循環加熱增強苦瓜素MMD-I對人類胰臟癌細胞的抗癌作用

Abstract

隨著醫療的進步，一些病症相繼被克服，而癌症成為了目前造成國人死亡最多的病症，癌細胞主要的治療手法為手術切除以及各種的化學治療、放射線治療，但若使用足夠有效的高劑量治療，會對人體造成各式的副作用。但天然草本萃取物大多數僅會對人體造成較小傷害，並且仍對癌細胞具有一定的抑制作用。除了藥物方面的研究，目前有許多團隊亦在進行熱療搭配化學或放射線治療的試驗，但傳統熱療方式易對癌症腫瘤四周的正常細胞造成不可逆的傷害，也因此本研究欲驗證具有週期性穿插降溫過程的循環加熱技術，是否可於減少正常細胞受損的同時，仍使患部的癌細胞能受到有效的熱刺激。本研究旨在探討苦瓜素MMD-I輔以循環加熱對於胰臟癌細胞的協同抑制效果，並進行傳統加熱與循環加熱對人類正常及胰臟癌細胞的影響比較。實驗中採用PANC-1與H6c7細胞株分別模擬人體內的胰臟癌細胞及正常胰管細胞，以MTT試驗測試在各種處理之下的PANC-1與H6c7細胞存活率，並針對MMD-I協同循環加熱以流式細胞術測定細胞凋亡比例與氧化壓力、西方墨點法定量細胞中Bax、Bcl-2、PARP、cleaved PARP等細胞凋亡相關的指標蛋白含量。實驗結果顯示，苦瓜素MMD-I與循環加熱均具有抗癌作用，且循環加熱與MMD-I協同抑制癌細胞的效果較傳統加熱為佳，並在顯微鏡影像中可明顯看到PANC-1細胞外觀於處理後有凋亡的表現。而進一步的以流式細胞術的螢光檢測細胞狀態，結果顯示PANC-1細胞株再經過藥物與循環加熱協同處理過後，其細胞內部氧化壓力與產生凋亡現象細胞的比例皆有明顯的上升。最後以西方墨點法來定量PANC-1細胞經處理過後，其內部各種凋亡相關的指標蛋白冷光訊號，透過Bax / Bcl-2以及cleaved PARP / PARP的蛋白訊號強度比例來推斷細胞凋亡傾向，結果顯示苦瓜素MMD-I協同循環加熱確實有促使PANC-1細胞進行凋亡反應，且較單獨藥物或加熱處理的組別有明顯的增強抑制癌細胞的效果。總結本研究的實驗結果，我們發現循環加熱技術並不會對正常胰臟H6c7細胞造成太大的傷害，且與苦瓜素MMD-I的協同抑制PANC-1胰臟癌細胞表現較傳統加熱為佳。而苦瓜素MMD-I輔以循環加熱的協同處理能透過增加細胞氧化壓力的方式促使PANC-1胰臟癌細胞凋亡，且其對於抑制人類胰臟癌細胞株的效果較單獨藥物或循環加熱處理的組別有顯著的提升。本研究所進行的此種藥物與循環加熱協同的癌細胞抑制效果，未來是有潛力應用於胰臟癌的治療。

To date, cancer has become one of the major diseases that causes the death of Taiwanese people. The current anticancer therapies now are the traditional surgical excision, chemotherapy, radiotherapy, and so on. However, if we tend to effectively medicate with high dosage medicine, the side effects are so intense that patients can barely endure. Nowadays, many research teams have gradually put an eye on the herbal medicines, which are commonly known to cause milder side effects to patients, but still can efficiently suppress many kinds of cancer cells. Besides the evolution of medicament, hyperthermia (HT) treatment is also gradually drawing the attention of many medical professions. However, traditional HT treatment will unavoidably cause the irreversible damage to normal tissues. Therefore, for fighting cancer in this thesis, we replace HT treatment with a new methodology - thermal cycling-hyperthermia treatment (TC-HT), which is periodically interspersing low-temperature process in the high-temperature traditional HT treatment, to investigate whether it is possible to decrease the damage on normal tissues while still being able to provide sufficient thermal dosage to kill cancer cells. This study is designed to research if the TC-HT combined with Momordicin I (MMD-I) can inhibit human pancreatic cancer cells, and compare the effects of HT and TC-HT on human normal cells and pancreatic cancer cells. In this thesis, we used PANC-1 and H6c7 cell lines as the human pancreatic cancer and human pancreatic duct epithelial cell models, respectively. MTT assay was adopted for the cell viability measurements of PANC-1 and H6c7 cells under various treatments. Then we focused on the synergy of MMD-I and TC-HT technique. Flow cytometry was used for measuring cell apoptosis rate and reactive oxygen species quantity. Western bolt analysis was taken for measuring the expression of apoptosis-related proteins such as Bax, Bcl-2, PARP, and cleaved PARP. Our experimental data showed that both MMD-I and TC-HT inhibited PANC-1 cells, and the effect of synergistic combination of the two was superior than that of MMD-I treated with conventional HT. On the other hand, the results of flow cytometry and western bolt analyses also showed that the combined treatment of MMD-I and TC-HT, comparing to the single treatment groups, was able to significantly increase the portion of apoptosis cells, reactive oxygen species quantity, and the expressions of apoptosis-related proteins. In summary, this study demonstrates that MMD-I combined with TC-HT exhibited better effect in synergistically inhibiting PANC-1 cells than with conventional HT treatment, while TC-HT method was found to cause much less damage on H6c7 cells which we adopted as the normal pancreatic cell model. And we further find that the combination treatment of MMD-I and TC-HT can increase intracellular level of ROS via heat-sensitizing effect, and further causing the cell apoptosis. The study suggests that MMD-I and TC-HT combination would be potentially used as an effective therapeutic method for combating human pancreatic cancer.