探討乳漿蛋白濃縮物於乳癌治療之分子機制

乳癌為我國女性癌症發生率第一名，死亡率第四名，且乳癌的高復發率、高轉移性以及抗藥性依舊是一個棘手的問題。目前，乳癌病人的五年存活率日益增高; 然而，由於台灣女性乳癌發病有年輕化的趨勢，面對乳癌治療的新挑戰將是如何提高病後福祉以及改善長期預後。先前研究指出：乳癌細胞的氧化還原狀態與治療的感受性與以及抗藥性有關。其中，活性氧分子（Reactive oxygen species; ROS）參與乳癌的起始和進展；穀胱甘肽（Glutathione; GSH）則在細胞抗氧化系統扮演重要的角色。而乳漿蛋白濃縮物(whey protein concentrate; WPC)是一種富含合成GSH前驅原料的抗氧化物。本研究分為以下幾個部分：第一部份：探討乳漿蛋白濃縮物對2, 2-二羟甲基丁酸（7, 12-dimethylbenz(a)anthracene; DMBA）所誘導的大鼠乳腺腫瘤的影響。第二部份：探討合併乳漿蛋白濃縮物與mTOR抑制劑於三陰性乳癌細胞的治療效用與機制。第三部份：探討合併乳漿蛋白濃縮物與mTOR抑制劑於賀癌平(trastuzumab)抗藥性乳癌細胞的治療效用與機制。在第一部分研究中，我們評估乳漿蛋白濃縮物對DMBA誘導大鼠乳腺腫瘤的影響。DMBA會促使細胞轉型 (transformation)，可用來模擬乳腺腫瘤發生的起始和進程。實驗結果顯示：WPC (0.334 g / kg) 提高大鼠肝臟組織92% GSH含量 (與控制組相比；P < 0.05)，並伴隨著Bax / Bcl-2與cleaved caspase-3/procaspase-3的降低 (抑制細胞凋亡)。在DMBA誘導乳腺腫瘤的大鼠中，WPC降低大鼠的腫瘤組織47% GSH 含量(與DMBA組相比；P < 0.05)，且提高Bax / Bcl-2與cleaved caspase-3/procaspase-3 (促進細胞凋亡)。因此，本部分的研究結果揭示：WPC可以選擇性地提高正常組織的GSH含量且消耗腫瘤組織中的GSH含量並伴隨著細胞凋亡分子狀態的改變。在第二部分研究中，我們進一步探討合併乳漿蛋白濃縮物與mTOR抑制劑於三陰性乳癌細胞的治療效用與機制。研究結果發現：三陰性乳癌細胞株MDA-MB-231對雷帕黴素 (rapamycin) 不敏感 (insensitive)，並且具有比非致瘤的乳腺上皮细胞株MCF-10A更高含量的GSH與ROS。然而，合併WPC與rapamycin，可降低MDA-MB-231細胞對rapamycin的最大半數抑制濃度 (IC50)；且此組合可降低細胞GSH含量並減少Nrf2進入細胞核。此外，WPC可以激活GSK3?? / mTOR信號傳導。同時，研究亦發現：GSK3?珧捋PWPC所介導的Nrf2的還原與mTOR的活化。總結來說，WPC透過改變細胞的氧化還原狀態並且激活GSK3?? / mTOR信號，提高MDA-MB-231細胞株對rapamycin的感受性。此結果提供乳腺癌治療的新型策略，以及可能影響三陰性乳癌mTOR抑制劑抗藥性的關鍵途徑。第三部分研究，我們建立trastuzumab抗藥性的乳癌細胞株，並且探討mTOR抑制劑以及合併乳漿蛋白濃縮物於trastuzumab抗藥性乳癌細胞發展的影響與機制。研究結果發現：trastuzumab抗藥性發展過程中，細胞PTEN表現量下降並且活化mTOR / S6RP。此外，抗藥性細胞的GSH與ROS含量亦增加。Rapamycin在抗藥性細胞發展初期能有效抑制癌細胞的侵襲力 (invasiveness) 與上皮-間質細胞轉化能力 (epithelial-mesenchymal transition; EMT); 然而，rapamycin無法抑制癌幹細胞的形成。透過合併rapamycin與WPC使用，可以激活GSK3?牷A抑制Snail表現量，進而抑制EMT，並且阻斷癌細胞自我更新能力 (self-renew)。這可能與抑制trastuzumab抗藥性發展有關。本研究結果闡明了合併抗氧化劑與mTOR抑制劑對乳腺癌治療的療效與機制。透過抗氧化劑補充可改善正常細胞的自我保護能力，並增加癌細胞對治療藥物的感受性以及抑制抗藥性的發展。合併抗氧化劑與mTOR抑制劑的組合方式可應用於乳腺癌患者的不同受體狀態，進一步開發出乳腺癌臨床治療的新策略。

關鍵字

乳癌；乳漿蛋白濃縮物；雷帕黴素受體抑制劑；賀癌平抗藥性

並列摘要

Breast cancer is the leading causes and the fourth mortality rate of cancer among woman in Taiwan. The high recurrence rate, highly metastatic ability, and resistant to drug of breast cancer are still a sticky problem. Currently, the five-year survival rate of breast cancer patients is on the rise; however, with the trend of younger women suffering from breast cancer in Taiwan, a new challenge in the treatment of breast cancer will be to improve the patients’ post-illness well-being and long-term prognosis. Studies have indicated that the cellular redox status are related with the susceptibility and resistance to treatment. Among them, reactive oxygen species (ROS) is involved in the processes associated with initiation and development of breast cancer, and glutathione (GSH) plays an important role in antioxidant defense. Whey protein concentrate (WPC), a precursor of GSH, which is used as an antioxidant. This study was conducted the following parts: Part I. Explore the effects of WPC in rats with mammary tumors induced by 7, 12-dimethylbenz(a)anthracene (DMBA). Part II. Explore the effects of the combination of WPC with mTOR inhibitors on triple-negative breast cancer cell line. Part III. Explore the effects of the combination of WPC with mTOR inhibitors on trastuzumab-resistant breast cancer cell. In Part I study, we investigated the effects of WPC on rats with mammary tumors induced by DMBA. DMBA treatment results in cellular transform that mimic the initiation and promotion of carcinogenesis of breast tissue. The results indicate that WPC (0.334 g/kg) supplementation significantly increased the liver GSH levels by 92% (versus Control group; p < 0.05), and were accompanied by low Bax/Bcl-2 ratio and cleaved caspase-3/procaspase-3 ratio (inhibit apoptosis) in DMBA-treated rats. Furthermore, tumor GSH levels were decreased by 47% in WPC-supplemented rats (versus DMBA group; p < 0.05), which resulted in increased Bax/Bcl-2 ratio and cleaved caspase-3/procaspase-3 ratio (promote apoptosis). The results suggest that supplementation with WPC could selectively increase normal tissues GSH levels but deplete tumor GSH levels, while modulate apoptosis-related proteins. In Part II study, we further investigated the anticancer mechanism of the combination of WPC and mTOR inhibitor therapy in triple-negative breast cancer (TNBC). We found that MDA-MB-231 cells are insensitive to rapamycin and exhibit higher GSH and ROS levels than non-tumorigenic MCF-10A cells. However, for MDA-MB-231 cells, the half maximal inhibitory concentration (IC50) of rapamycin was lower when this drug was administered in combination with WPC than when used alone. Furthermore, combining WPC with rapamycin depleted GSH levels and reduced nuclear factor erythroid 2 (NFE2)-related factor 2 (Nrf2) nuclear accumulation. In addition, WPC activated GSK3??/mTOR signaling, and GSK3?? appeared to be involved in the WPC-mediated Nrf2 reduction and mTOR activation. To sum up, WPC induce rapamycin sensitivity in MDA-MB-231 cells by altering cells’ redox state and activating GSK3??/mTOR signaling. These results not only suggest a novel therapeutic approach for breast cancer treatment, but also provide insight into the critical pathways affecting the resistance to mTOR inhibition observed in a subgroup of TNBC patients. In Part III study, we established the trastuzumab-resistant breast cancer cell lines, and investigated the effect mechanism of mTOR inhibitor and the combination of WPC and mTOR inhibitor on the Herceptin resistance development of breast cancer cells. The results showed that during the development of resistance to trastuzumab, a decreased expression of PTEN and an activated mTOR/S6RP were observed. In addition, GSH and ROS levels of trastuzumab-resistant cells were increased. Rapamycin could effectively inhibit cancer cell invasiveness and epithelial-mesenchymal transition (EMT) in the early development of drug-resistant cells; however, rapamycin did not inhibit cells stemness. Of note, the combination of rapamycin and WPC activated GSK3β and inhibited the expression of Snail, thereby inhibiting EMT and blocking the self-renewal ability of cancer cells. These results might be related to the abolishment of the resistance development. The results clarify the effects of the combination antioxidant with mTOR inhibitor on breast cancer treatment. By using antioxidant supplements improve the self-protection of normal cells; confer cancer cells susceptibility to treatment and inhibit the development of drug resistance. The combination antioxidant with mTOR inhibitor can be applied to different receptor status of breast cancer patients, providing further development of new strategies of clinical treatment for breast cancer.

並列關鍵字

breast cancer ； whey protein concentrate ； mTOR inhibitor ； trastuzumab resistance

參考文獻

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探討乳漿蛋白濃縮物於乳癌治療之分子機制

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