草本多酚結合非侵入式物理性刺激對於胰臟癌細胞影響之研究

Abstract

胰臟癌是一種高度惡性的腫瘤，存活率更是所有癌症中最低。除了手術，現行的醫學治療方式往往涉及化學療法和放射線治療，這些傳統療法還可能引發不必要的副作用。因此，近來人們積極開發及應用新型治療方法來取代現行可能會產生許多副作用的傳統療法。在本論文中，我們採取草本多酚結合物理性刺激的方法作為一種溫和的治療方式，並研究這樣的結合治療對人胰臟癌細胞株PANC-1的效應及其協同作用。 在第一個結合治療的主題中，我們提出了一種基於非接觸式電刺激的物理方法，應用此低強度脈衝電場 (low-intensity pulsed electric field; LIPEF)與施加薑黃素(curcumin) 的組合作用於PANC-1細胞上。實驗結果顯示，非接觸式LIPEF在施加特定強度後可顯著地增強低劑量薑黃素的細胞毒殺作用所引起之外源性和內源性細胞凋亡途徑。值得注意的是，相同的結合療法對於非癌性的HEK293細胞幾乎沒有產生毒殺作用。此外，藉由LIPEF引起的電誘發內吞作用(electro-endocytosis)增加PANC-1細胞對於薑黃素吸收的結果，不僅提供了薑黃素抗癌活性增強的直接性證據，也揭示了LIPEF與薑黃素的協同作用機制。 針對第二個研究主題，我們採用溫度循環熱療 (thermal-cycling hyperthermia; TC-HT) 此一新方法結合兒茶素 (EGCG) 或氯原酸 (CGA) 並探討其組合對於PANC-1細胞之毒殺影響。結果顯示，TC-HT結合CGA以及TC-HT 結合EGCG的這兩種組合都能顯著地發揮對PANC-1細胞的抗癌作用，然而在單一藥劑或TC-HT單獨處理後並未能引起此種效果；其中協同活性是經由G2 / M期的細胞週期停滯和氧化壓力介入導致粒腺體調控凋亡路徑被激活所致。此研究顯示TC-HT可增加PANC-1細胞對抗癌藥劑的敏感性，從而產生CGA或EGCG的熱增強之細胞毒殺作用。 最後，本論文的目標是要探討應用兩種物理刺激TC-HT和LIPEF搭配CGA對於PANC-1細胞處理之影響。研究發現這種三聯組合處理顯著地抑制了胰臟癌細胞的生長，其中在24小時後細胞存活率僅剩餘約20％。實驗結果顯示此三聯組合處理能使PANC-1細胞週期停滯於G2 / M期及引起細胞凋亡，我們發現這是與p53的表現上升及其下游蛋白p21和Bax的增加有關。研究進一步發現細胞凋亡與粒線體路徑有關，包括Bcl-2蛋白表現減少，粒線體膜電位降低，啟動caspase-9和PARP的活化。此外，我們發現透過三聯組合處理會造成細胞內氧化壓力的增加;而抗氧化劑N-acetyl-cysteine (NAC) 能顯著抑制三聯組合處理所誘導出之細胞毒性，並同時抑制p53和p21蛋白的表現上升以及Bax / Bcl-2比例的增加，這些結果顯示出TC-HT和LIPEF結合CGA在PANC-1細胞產生的細胞毒性是因氧化壓力誘發p53訊息路徑的活化所導致。 總結本論文中的三個研究中，非接觸性LIPEF和TC-HT結合草本帶來的協同效益均顯示了作為癌症治療中物理方法的潛力和可行性，再加上草本對於病患而言具有較好的耐受性，相信將草本與LIPEF和/或TC-HT聯合用藥是一種具有前景的新穎胰臟癌治療策略。

Pancreatic cancer is one of highly lethal cancers, with the lowest survival rate among all human cancers. Despite the current availability of many modulations against cancer, these treatments generally involve chemotherapy and radiotherapy, which are associated with undesired side effects. Recently, many researchers have focused on the development and applications of alternative cancer treatments. In this dissertation, the combination of herbal polyphenol with physical stimuli as a mild treatment modality was adopted to study their cooperative anticancer effects on human pancreatic cancer PANC-1 cells. Firstly, we present a non-invasive treatment comprising of exposure of PANC-1 cells to curcumin coupled with the low-intensity pulsed electric field (LIPEF). The results showed that non-contact LIPEF at certain intensity could enhance the anticancer effect of low-dose curcumin on PANC-1 cells through both extrinsic and intrinsic caspase pathways. This combination approach, by contrast, was found to be less toxic toward non-cancerous human HEK293 cells. Furthermore, the increase of curcumin uptake in PANC-1 cells via electro-endocytosis revealed the mechanism of combination action for electrical enhancement of curcumin’s anticancer activities. Secondly, a novel methodology, termed thermal-cycling hyperthermia (TC-HT), to enhance the cytotoxicity of epigallocatechin gallate (EGCG) and chlorogenic acid (CGA) in PANC-1 cells was illustrated. Our results demonstrated that combination of TC-HT plus CGA and TC-HT plus EGCG significantly exerted the anticancer activites against PANC-1 cells, while any of the single treatment did not induce such effects. The synergistic activity was attributed to the cell cycle arrest at the G2/M phase and the induction of reactive oxygen species (ROS)-dependent mitochondria-mediated apoptosis. The finding of our study indicates that the TC-HT may sensitize PANC-1 cells to anticancer agents, thereby yielding thermal enhancement of cytotoxicity of CGA or EGCG. Finally, the TC-HT and LIPEF were both employed to combine with CGA in the treatment of PANC-1 cells. It was found that this triple combination can impede the proliferation of human pancreatic cancer PANC-1 cells, with only about 20% viable cells left after 24 h, while being non-toxic to normal human pancreatic duct H6c7 cells. The synergistic effect of this triple treatment could be achieved through up-regulation of p53 and coupled with increased expressions of downstream proteins p21 and Bax, which resulted in G2/M phase arrest and cell death. Further mechanism investigations revealed that the cytotoxic activity could be related to mitochondrial apoptosis, characterized by the reduced level of Bcl-2, mitochondrial dysfunction, and sequential activation of caspase-9 and PARP. In addition, we found that intracellular ROS production was increased by triple treatment. Notably, the triple treatment-induced cytotoxic effects and the elevated expression of p53 and p21 proteins, as well as the increased Bax/Bcl-2 ratio, all could be alleviated by the ROS scavenger, N-acetyl-cysteine (NAC). To sum up, the studies have unambiguously shown the potential and feasibility of the non-contact LIPEF and TC-HT as physical remedies combined with herbs in pancreatic cancer treatment. It is known that the herbal agents often possess much gentle and tolerable properties, it can be suggested the combination use of herb medicine with LIPEF and/or TC-HT could be the novel and promising strategy for pancreatic cancer treatment.