抑制胸苷酸激酶及DNA拓樸異構酶II促進癌細胞死亡

Creating Synthetic Lethality in Cancer Cells by Inhibiting Thymidylate Kinase and DNA Topoisomerase II

胡春美(Chun-Mei Hu)

指導教授 ： 張智芬

國立臺灣大學/醫學院/生物化學暨分子生物學研究所/博士(2010年)

https://doi.org/10.6342/NTU.2010.02634

摘要

胸苷三磷酸(dTTP)的供給是受嚴密的機制所調控，且對DNA合成及修補非常重要的過程。細胞內有兩條路徑可以提供胸苷三磷酸(dTTP)的合成，分別是新生成和回收路徑。無論是經由新生成或是回收路徑形成的胸苷酸(dTMP)都必須經由胸苷酸激酶(thymidylate kinase, TMPK)進一步的磷酸化形成胸苷二磷酸(dTDP)，再透過核苷二磷酸激酶(NDK)的磷酸化最後完成dTTP的合成。抑制胸苷三磷酸(dTTP）的新合成路徑，已經是許多癌症化學治療藥劑發展的重要策略。

因為胸苷酸激酶(TMPK)在胸苷三磷酸(dTTP)新生成和回收路徑上都扮演重要的角色，我測試是否抑制胸苷酸激酶(TMPK)的表現，能夠降低胸苷三磷酸 (dTTP)的含量並促使癌細胞敏感於基因毒化物的毒殺能力。我使用p53功能正常和缺失的HCT-116結腸癌細胞作為研究材料，並利用以慢病毒為載體的shRNA去抑制TMPK的表現。結果顯示：表現TMPK shRNA可有效降低結腸癌細胞中胸苷酸激酶(TMPK)與胸苷三磷酸(dTTP)的含量。結合TMPK shRNA與廣泛使用於癌症化學治療的試劑–小紅莓(doxorubicin)，則可顯著增加p53功能正常和缺失的HCT-116結腸癌細胞對於小紅莓(doxorubicin)的敏感度。相對而言，若只是降低胸苷三磷酸（dTTP）新合成路徑中重要酵素—胸苷酸合成酶(thymidylate synthase, TS)的表現量與結合小紅莓(doxorubicin)的治療，並無法增進p53功能缺失的結腸癌細胞走向死亡，因為回收路徑的胸腺嘧啶激酶(thymidine kinase1, TK1)會彌補新合成路徑中胸苷酸合成酶(TS) 表現量的降低，以維持細胞中胸苷三磷酸(dTTP)的含量，導致治療效果不佳。因此，專一的抑制胸苷酸激酶(TMPK)的功能並結合低劑量小紅莓(doxorubicin)的治療，是一種非常有用的策略，可使癌細胞更容易被化療藥物所毒殺且不受p53功能影響。

因為小片段干擾RNA的應用仍然受限於其遞送的效率，因此利用小分子去抑制胸苷酸激酶(TMPK)的活性應是較為可行的策略。但目前針對人類胸苷酸激酶(TMPK)並沒有專一性高的抑制劑，因此為了去尋找專一性高的人類胸苷酸激酶(TMPK)抑制劑。我建立一種新方法適用於高效率藥物篩選，透過此新方法，篩選到一個化合物，命名為H9805，它可以有效的降低人類TMPK的活性，其IC50為0.61 ± 0.02 μM。更進一步，用H9805化合物處理各種癌細胞，結果發現：H9805化合物可以有效降低各種癌細胞內胸苷三磷酸(dTTP)的含量。MTS分析和克隆形成分析都顯示:經過H9805化合物處理的細胞，增加低劑量小紅莓所誘導的細胞死亡。這些研究資料暗示:H9805化合物是一個有潛力的前驅化合物，可以應用在促進癌細胞對於化學治療之毒殺作用。

總結，本論文的工作，闡明了新生成和回收路徑供給胸苷三磷酸(dTTP)，對於DNA修復過程的重要性，以及提供一種治療癌症的新洞見，抑制胸苷酸激酶(TMPK)的活性可增加癌細胞對於化學治療的敏感度，而有效促使癌細胞死亡。

關鍵字

胸苷三磷酸, 胸苷酸激酶, 小紅莓, 合成致死

並列摘要

Intracellular supply of deoxythymidine triphosphate (dTTP) is a highly regulated process and is important for DNA replication and repair processes. Supply of dTTP in the cells is controlled by de novo and salvage pathways, by which dTMP is synthesized. Subsequent phosphorylation of dTMP by thymidylate kinase (TMPK) gives dTDP, which is then converted to dTTP by dNDP kinase for DNA synthesis. Targeting the de novo pathway of dTTP synthesis has been one dogma for chemotherapeutic drug development.

Given the emerged role of TMPK in de novo and salvage pathways, I tested whether TMPK knockdown is able to deplete dTTP level and sensitize cancer cells to genotoxic insults. By using p53(+/+) and p53(-/-) HCT-116 colon cancer cells and lentiviral-based shRNA to silence TMPK, the experimental data showed that TMPK knockdown was sufficient to decrease dTTP level. In combination with a widely used chemotherapeutic agent, doxorubicin, it was found that silencing of TMPK significantly increased doxorubicin sensitivity dramatically in p53-proficient, -null HCT-116 cells. In contrast, silencing of thymidylate synthase (TS) that blocks the de novo pathway was incapable of sensitizing p53-null HCT-116 cells to doxorubicin-induced apoptosis because of the compensation by the salvage pathway. Thus, specifically blocking TMPK function and combined with low-dose doxorubicin treatment will be a very useful strategy for chemosensitization in killing cancer cells regardless of the p53 status.

Since the siRNA application is still limited by its delivery efficiency, it would be more feasible to use small molecule to block TMPK function. Since a specific inhibitor of human TMPK is still lacking, I started to search for specific hTMPK inhibitor. A new assay suitable for high-throughput screening of TMPK inhibitors was established and allowed the identification of a potential compound, named H9805, which inhibits TMPK with IC50 0.61 ± 0.02 μM. Furthermore, treatment with H9805 decreased cellular dTTP level in various cancer cell lines. Both MTS assay and colonies formation analysis showed that H9805 treatment enhanced doxorubicin-induced cell death. These data collectively suggest that H9805 compound is a potential lead compound for the use of chemosensitization.

In summary, this thesis work illuminates the importance of dTTP supply from de novo and salvage pathway in DNA repair process and provides new insights into targeting TMPK for chemosensization.

並列關鍵字

dTTP, TMPK. doxorubicin, synthetic lethality

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