急性鎘中毒活化之蛋白質降解系統與第二型拓樸異構酶調控DNA損壞之探討

Abstract

鎘，具有毒性及致癌性，是一種有硫化巨分子能力的過渡金屬。然而目前為止對於其毒性及致癌性的詳細機轉並無一致性的報導，因此我們試圖利用組織培養系統來探討鎘所造成的毒性之可能機制。有趣的是，我們發現在暴露於鎘之後，DNA拓樸異構酶的蛋白質表現量有減少的跡象，意味著鎘或許可以活化細胞中蛋白質降解系統(proteolytic systems)。此外，我們利用許多不同的蛋白酶抑制劑，來探討在鎘所誘發的蛋白質降解及細胞毒性中各種蛋白質降解酶可能扮演的角色。初步的研究結果指出鎘所誘發的蛋白質降解可能與26S proteasome、metalloproteases、serine proteases、cysteine proteases以及活性氧分子(reactive oxygen species, ROS)等有關。另外，我們亦發現鎘會造成DNA斷裂且隨著處理濃度的增加而DNA斷裂的情形會更為嚴重；同時亦證實第二型拓樸異構酶可能參與在其中，並且反映於鎘所造成之細胞毒性。綜合上述結果，我們證實並討論鎘誘發之蛋白質降解與第二型拓樸異構酶造成DNA斷裂以及活性氧分子對於鎘所引發的細胞毒性之可能重要性。

Cadmium (Cd2+), a macromolecule-thiolating metal, exhibits cytotoxic and carcinogenic activities. However, no unified mechanism for Cd2+-mediated toxicity and/or carcinogenicity has been reported or proposed. Here, we explored the underlying molecular mechanism(s) of Cd2+-induced toxicity in a tissue culture system. Surprisingly, we have found that the protein levels of DNA topoisomerases were greatly reduced upon cellular exposure to Cd2+, suggesting that Cd2+ might activate cellular proteolytic systems. Moreover, the proteolytic systems involved in the cadmium-induced degradation and cytotoxicity were investigated with multiple protease inhibitors. Our results suggest the potential involvements of 26S proteasome, metalloproteases, serine and cysteine proteases as well as reactive oxygen species (ROS) in Cd2+-activated proteolysis. In addition, we also found that Cd2+ induces the formation of chromosomal DNA fragmentation in a dose-dependent manner. Interestingly, the formation of these Cd2+-induced DNA breakages can be partially antagonized by a lot of TOP2 catalytic inhibitors such as ICRF-193, aclarubicin, merbarone and novobiocin. It appears that TOP2 might play a role in Cd2+-induced DNA breakage during acute cadmium stress. Furthermore, we showed that TOP2-mediated DNA breakage contributes to the cytotoxic action of Cd2+ in addition to Cd2+-induced proteases and ROS. The importance of Cd2+-induced proteolytic systems, TOP2-mediated DNA breakage and ROS formation in its cytotoxicity was discussed.