吲哚與比咯苯偶氮駢共軛抗癌試劑 (Indole carboxylate-PBD hybrid）對A375黑色素細胞瘤之促細胞凋亡、抗癌細胞轉移與抗血管新生作用

Apoptosis-inducing, Anti-migratory, and Anti-angiogenic Effects of a Novel DC-81-indole Conjugate Agent on Cultured Human Melanoma A375 Cells

謝銘珠(Ming-Chu Hsieh)

指導教授 ： 王志鉦

高雄醫學大學/藥學院/藥學研究所/博士(2011年)

摘要

比咯苯偶氮駢 (Pyrrolo [1,4]benzodiazepines，簡稱PBDs) 為一純化自鏈黴菌屬 (Streptomyces sp.) 之多環化合物，可與染色體產生交叉鍵結 (cross linkage)，進而影響DNA的合成，為一強效抗癌抗生素，先前的研究顯示吲哚與 PBDs共軛試劑會誘發黑色素瘤細胞凋亡，並且抑制癌細胞轉移到老鼠肺部，本研究的目的主要是觀察新型吲哚與 PBDs間含四個碳鏈結之共軛試劑 (IN4CPBD) 對黑色素瘤細胞的生理活性，並和原型 DC 81進行活性比對，同時確認IN4CPBD促進癌細胞凋亡、抗癌細胞轉移與抗血管新生的生理效應與藥理作用機轉。研究結果顯示， IN4CPBD的IC50約為0.25?嵱，其細胞死亡機轉為活化P53與P21，造成細胞停滯在 G1/S 期，繼而抑制Bcl-2表現並促進Bax表現，破壞粒線體的結構造成膜內外電位差下降，活化Caspase活性後，引起DNA片斷化而致細胞凋亡。癌症轉移與腫瘤細胞移動 （cell migration） 有正相關，SDF1-?捋冗乜375 細胞移動，當細胞以不同的 kinase 抑制劑前處理，將造成特定訊息傳遞路徑被阻斷，故吾人確認了 Rho/ROCK、Akt、ERK1/2 及 p38 MAPK 等訊息傳遞路徑，參與了 SDF1-?悕珨今o之黑色素瘤細胞生長與移動。SDF1-?捋今o之黑色素瘤細胞生長與移動會被IN4CPBD有效抑制，主要機轉為降低黑色素瘤細胞中 VEGF 基因及蛋白表現，同時抑制 MMP-9 膠原水解的活性，此結果意味IN4CPBD具潛在抗血管新生作用，呼應本研究室先前在動物模式中的研究，並進一步提出IN4PBDs抑制黑色素細胞瘤轉移的合理藥理作用機制，IN4CPBD抑制癌細胞轉移的機轉應是透過抑制抗血管新生與細胞移動促成，故IN4PBDs為具潛力的抗癌藥物可期待發展應用於臨床治療。

關鍵字

比咯苯偶氮駢 ； 黑色素細胞瘤 ； 癌細胞轉移

並列摘要

Pyrrolo[2,1-c][1,4]benzodiazepine (PBD) chemicals are antitumor antibiotics inhibiting nucleic acid synthesis. An indole carboxylate-PBD hybrid with six-carbon spacer structure (IN6CPBD) has been previously demonstrated to induce melanoma cell apoptosis and reduce metastasis in mouse lungs. This study aimed at investigating the efficacy of the other hybrid compound with four-carbon spacer (IN4CPBD) and elucidating its anti-metastatic mechanism. Human melanoma A375 cells with IN4CPBD treatment underwent cytotoxicity and apoptosis-associated assays. Transwell migration assay, Western blotting, and ELISA were used for mechanistic study. IN4CPBD exhibited potent melanoma cytotoxicity through interrupting G1/S cell cycle progression, increasing DNA fragmentation and hypodipoidic DNA contents, and reducing mitochondrial membrane potential. Caspase activity elevation suggested that both intrinsic and extrinsic pathways were involved in IN4CPBD-induced melanoma apoptosis. IN4CPBD up-regulated p53 and p21, thereby concomitantly derailing the equilibrium between Bcl-2 and Bax levels. Transwell migration assay demonstrated that stromal cell-derived factor-1???? (SDF1-??) stimulated A375 cell motility, while kinase inhibitors treatment confirmed that Rho/ROCK, Akt, ERK1/2, and p38 MAPK pathways were involved in SDF1-??-enhanced melanoma migration. IN4CPBD not only abolished the SDF1-??-enhanced chemotactic motility but also suppressed constitutive MMP-9 and VEGF expression. Mechanistically, IN4CPBD down-regulated Akt, ERK1/2, and p38 MAPK total proteins and MYPT1 phosphorylation. In conclusion, beyond the fact that IN4CPBD induces melanoma cell apoptosis at cytotoxic dose, the interruption in the VEGF expression and the SDF1-??-related signaling at cytostatic dose may partially constitute the rationale for its in vivo anti-metastatic potency.

並列關鍵字

SDF-1α ； Melanoma cells ； IN4CPBD ； Transwell migration ； Angiogenic factor

參考文獻

Ahmed Kamal, O. Srinivas, P. Ramulu, G. Ramesh, P. Praveen Kumar and M. Shiva Kumar (2004). Synthesis and DNA binding affinity of novel A-C8/C-C2-exo unsaturated alkoxyamido-linked pyrrolo[2,1-c][1,4]benzodiazepine dimers Bioorganic & Medicinal Chemistry 12: 4337-4350.

Ahuja V, b; Dieckgraefe, Brian Kb; Anant, Shrikantb,c (2006). Molecular biology of the small intestine. Current Opinion in Gastroenterology 22: 90-94.

April C. Childs SLP, Amie J. Dirks, Tracey Phillips, and Christiaan Leeuwenburgh ( 2002). Doxorubicin Treatment in Vivo Causes Cytochrome c Release and Cardiomyocyte Apoptosis, As Well As Increased Mitochondrial Efficiency, Superoxide Dismutase Activity, and Bcl-2:Bax Ratio. CANCER RESEARCH 62: 4592-4598.

Arkenau HT, Kefford R, Long GV (2010). Targeting BRAF for patients with melanoma. British Journal of Cancer 104: 392-398.

Baraldi PG, Cacciari B, Guiotto A, Leoni A, Romagnoli R, Spalluto G et al (1998). Design, synthesis and biological activity of a pyrrolo [2,1-c][1,4]benzodiazepine (PBD)-distamycin hybrid. Bioorg Med Chem Lett 8: 3019-3024.