睪丸癌流行病學與PNU-74654經由抑制TNFR1/IKB Alpha/p65途徑促使睪丸癌細胞凋亡之研究

The Study of the Epidemiology of Testicular Cancer and PNU-74654 Suppressing TNFR1/IKB Alpha/p65 Signaling and Inducing Cell Death in Testicular Cancer Cells

陳文榮(Wen-Jung Chen)

指導教授 ： 李宗賢

中山醫學大學/醫學院/醫學研究所/博士(2022年)

https://doi.org/10.6834/csmu202200203

摘要

研究目的：睪丸癌是一種在全世界罕見的惡性腫瘤，但它卻是15至44歲男性間最常見的癌症。我們首先探究睪丸癌之死亡率與發生率比值 (mortality-to-incidence ratio, MIR) 與不同國家健康照護差異如健康照護支出及健康照護系統排名的關係。細胞內部Wnt/β-catenin訊息路徑在許多細胞的功能扮演重要的角色，且在腫瘤的生成及進展也有舉足輕重的影響。PNU-74654是一種小分子，它是β-catenin與轉錄因子4 (Transcription factor 4, TCF4) 間的結合抑制劑。研究後半部我們評估PNU-74654在兩種睪丸癌細胞株，NCCIT與NTERA2的治療角色。
研究方法及資料：我們先使用Spearman’s rank correlation coefficient (CC) 來分析睪丸癌之死亡率與發生率比值與不同國家間健康支出占國內生產毛額比值 (total expenditures on health/gross domestic product, e/GDP) 及世界衛生組織健康照護系統排名的關係。接下來我們使用細胞存活率分析 (MTT assay) 來偵測細胞毒性及細胞生長，並藉由流式細胞分析 (flow cytometry analysis) 來偵測細胞週期的數量及細胞凋亡的比例。另外我們使用蛋白質陣列 (protein array) 及西方墨點法 (Western blotting) 來研究與細胞凋亡相關之蛋白質的變化藉以了解PNU-74654可能的作用機制。
研究結果： 流行病學部分最終納入57個國家加以分析。整體來說，已開發國家及地區有相對較高的發生率，但也有相對較有利的死亡率與發生率比值。歐洲的發生率最高，而最高的死亡率與發生率比值則在非洲。擁有較佳健康支出占國內生產毛額比值及較佳世界衛生組織健康照護系統排名的國家也會擁有較佳的死亡率與發生率比值。PNU-74654會造成sub G1期細胞、赫斯特染色 (Hoechst stain) 陽性率及膜聯蛋白V/PI (Annexin V/PI) 陽性率的增加，亦即會減少睪丸癌細胞的活性及引發細胞凋亡。NCCIT及NTERA2兩種細胞株經PNU-74654處理過後，顯示TNFR1/IKB alpha/p65路徑會受到抑制及與細胞凋亡之執行階段有關。
結論與建議：睪丸癌之死亡率與發生率比值會因不同國家或地區間健康照護支出及健康照護系統排名不同而有所差異。另外我們的研究也證實了PNU-74654可以藉由與細胞凋亡之執行階段相關之機制及抑制TNFR1/IKB alpha/p65路徑而導致睪丸癌細胞的凋亡。因此，PNU-74654有潛力可以成為睪丸癌治療的另一個新方向。

關鍵字

睪丸癌 ； 死亡率與發生率比值 ； PNU-74654 ； TNF接受體 ； 細胞凋亡

並列摘要

Objective：Testicular cancer (TC) is a rare malignancy worldwide, nonetheless, it is the most common malignancy in males aged 15–44 years. Firstly, we study the association between mortality-to-incidence ratio (MIR) for testicular cancer and health care disparities, including differences in expenditures and health system rankings among different countries. The Wnt/β-catenin signaling pathway mediates numerous essential cellular functions and has potentially important effects on tumorigenesis and cancer progression. The search for drugs to inhibit this pathway has identified a small molecule, PNU-74654, as an inhibitor of the β-catenin/TCF4 (transcription factor 4) interaction. We subsequently evaluated the therapeutic role of PNU-74654 in two TC cell lines, NCCIT and NTERA2.
Methods and Materials：We used the Spearman’s rank correlation coefficient (CC) to analyze the correlation between testicular cancer MIRs and both total expenditures on health/gross domestic product (e/GDP) and the World Health Organization’s (WHO) health system rankings. The MTT assay was then used to detect cytotoxicity and cell growth. A flow cytometer was used to determine the cell cycle population and apoptosis percentage. Potential pathways were evaluated by protein arrays and Western blots.
Results：After screening the data for quality and missing information, 57 countries were chosen for analysis. Generally, developed countries and regions had relatively high rates of incidence, but with a favorable MIR. Among the continents, Europe had the highest incidence rates, whereas the highest MIRs were in Africa. Globally, favorable testicular cancer MIRs were observed in countries with both a high e/GDP and a good WHO ranking. PNU-74654 decreased cell viability and induced apoptosis of TC cells, with significant increases in the sub G1, Hoechst-stained, Annexin V-PI-positive rates. PNU-74654 treatment of both TC cell lines inhibited the TNFR1/IKB alpha/p65 pathway and the execution phase of apoptosis, as shown by protein arrays and Western blotting.
Conclusion and Suggestion：In conclusion, the MIR for testicular cancer varies in countries and regions based on both their total health expenditure and their health care system ranking. Our findings also demonstrate that PNU-74654 can induce apoptosis in TC cells through mechanisms involving the execution phase of apoptosis and inhibition of TNFR1/IKB alpha/p65 signaling. Therefore, small molecules such as PNU-74654 may be a potential new treatment strategy for TC.

並列關鍵字

testicular cancer ； mortality-to-incidence ratio ； PNU-74654 ； TNF receptor-1 ； apoptosis

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