BMVC-12C分子針對癌症細胞的選擇性毒殺之機制探討

近年來，螢光小分子應用研究逐漸受到科學家們的重視，本實驗室新合成一個螢光小分子-BMVC-12C，相較於正常的纖維母細胞株，若把此小分子與癌細胞混合，會觀察到6-10倍的螢光增強，此外，藉由雷射共軛焦的幫助，我們發現在正常細胞株內，BMVC-12C主要聚集於溶酶體(Lysosome)中，但癌症細胞中，BMVC-12C卻會離開溶酶體，進而聚集在粒線體(Mitochondria)。藉由BMVC-12C的螢光性質與位置的不同，可區分正常與癌症細胞株，並應用BMVC-12C於偵測早期癌症細胞研究上面。研究顯示BMVC-12C能夠針對癌症細胞有毒殺性，且此毒殺性與在粒線體中BMVC-12C的聚集含量有正比關係，若粒線體中的BMVC-12C愈多，則毒殺效果愈好。由於BMVC-12C會聚集與癌症細胞的粒線體中，為研究此小分子對於粒線體的影響，我們發現BMVC-12C會破壞癌症細胞的粒線體功能，進而產生毒殺作用。故BMVC-12C小分子除了可以應用於臨床的癌症細胞偵測外，更有機會成為新一代的抗癌藥物分子。本研究結果顯示BMVC-12C的毒殺作用與螢光性質，皆會被位於粒線體膜上，抑制通透轉運孔洞的抑制劑-環孢靈(Cyclosporin A, CsA)所影響，此結果暗示BMVC-12C 的作用機制與粒線體上面的通透轉運孔洞有密切關聯性，我們認為BMVC-12C應可穿透此孔洞，進而與粒線體的基質內的DNA作用。由於CD與NMR光譜皆顯示粒線體DNA可能具有G4的四股結構，而我們也利用反轉錄聚合酶(RT-PCR)的實驗，證實BMVC-12C可抑制粒線體DNA的複製。故於此論文中，我們提出一個假設性的作用機制模型，我們認為BMVC-12C針對癌症細胞的抑制機制，是由於BMVC-12C可穩定粒線體DNA上面的G4四股結構，使粒線體DNA的複製作用被抑制，進而導致癌症細胞的死亡。

關鍵字

BMVC-12C ；選擇性毒殺作用；螢光小分子；粒線體； G4股結構

並列摘要

In recent years, fluorescence probe have been widely used in the studies of biological systems. We have synthesized a new fluorescence probe, 3,6-Bis(1-methyl-4-vinylpyridium iodide)-9-(1-(1-methyl-Piperidinium iodide) dodecyl) carbazole (BMVC-12C), which can be used to distinguish cancer and normal cells due to its 6-10 times enhanced fluorescence in cancer cells compare to normal cells. Confocal imaging revealed that BMVC-12C is mainly trapped in lysosomes of normal cells, but escapes from lysosomes and targets to mitochondria of cancer cells. In addition, we discovered that the selective cytotoxicity of BMVC-12C is highly correlated to mitochondria overlay percentage based on the statistic result of several cell lines. The higher the mitochondria overlay percentage, the higher the toxicity. The accumulation of BMVC-12C in mitochondria of cancer cells is capable of disrupting mitochondria functions. Thus, BMVC-12C has the potential not only to be used as a fluorescence tumor marker for lighting up cancer cells in clinical cancer diagnosis but also act as a mitocan for cancer treatment. To further verify the targets of BMVC-12C in mitochondria of cancer cells, we used cyclosporin A (CsA), a specific mitochondria inner membrane permeability transition pore inhibitor, and found both fluorescence and cytotoxicity of BMVC-12C were reduced by CsA preincubation. This result suggested that it is possible that BMVC-12C can pass through mitochondria inner membrane and targets mitochondria DNA. Moreover, the existence of secondary G-quadruplex (G4) structures within mtDNA genes were confirmed by Circular Dichroism (CD) and Nuclear Magnetic Resonance (NMR) Spectroscopy. Furthermore, these mitochondrial gene functions were suppressed by BMVC-12C confirmed by RT-PCR experiments. In conclusion, this work lead to a proposed model in which BMVC-12C can induce cell death and mitochondria dysfunctions by stabilizing secondary G4 structures within mitochondria DNAs.

並列關鍵字

BMVC-12C ； selective cytotoxicity ； fluorescence ； Mitochondria ； G4 structure

參考文獻

cancer diagnosis and photodynamic therapy. in IAMS, National Tsing

1. Warburg, O. (1956) On the origin of cancer cells. Science (New York, N.Y 123, 309-314

2. Simonnet, H., Alazard, N., Pfeiffer, K., Gallou, C., Beroud, C., Demont, J., Bouvier, R., Schagger, H., and Godinot, C. (2002) Low mitochondrial respiratory chain content correlates with tumor aggressiveness in renal cell carcinoma. Carcinogenesis 23, 759-768

3. Arora, K. K., Parry, D. M., and Pedersen, P. L. (1992) Hexokinase receptors: preferential enzyme binding in normal cells to nonmitochondrial sites and in transformed cells to mitochondrial sites. Journal of bioenergetics and biomembranes 24, 47-53

4. Steinberg, R. A. (1984) Cyclic AMP-dependent phosphorylation of the precursor to beta subunit of mitochondrial F1-ATPase: a physiological mistake? The Journal of cell biology 98, 2174-2178

國際替代計量

BMVC-12C分子針對癌症細胞的選擇性毒殺之機制探討

全文下載

主題瀏覽