多重抗藥性(Multidrug resistance, MDR)一直是乳癌治療上的一個主要的障礙。而造成多重抗藥性的機轉主要來自於依賴ATP的藥物外排泵(ATP-binding cassette (ABC) transporter proteins)活性的增加或是過度表現，造成藥物在細胞內的濃度下降。對於doxorubicin的抗藥性就是例子之一。Sulbactam是一種β-內醯胺酶(β-lactamase)的抑制劑，通常會和β-內醯胺合併使用來治療細菌感染。Sulbactam也可單獨用來治療鲍氏不動桿菌(Acinetobacter baumannii)造成的感染，其機轉是透過抑制鲍氏不動桿菌ABC transporter和轉錄相關蛋白的表現。這是第一篇報導sulbactam在哺乳類細胞上的效果的研究。我們以乳癌細胞為模型系統來測試sulbactam是否會影響癌細胞。我們以MTT來測試doxorubicin外加sulbactam對乳癌細胞存活的影響。以液相層析串聯式質譜儀來測定乳癌細胞在doxorubicin單獨作用和sulbactam合併doxorubicin作用過後有改變的蛋白。接著以即時聚合酶鏈鎖反應分析以doxorubicn外加或沒外加sulbactam處理過後，ABC transporter的mRNA表現量的差異。並以doxorubicin排出實驗測量doxrubicin的排出是否有受到sulbactam的影響。最後以電腦模擬分析sulbactam和ABC transporter的ATP接合位的互動。以上實驗結果顯示，sulbactam增加了doxorubicin對乳癌細胞的毒性。Doxorubicin和sulbactam合併作用會使ABC transporter、和轉錄及轉譯起始相關的蛋白表現量都減少。ABC transporter的mRNA表現量在doxorubicin及sulbactam的處理過後也下降了。Doxorubicin排出實驗顯示sulbactam處理過後的細胞，doxorubicin的排出會被抑制。電腦模擬的結果則顯示sulbactam會和ATP競爭結合到ABC transporter的ATP接合部位，進而抑制ABC transporter的功能。總而言之，Sulbactam和doxorubicin合併使用能增加doxorubicin對於乳癌細胞的毒殺性。這樣的結果是藉由抑制和轉錄和轉譯相關的蛋白和ABC transporters的表現，並抑制ABC transporters的功能使doxorubicin在細胞內的時間延長，此作用可能是藉由sulbactam和ATP競爭ABC transporter的ATP-docking sites。因此doxorubicin和sulbactam合併使用可以降低doxorubicin施用的濃度，進而避免doxorubicin的副作用。

Background: Multidrug resistance (MDR) is a major obstacle in breast cancer treatment. The predominant mechanism underlying MDR is an increase in the activity of adenosine triphosphate (ATP)-dependent drug efflux transporters. Sulbactam, a β-lactamase inhibitor, is generally combined with β-lactam antibiotics for treating bacterial infections. However, sulbactam alone can be used to treat Acinetobacter baumannii infections because it inhibits the expression of ATP-binding cassette (ABC) transporter proteins. This is the first study to report the effects of sulbactam on mammalian cells.
Methods: We used the breast cancer cell lines as a model system to determine whether sulbactam affects cancer cells. The cell viabilities in the present of doxorubicin with or without sulbactam were measured by MTT assay. Protein identities and the changes in protein expression levels in the cells after sulbactam and doxorubicin treatment were determined using LC-MS/MS. Real-time reverse transcription polymerase chain reaction was used to analyze the change in mRNA expression levels of ABC transporters after treatment of doxorubicin with or without sulbactam.. The efflux of doxorubicin was measures by the doxorubicin efflux assay. Computer simulation was used to analyze the interaction of sulbactam and the ATP-docking sites of ABC transporter proteins.
Results: MTT assay revealed that sulbactam enhanced the cytotoxicity of doxorubicin in breast cancer cells. The results of proteomics showed that ABC transporter proteins and proteins associated with the transcription and initiation of translation were reduced. The mRNA expression levels of ABC transporters were also decreased when treated with doxorubicin and sulbactam. The doxorubicin efflux assay showed that sulbactam treatment inhibited doxorubicin efflux. The computer simulation showed that sulbactam may compete with ATP for the ATP-docking sites of ABC transporter proteins, thus inhibiting their function.
Conclusions: The combination of sulbactam and doxorubicin can enhance the cytotoxicity of doxorubicin in the breast cancer cells by inhibiting the expressions of transcription and initiation of translation associated proteins and ABC transporter proteins, reducing the function of ABC transporters to retain doxorubicin in the cells, and competing with ATP for the ATP-docking sites of the ABC transporter proteins to enhance the cytotoxicity of doxorubicin in breast cancer cells. Therefore, co-treatment of doxorubicin and sulbactam may decrease the prescribed dose of doxorubicin to avoid the adverse effects of doxorubicin.

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