HSP90抑制劑17-DMAG合併放射線治療肝癌之評估

Evaluating the combination of HSP90 inhibitor, 17-DMAG, and radiation for liver cancer treatment

潘俊仁(Jun-Jen Pan)

指導教授 ： 劉俊仁

臺北醫學大學/醫學科技學院/醫學檢驗暨生物技術學系/碩士(2011年)

https://doi.org/10.6831/TMU.2011.00048

摘要

肝癌是台灣常見且預後差的惡性腫瘤之一，現行的治療方式有外科手術、局部脫落、肝動脈化學栓塞、標靶藥物、化學治療及放射線治療等。即便當前的治療方式眾多，但肝癌病人的五年存活率仍僅有3-5%。因此，利用合併的治療方式以改善單一療法的缺點，成為肝癌治療的一個希望。游離輻射是臨床上治療許多癌症的方式之一，透過放射線的攻擊使雙股DNA斷裂，誘使細胞死亡。而癌細胞亦可以透過活化DNA修復蛋白，對放射線治療產生抗性，使癌細胞難以清除。過去的研究指出，熱休克蛋白HSP90調節許多DNA修復蛋白的折疊，為了瞭解HSP90否有參與癌細胞對放射線治療所產生的抗性，故於放射線給予的同時合併HSP90的抑制劑17-DMAG (17-Dimethyaminoethylamino demethoxygeldanamycin)，觀察以下實驗。我們先以放射線合併HSP90的抑制劑處理肝癌細胞株HepJ5，觀察其細胞聚落的形成，同時合併具有較好的抑制效果。透過細胞週期的觀察，單獨處理17-DMAG或放射線後，皆可使細胞停滯在G2/M phase，停滯現象分別發生於處理後24至48小時及6至24小時。故設計了三種合併方式，其中於放射線同時合併HSP 90抑制劑有最佳的細胞生長抑制率。在西方點墨法以相同的結合方式觀察DNA修復蛋白的表現量，放射線處理後，Chk1、p-Cdc2、p-Cdc25c、RAD51、AKT及HIF1α等蛋白表現量增加;合併HSP 90抑制劑後，均受到抑制。更進一步於p-Histone H3染色的觀察，證實17-DMAG確實干擾細胞週期於G2/M phase的停滯調節，而影響了DNA修復機制。因此，在本篇的研究認為，HSP 90抑制劑可以作為一放射線治療的增敏劑。

關鍵字

熱休克蛋白,放射線治療,肝癌

並列摘要

Hepatocellular carcinoma (HCC) is one of the most common cancers with poor prognosis in Taiwan. Current therapeutical strategies include surgery, local ablation , transarterial chemoembolization(TACE), target therapy, chemotherapy and radiotherapy. Even though progress has been achieved for HCC treatment, the overall five-year survival rate has remained steady at 3-5 %. So combinative therapy which can improve problems in single agent treatment becomes a light for HCC.
Ionizing radiation(IR) is one of the therapeutic strategies to cancers. Cell death induced by ionizing radiation is known to associate with the DNA double strand breaks (DSBs). However, radiation stress will also induce protection me-chanisms to lead radio-resistance. Previous studies showed that heat shock protein 90 (HSP90) could regulate many oncoproteins’ folding which might mediate DNA repair. Therefore synergistic effect of HSP90 inhibitor, 17-DMAG (17-Dimethy- aminoethylamino demethoxygeldanamycin), on radiotherapy was evaluated.
Through the pretest, concurrent IR and 17-DMAG in colony formation dis-played the benefit of combinative treatment. And cell cycle of Hep-J5 would arrest at G2/M phase after 24-48 and 6-24 hours of 17-DMAG and radiation treatment respectively. Therefore, three combination schedules (A) concurrent IR and 17-DMAG treatment for 48 hours ; (B) after 6 hours of IR combine 17-DMAG for 48 hours ; and (C) before IR pre-treat with 17-DMAG for 48hr, were designed to evaluate the optima combination effect. With low dose radia-tion, 17-DMAG could markedly enhance the growth inhibitory effect through schedule (A), when compared to the IR and drug alone. In vitro western blot analysis showed that, upon IR, checkpoint kinase 1 (Chk1), phospho-cyclin- de-pendent kinase 1 (p-Cdk1 /p-Cdc2), p-Cdc25c, RAD51, AKT/protein kinase B (PKB), and Hypoxia -inducible factor 1α (HIF1α) might increase as a protection mechanism for HCC cell. However, after combine with 17-DMAG these proteins drop to the lower level. Further analysis by phospho-Histone H3 staining proved that 17-DMAG interfered the G2/M checkpoint arrest mechanism which functions in DNA damage repair signaling activation. Herein, all the data collected demonstrated that HSP90 inhibitor might be a good chemoradiosensitizer agent.

並列關鍵字

HSP90 inhibitor, 17-DMAG, radiation,liver cancer

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