循環加熱增強松果菊苷對人類胰腺癌細胞的抗癌作用

Abstract

癌症長年以來一直是人類的死亡主因之一，其中胰臟癌更是因為其難以早期 發現的特性，導致確診時都已經是晚期。儘管能透過手術切除或是化療用藥抑制 腫瘤的發展，然而這些方法均會帶來相當嚴重的副作用。有鑒於近年來中藥在抗 癌領域受到重視，也陸續證實了各類中藥對抗癌症與腫瘤的效果，我們可以把中 藥作為一個具有相當潛力的化療藥物替代品。除了中藥之外，近年來使用物理刺 激對癌變細胞進行治療也逐漸被引入醫療領域之中，相對於藥物，物理刺激可以 更加有針對性的作用於特定身體部位，不會像化療藥物，一經使用，藥效和其副 作用就會蔓延全身。 在本研究中，我們團隊使用一特殊的循環加熱療法，在此療法中讓細胞在一 高溫階段與一低溫階段週期性的連續循環，其優點在於相比起傳統的熱療法，在 相同強度的熱刺激下，被治療區域其周圍的非癌細胞，較不易受到傷害。尤其， 目前已知有部分天然草本萃取物在熱療法作用下會產生協同效果，可進而提升藥 效的同時降低藥物濃度，讓藥物對非癌細胞的傷害降低，故透過此特殊的循環熱 療技術，我們希望能更進一步地減少治療過程中對非癌細胞造成的傷害。在我們 的研究中，我們選用生長於沙漠的特殊寄生植物，肉蓯蓉中的有效成分，松果菊 苷與循環加熱療法作用，通過 MTT 與光學顯微鏡的結果顯示，松果菊苷與循環加 熱同時作用會對胰臟癌 PANC-1 細胞產生協同作用之效果，並且使加熱後的藥效 幾近等同於未加熱的三倍藥效，效果顯著。通過流式細胞儀的檢測，在松果菊苷 與循環加熱一同作用的條件下，胰臟癌 PANC-1 細胞內氧化壓力與凋亡的訊號比 起單獨加入藥物或物理刺激時要更加明顯。此外，在西方墨點法的蛋白分析中， 藥物與物理刺激一同作用後，亦在 Bax/Bcl-2 的拮抗蛋白或是 PARP 蛋白的分析結 果中，顯示出與其他條件相比更強的凋亡訊號。由此推斷，松果菊苷與循環加熱 的條件下，胰臟癌 PANC-1 細胞內的氧化壓力增加，使得 Bax/Bcl-2 的蛋白拮抗中，Bax 蛋白表現增加，進而推動下游蛋白 PARP 被裁切，引起凋亡。此外，對於非癌 細胞 H6c7，我們發現松果菊苷或是循環加熱均不會對細胞存活率產生明顯影響， 然而傳統熱療法的作用下，卻會使 H6c7 非癌細胞也受到熱作用的大幅傷害。 本研究首度提出了松果菊苷與循環加熱療法對胰臟癌 PANC-1 細胞的協同抗 癌效果，藉由循環加熱法可以大幅降低松果菊苷所需劑量，同時也能改善非癌細 胞 H6c7 被過去傳統熱療法刺激而產生的傷害，因此本論文結果說明將草本萃取物 與循環加熱結合是很好的治療策略。未來期望能在不同的草本萃取物或癌細胞上 同樣達到顯著的抗癌效果，為癌症病患帶來更安全有效的治療方案。

Cancer has been one of the leading causes of death for many decades. Among all, pancreatic cancer is even more difficult to detect in its early stage, resulting in a very low cure rate. Although tumors can be removed by surgery or inhibited by chemotherapy, these methods may cause serious side effects. In view of the importance regarding the application of traditional Chinese medicines (CM) against cancers in recent years, and the gradual confirmation of the effectiveness of various types of CM in fighting cancer and tumors, CM is a promising alternative to chemotherapy drugs. In addition to CM, the use of physical stimulation to treat carcinoma cells has been gradually introduced into the medical field in recent years. Physical stimulation can be targeted to specific parts of the body, unlike chemotherapy drugs, which, once used, harm both carcinoma cells and normal cells. In this study, our team used a novel cyclic hyperthermia (HT) where cells were exposed to two phase cycling system, a high temperature phase applied for killing cancer cells, and a lower temperature phase used for securing the survival of normal cells. This novel HT treatment, which we named as thermal cycling HT (TC-HT), was found to cause less damage on the normal cells compared to the conventional HT with the same thermal dosage. On the other hand, there have been some natural botanical extracts which can perform synergistic effect with thermal stimulation, not only increasing the anti-cancer efficacy of these natural compounds but also lowering the required dosage for cancer cure, and thus decreasing the damage to normal cells. In our study, echinacoside (Ech), the active ingredients of a special parasitic plant (Cistanche deserticola) grown in the desert, was selected to be used in combination with TC-HT. The results of MTT and the observations using optical microscope showed that there were strong synergistic anti-cancer effects on pancreatic PANC-1 cancer cells when using the combined TC-HT and Ech treatment. We find that such TC-HT synergistic effect makes the anti-cancer effect of Ech compound nearly equal to three times of that of unheated one. The intracellular oxidative stress and apoptotic signal using combination treatment were found to be more pronounced than single Ech or TC-HT treatment. In addition, the western blot analysis showed that Bax/Bcl-2 ratio and cleaved PARP proteins under the combination treatment were enhanced significantly compared to both single treatments, indicating a more potent apoptotic signal tendency. Thus, it can be inferred that the combination treatment increased the oxidative stress in PANC-1 cancer cells, increasing the Bax protein expression, which consequently caused the downstream protein PARP to be trimmed, eventually causing cell apoptosis. Furthermore, for normal pancreatic H6c7 cells, the study showed that neither Ech nor TC-HT had any effect on cell viability, but the conventional HT caused significant damage on H6c7 normal pancreatic cells. This study presents the synergistic anti-cancer effects of Ech in combination with TC-HT for the first time. The results showed that TC-HT notably increased the anti-cancer effect of Ech on PANC-1 cells by its unique periodic heating process. The required dosage of Ech in combination with TC-HT to achieve the same inhibition effect on PANC-1 cells was reduced to one third of the original Ech dosage. Moreover, the combined treatment also can prevent H6c7 normal cell damage caused by traditional HT thermal stimulation. Result of this thesis points out that the combination of herbal extracts and TC-HT is a great strategy on anti-cancer treatment. In the future, it is expected that employing such unique combination treatment, the significant anti-cancer effect can also be achieved on many cancers applied with different herbal extracts, which brings safer and more effective therapeutic options for cancer patients.