘤活性及機制的研究，儘管仍需更進一步的研究，我們目前的結果顯示開發此三萜類皂素做為抗癌藥物的潛。 在我們研究室先前利用同一異種移植的BRAF突變且抗PLX4032之黑色素瘤小鼠 模式中，從菊科 (Asteraceae) 草藥地膽草 (Elephantopus scaber L.) 所萃取出的DET及其衍生物DETD-35能夠增進抗PLX4032腫瘤對PLX4032的敏感性。另外，DET及DETD-35能夠在異種移植小鼠模式中抑制A375LM5IF4g/Luc黑色素瘤細胞的肺轉移。本論文活體試驗結果研究DET及DETD-35抑制黑色素瘤肺轉移活性的相關機轉。我們發現自由基螯合劑N-acetylcysteine (NAC)的預處理能夠壓制DET及DETD-35所誘發的細胞毒性及細胞凋亡。再者，DET及DETD-35能夠破壞A375LM5IF4g/Luc細胞的粒線體構形並導致粒線體功能喪失。更重要的是，DET及DETD-35能抑制轉移相關的分子標記N-cadherin 的表現量，而在NAC預處理後，能阻斷此現象，且在肺轉移密切相關的分子標記integrin 4亦可觀察到表現量下降，但NAC僅稍微阻斷。我們在細胞實驗觀察到DET及DETD-35的抗轉移活性也在活體試驗結果得到證實，其中包含在DET及DETD-35處理後的小鼠黑色素瘤轉移之肺臟組織觀察到N-cadherin的減少，以及細胞緊密連接蛋白claudin-1和ZO-1及血管新生標記CD31的表現量顯著減少。DET及DETD-35並抑制腫瘤細胞在肺臟所引發的巨噬細胞及嗜中性白血球的浸潤。DET及DETD-35的免疫調節潛力也能夠透過減低的肺臟組織中COX-2及PD-L1表現量受到證實。本研究揭露了DET及DETD-35抑制肺轉移黑色素瘤的活性，其中一部分是透過影響活性氧類相關的細胞凋亡、轉移型態的調控以及肺腫瘤微環境的調節。 總結來說，此篇論文所研究的內容揭露了植物皂素CUMA及倍半萜內酯DET/DETD-35被開發做為抑制黑色素瘤藥物的潛力。

Mutated proto-oncogene BRAF is a bona fide therapeutic target for melanomas. Regrettably, melanoma acquires resistance to BRAF inhibitors, e.g., vemurafenib (PLX4032) casting doubt on this promising melanoma targeted therapy. BRAF-mutant metastatic melanoma is still challenging and new treatment modalities for advanced melanoma patients are of urgent need to elicit durable response or minimize current adverse effects in therapy. The objective of this dissertation was to investigate the anti-melanoma effect of phyto-saponin cumingianoside A (CUMA) and phyto-sesquiterpene lactone DET and its semi-synthetic derivative DETD-35 against BRAF-mutant PLX4032-resistant or metastatic melanoma, and to explore the molecular mechanisms. First, in this study we established the protocol to isolate triterpenoid saponin (CUMA) from the leaves and twigs of Dysoxylum cumingianum (Meliaceae) and addressed CUMA effect against PLX4032-resistant BRAFV600E mutant melanoma A375-R in vitro and in vivo. Our data showed that CUMA treatment inhibited A375-R melanoma cell proliferation in a time- and dose-dependent manner. CUMA also suppressed the expression of CDK1/cyclin B1 complex and led to G2/M-phase arrest of A375-R cells. Furthermore, CUMA treatment resulted in induction of apoptosis as shown by the increased activation of caspase 3 and caspase 7, and the proteolytic cleavage of poly(ADP-ribose) polymerase (PARP). We also observed that CUMA induced autophagy-like activity in A375-R cells, as shown by the increased expression of autophagy-related genes and increased formation of autophagosomes. Moreover, we found that CUMA treatment induced ER stress response and co-treatment with an ER stress inhibitor (4-PBA) could attenuate apoptosis induced by CUMA. Notably, orally administered CUMA as a single agent or in combination with PLX4032 exhibited strong tumor growth inhibition in a PLX4032-resistant A375-R xenograft mouse model, and with little toxicity to organs. This is the first report to explore the anti-melanoma activity of CUMA in vitro and in vivo mechanistically, and albeit further research is required, our results imply that this triterpenoid saponin may be suitable for further research and development. Our lab demonstrated previously that DET isolated from the medicinal herb Elephantopus scaber L. (Asteraceae) and DETD-35 overcome PLX4032 acquired resistance and sensitized PLX4032 in the same xenograft mouse model. Also, DET and DETD-35 significantly suppressed the metastatic potential of A375LM5IF4g/Luc lung-seeking melanoma cells in xenograft mice. As a continuing work, the mechanisms underlying the anti-metastatic activities of DET and DETD-35 against A375LM5IF4g/Luc cells were explored. We observed that pretreatment with ROS scavenger N-acetylcysteine (NAC) blunted DET- and DETD-35 induced cytotoxicity and apoptosis in cells. Moreover, DET and DETD-35 impaired A375LM5IF4g/Luc mitochondrial integrity and caused mitochondrial bioenergetic dysfunction. Furthermore, DET- and DETD-35-treatment lead to down-regulation of EMT-related marker N-cadherin which expression was reversed to the basal level by NAC pretreatment. The level of lung specific metastasis marker integrin 4 was also decreased by both compounds and partially reversed by NAC pretreatment. The anti-metastatic properties of DET and DETD-35 observed in vitro were supported by the decreased N-cadherin expression levels in melanoma metastatic lung tissues of mice treated with DET and DETD-35. Significant decrease in the levels of tight junction proteins claudin-1 and ZO-2 and angiogenesis marker CD31 were also observed in the same treated lung tissues. Furthermore, the significant tumor-elicited macrophage and neutrophils infiltration into mice lungs was suppressed by DET and DETD-35. The immunomodulatory potential of both compounds was also evident by the decreased COX-2 and PD-L1 expression in lung tissues compared to tumor control mice. Altogether, this study gives mechanistic insight into the bio-efficacy of DET and DETD-35 against lung metastatic melanoma which might be in part thorough ROS-related apoptosis and metastatic cell phenotype modulation and lung tumor microenvironment regulation. In summary, the study from this dissertation sheds a light on the potential of phytocompounds saponin CUMA and sesquiterpene lactone DET/DETD-35 in the treatment of melanoma.