循環加熱增強異硫氰酸烯丙酯AITC對人類胰臟癌細胞的抗癌作用

Abstract

胰臟癌為目前臨床上預後最差之惡性腫瘤之一，其高度侵襲性與對傳統化學治療及放射治療之低反應性，使整體存活率長期維持在較低水準。因此，發展具有效性且副作用較低之輔助治療策略，仍為胰臟癌研究的重要課題。異硫氰酸烯丙酯 (allyl isothiocyanate, AITC) 為十字花科植物中常見之具有生物活性之天然化合物，過去研究顯示其具有抑制癌細胞增殖、誘發細胞凋亡及氧化壓力之潛力。然而，AITC於實際應用上仍受限於溶解度不佳、穩定性不足等問題，限制其進一步發展為抗癌治療的手段。 近年來，熱療被視為一種有潛力的癌症輔助治療方式，其中本實驗室提出的循環加熱 (thermal cycling-hyperthermia, TC-HT) 相較於傳統持續高溫熱療，具有對正常細胞傷害較低等優點，並被認為可能透過調控細胞內壓力反應而增強藥物治療效果。然而，TC-HT是否能與AITC產生協同作用，以及其可能涉及之分子機制，仍有待進一步探討。因此，本研究旨在評估TC-HT與AITC聯合處理對胰臟癌細胞之抗癌效果，並分析其對細胞活性、氧化壓力、細胞凋亡及相關蛋白表現之影響。 本研究以人類胰臟癌細胞株PANC-1作為體外細胞模型，分別施予AITC、TC-HT以及兩者聯合處理，以比較不同處理條件下對癌細胞之影響。細胞存活率透過MTT試驗進行評估，細胞凋亡與細胞內活性氧化物 (reactive oxygen species, ROS) 水平之變化則以流式細胞術進行分析，並進一步利用西方墨點法檢測與ROS調節相關蛋白磷酸化ERK與磷酸化CREB，以DNA保護相關蛋白MTH1之表現變化。 研究結果顯示，AITC或TC-HT單獨處理僅對PANC-1細胞存活率產生有限抑制效果，然而兩者合併處理可顯著降低細胞存活率，並明顯提升細胞凋亡比例，顯示AITC與TC-HT聯合處理可以增強抑制癌細胞活性之效果。此外，聯合處理組的細胞內ROS水平顯著上升，且其增加幅度高於任何單一處理組，顯示TC-HT可放大AITC所誘發之氧化壓力反應。在蛋白水平的分析方面，AITC搭配TC-HT處理可降低磷酸化ERK與磷酸化CREB的表現量，並伴隨MTH1表現量下降，此結果顯示AITC搭配TC-HT的處理方式可能透過干擾調控氧化壓力之相關訊號路徑與DNA的保護機制，使癌細胞對氧化性損傷更為敏感。 綜合上述結果，本研究顯示TC-HT可有效增強低劑量AITC對PANC-1細胞之抗癌效應，並指出此聯合處理對於氧化壓力及DNA穩定性調控的干擾可能參與其中。本研究成果提供AITC結合TC-HT之抗癌效果的實驗依據，顯示藉由TC-HT的輔助有望克服AITC於臨床上使用的困難，在胰臟癌的治療上具有潛在應用價值，亦可作為後續相關研究之參考基礎。

Pancreatic cancer is one of the most aggressive malignancies and is associated with poor prognosis. Due to its high invasiveness and low responsiveness to conventional chemotherapy and radiotherapy, overall survival rates remain persistently low. Therefore, the development of effective adjuvant therapies with less adverse effects remains an important issue in pancreatic cancer research. Allyl isothiocyanate (AITC), a naturally occurring bioactive compound commonly found in cruciferous vegetables, has been reported to exhibit anticancer potential, including the inhibition of cancer cell proliferation and the induction of apoptosis and oxidative stress. However, the application of AITC is limited by its poor solubility and insufficient stability, which restrict its further development as an anticancer therapeutic agent. In recent years, hyperthermia has been considered a promising adjuvant cancer treatment strategy. Thermal cycling–hyperthermia (TC-HT), proposed by our laboratory, has advantages over conventional hyperthermia treatment, including reduced damage to normal cells, and is thought to enhance therapeutic efficacy by modulating cellular stress responses. However, whether TC-HT can provide synergistic effects with AITC and the underlying molecular mechanisms remain unclear. Therefore, this study aimed to evaluate the anticancer effects of combined TC-HT and AITC treatment on pancreatic cancer cells and to analyze its effects on cell viability, oxidative stress, apoptosis, and the expression of related proteins. In this study, we used the human pancreatic cancer cell line PANC-1 as an in vitro model. Cells were treated with AITC, TC-HT, or a combination of both treatments to evaluate their entire cancer effects. Cell viability was assessed using the MTT assay, while apoptosis and intracellular reactive oxygen species (ROS) levels were analyzed by flow cytometry. In addition, Western blot analysis was performed to examine the expression levels of ROS-regulating proteins phosphorylated ERK and phosphorylated CREB, and the DNA protective protein MTH1. The results demonstrated that either AITC or TC-HT exerted only limited inhibitory effects on PANC-1 cell viability. In contrast, the combined treatment significantly reduced cell viability and markedly increased apoptotic cell populations, indicating a synergistic effect between AITC and TC-HT. Moreover, ROS levels were significantly elevated in the combined treatment group, with a greater increase than that observed in either treatment alone, suggesting that TC-HT amplified AITC-induced oxidative stress. At the protein level, the combined treatment reduced the expression of phosphorylated ERK and phosphorylated CREB, accompanied by a decrease in MTH1 expression, implying increased susceptibility of cancer cells to oxidative damage. Collectively, these findings indicate that TC-HT effectively enhances the anticancer effects of low-dose AITC in PANC-1 cells and may help overcome the limitations associated with AITC application.