色胺酮在抗藥性乳癌細胞株MCF-7/adr抑制多重抗藥性之機制

The Mechanisms of Tryptanthrin Reversing Multidrug Resistance in a Breast Cancer Cell Line MCF-7/adr

尤松材(Sung-Tsai Yu)

指導教授 ： 陳燕惠

國立臺灣大學/醫學院/藥學研究所/博士(2009年)

https://doi.org/10.6342/NTU.2009.02603

摘要

化學治療是治療癌症常用且有效的療法，但是腫瘤細胞對抗癌藥物產生抗藥性是化學治療失敗的主要原因。而造成癌細胞多重抗藥性(multidrug resistance；MDR)的主因是細胞內某些酵素和蛋白質在量或功能上的改變，基本上可以分為輸送子(transporter)和非輸送子(non-transporter)路徑。輸送子產生的多重抗藥性主要是藉由ATP-binding cassette (ABC)蛋白的大量表現，改變藥物在細胞內的濃度；而穀胱甘肽硫轉移酶π (glutathione S-transferase π ; GSTπ)的過度表現而增加藥物代謝和藉由第二型拓樸異構酶(Topoisomerase II; Topo II)突變或降低表現量使藥物失去作用目標等二者，則是屬於非輸送子的路徑。
色胺酮(tryptanthrin)為一種indoloquinazoline類衍生物，根據以往的研究顯示對腫瘤細胞及多種真菌、細菌具有殺傷和抑制作用，抗發炎和調節免疫功能等藥用價值。我們利用兩種乳癌細胞株MCF-7/wt及對艾黴素(doxorubicin)有抗藥性的細胞MCF-7/adr，來研究色胺酮對癌細胞多重抗藥性的影響。由逆轉抗藥性的活性測試顯示，色胺酮具有明顯加強doxorubicin 細胞毒性的能力。利用羅丹明123(rhodamine 123)螢光染劑來測試色胺酮是否直接影響 P-gp的功能表現，發現羅丹明123在細胞內的累積量增加將近二倍。藉由定量聚合酵素鏈鎖反應(real-time PCR)實驗，我們證明10-6M色胺酮能降低MCF-7/adr細胞多重抗藥性基因1(MDR1，轉錄P-gp) mRNA的表現。觀察色胺酮對MDR1基因啟動子與轉錄因子結合能力的影響，發現色胺酮能藉由增加NF-κB/p65和c-Fos複合物與MDR1基因啟動子上CAAT區域結合能力，而負向控制MDR1基因的表現。此外，我們也發現色胺酮可縮短MCF-7/adr細胞內突變型p53蛋白的半衰期，增加細胞內野生型p53蛋白的比例，而抑制MDR1基因的表現。
在非transporter路徑方面，我們發現色胺酮也可以抑制穀胱甘肽硫轉移酶π基因的表現。藉由穀胱甘肽硫轉移酶活性分析，發現穀胱甘肽硫轉移酶活性的降低；但是色胺酮對第二型拓樸異構酶基因的表現並沒有看到明顯的影響。有研究報導穀胱甘肽硫轉移酶π蛋白能與c-Jun氨基末端激酶(JNK)結合而抑制c-Jun氨基末端激酶活性，從而影響細胞凋亡、增殖。經共同免疫沈澱法實驗進一步證實由於穀胱甘肽硫轉移酶π蛋白的減少，GSTπ-JNK結合的情形也降低，表示加入色胺酮後MCF-7/adr細胞中未結合的c-Jun氨基末端激酶比率增加。我們也發現當細胞加入艾黴素後此較高比率未結合的c-Jun氨基末端激酶蛋白容易被磷酸化，進而啟動JNK/SAPK (stress-activated protein kinase)路徑導致細胞凋亡。
由以上的結果發現色胺酮可以藉由增加NF-κB/p65和c-Fos複合物與MDR1基因啟動子上CAAT區域結合能力以及影響突變型p53蛋白的穩定，而抑制P-gp的表現。此外，在非輸送子機制方面，可以抑制穀胱甘肽硫轉移酶π蛋白的表現，釋放c-Jun氨基末端激酶使得此抗藥性細胞容易受抗癌藥物刺激而凋亡。目前臨床上使用的多重抗藥性抑制劑多為P-gp的受質，與抗癌藥物競爭P-gp的結合位，但色胺酮作用機制與傳統機制明顯不同，為逆轉癌細胞多重抗藥性的研究提供的新的方向。

關鍵字

乳癌 ； 多重抗藥性 ； 艾黴素 ； 色胺酮 ； 逆轉多重抗藥性 ； 多重抗藥性基因1 ； 穀胱甘肽硫轉移酶π ； c-Jun氨基末端激酶細胞凋亡

並列摘要

Development of agents to overcome multidrug resistance (MDR) is important in cancer chemotherapy. MDR arises from transporter and non-transporter based mechanisms during cancer chemotherapy. Transporter-based MDR mechanisms are mainly caused by the transport proteins of ATP-binding cassette (ABC) family. Overexpression of glutathione S-transferase π (GSTπ) and underexpression of Topoisomerase II (Topo II) are associated with multidrug resistance (MDR) phenotype through non-transporter pathway.
Up to date, few chemicals have been reported to down-regulate MDR1 gene expression. I evaluated the effect of tryptanthrin on P-glycoprotein (P-gp) mediated MDR in a breast cancer cell line MCF-7. When treated with doxorubicin, tryptanthrin at 10-6M could enhance cytotoxicity of doxorubicin leading to a 6.2-fold decrease in IC50, in MCF-7/adr cells. Tryptanthrin at 10-6M increased intracellular rhodamine 123 accumulation by 2 folds in MCF-7/adr cells, compared with the control. Using real-time PCR assay, tryptanthrin could depress overexpression of MDR1 gene. I observed reduction of P-gp protein in parallel with decreases in mRNA in MCF-7/adr cells treated with tryptanthrin. Tryptanthrin suppressed the activity of MDR1 gene promoter. Tryptanthrin also enhanced interaction of the nuclear proteins with the negatively regulatory CAAT region of MDR1 gene promoter in MCF-7/adr. It might result in suppression of MDR1 gene. In addition, tryptanthrin decreased the amount of mutant p53 protein with decreasing mutant p53 protein stability. It might contribute to negative regulation of MDR1 gene.
In terms of non-transporter pathway, tryptanthrin down-regulated GSTπ expression and reduced GSTπ protein and glutathione-S transferase activity, but had no effect on Topo II expression. Less production of GSTπ decomposed the protein-protein interactions of GSTπ and c-jun NH2-terminal kinase (JNK). The resulting free form JNK underwent phosphorylation upon elevated intracellular doxorubicin accumulation and subsequently activated JNK-mediated apoptosis. Tryptanthrin reverses MDR partly via modulating GSTπ-related pathway, a non-transporter pathway, in MCF-7/adr cells.
In conclusion, tryptanthrin exhibits MDR reversing effect by down-regulation of MDR1 gene and inhibition of GSTπ expression in conjuction with release of free JNK for activation of JNK/c-jun pathway. Results implicate that tryptanthrin may be a potential chemo-adjuvant agent, exhibiting MDR-reversing activity via transporter and non-transporter pathways.

並列關鍵字

apoptosis ； breast cancer ； doxorubicin ； GSTπ ； JNK ； MCF-7 ； MDR reversal ； MDR1 ； multidrug resistance ； p53 ； tryptanthrin

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