| 摘要: | 大腸直腸癌在全世界癌症死亡率仍高居第二名，因此對於新療法的需求依然急迫。人類核糖核苷酸還原酶（RRM1/RRM2）可以把ribonucleoside diphosphate 還原成 2’-deoxyribonucleoside diphosphate 在維持核糖核苷酸恆定上扮演重大角色。RRM2 對於大腸直腸癌是一個不良預後因子，我們分析癌症基因資料庫顯示RRM2 在大腸直腸癌患者組織有過度表現，因此可做為大腸直腸癌之抗癌藥物標的。多靶藥理學意指一個藥物可以作用於多個標靶，研究顯示大多數治療藥物均具備多靶活性。因此我們整合了生物資訊分析，包括基因表現分析、connectivity MAP（CMAP）、LINCS （Library of Integrated Cellular Signatures）、STITCH （Search Tool for Interactions of Chemicals）及 WebGestalt （WEB-based GEne SeT AnaLysis Toolkit）發展化學基因體學策略系統性預測多靶藥理學，並發展老藥新用。我們利用 CMAP 找尋新的 RRM2 抑制劑，結果顯示細胞週期依賴性蛋白激酶抑制劑GW8510 是一個有潛力的 RRM2 抑制劑，西方墨點法指出 GW8510 藉由促進RRM2 蛋白質降來抑制其表現，此外，GW8510 促進細胞自噬而誘發癌細胞死亡。大腸癌細胞對於 GW8510 的敏感性與 RRM2 表現量及內生性自噬活性有關，這部份的研究顯示 GW8510 可作為針對 RRM2 的大腸癌治療藥物。我們也利用化學基因體學策略發現兩個核酸類似物azacytidine（AZA）與 decitabine（DAC）之差異化分子機轉。雖然 AZA 與 DAC 是結構與藥理作用相似的 DNA 去甲基化藥物，但兩者在細胞內的代謝途徑卻不同。其中 AZA 可鑲嵌到 RNA，DAC 最終鑲嵌到 DNA，因此我們預期兩者之抗癌分子機轉應存在差異性，的確我們也觀察到兩者對人類大腸直腸癌細胞的不同毒性，其中 AZA 的毒殺性明顯高於DAC。藉由多靶藥理學的分析，我們發現 AZA 會暫時阻擋蛋白質合成並誘使急性細胞凋亡反應，而此反應可以被同時產生的自噬反應拮抗，反之，DAC 會藉由 p53 蛋白增加來使細胞週期中止在 G2/M 期。整體而言，我們的研究支持應用生物資訊策略預測多靶藥理及發展老藥新用是可行的。
Colorectal cancer (CRC) is the second leading cause of cancer-related death in the world. It is still urgent to develop novel therapeutics. Human ribonucleotide reductase (RRM1/RRM2) plays an essential role in converting ribonucleoside diphosphate to 2’-deoxyribonucleoside diphosphate to maintain the homeostasis of nucleotide pools. RRM2 is a prognostic biomarker and predicts poor survival of CRC. Bioinformatics analyses show that RRM2 was overexpressed in CRC and might be an attractive target for treating CRC. Polypharmacology (the ability of a drug to affect more than one molecular target) is considered a basic property of many therapeutic small molecules. Herein, we used a chemical genomics approach to systematically analyze polypharmacology by integrating several analytical tools, including the gene expression signature-based approach, connectivity MAP (CMAP), LINCS (Library of Integrated Cellular Signatures), STITCH (Search Tool for Interactions of Chemicals), and WebGestalt (WEB-based GEne SeT AnaLysis Toolkit). We attempt to search novel RRM2 inhibitors by using a gene expression signature-based approach, connectivity MAP (CMAP). The result predicted GW8510, a cyclin-dependent kinase (CDK) inhibitor, as a potential RRM2 inhibitor. Western blot analysis indicated that GW-8510 inhibited RRM2 expression through promoting its proteasomal degradation. In addition, GW-8510 induced autophagic cell death. In addition, the sensitivities of CRC cells to GW8510 were associated with the levels of RRM2 and endogenous autophagic flux. Our study indicates that GW8510 could be a potential anti-CRC agent through targeting RRM2. We also applied this approach to identify functional disparities between two cytidine nucleoside analogs: azacytidine (AZA) and decitabine (DAC). AZA and DAC are structurally and mechanistically similar DNA-hypomethylating agents. However, their metabolism and destinations in cells are distinct. Due to their differential incorporation into RNA or DNA, functional disparities between AZA and DAC are expected. Indeed, different cytotoxicities of AZA and DAC toward human colorectal cancer cell lines were observed, in which cells were more sensitive to AZA. Based on a polypharmacological analysis, we found that AZA transiently blocked protein synthesis and induced an acute apoptotic response that was antagonized by concurrently induced cytoprotective autophagy. In contrast, DAC caused cell cycle arrest at the G2/M phase associated with p53 induction. Taken together, our studies showed the potential of bioinformatics strategy in predicting polypharmacology and facilitating drug repositioning. |
