探討葛根對於乳腺腫瘤內皮細胞一氧化氮合成酶調控機轉之研究

乳癌一直以來就是女性國人好發率及致死率相當高的癌症之一，其危險因子像是高糖高脂的飲食習慣、初經過早及停經過晚、肥胖、生活壓力大和環境因子等等。雖然歐美等地對於乳癌與基因多型性已有相當廣泛的研究，但是本質上亞洲與歐美人種基因多型性的差異也是不容忽略的課題，因此研究台灣乳癌患者其體內基因多型性與乳癌之間相關性有其必要性。先前許多研究已指出氧化壓力在乳癌中扮演重要的角色，然而氧化壓力相關酵素其基因多型性的差異是否會造成個體對於疾病的感受性有所不同，值得我們進一步深入探討。因此在本研究中，我們將針對幾項問題進行研究，1. 內皮細胞一氧化氮合成酶(eNOS)其基因多型性與乳癌的相關性；2. 補充抗氧化物葛根是否能抑制大鼠乳癌生長及其體內氧化壓力的變化情形；3. 如果葛根能抑制乳癌生長的話，其過程是否透過調控血管新生相關因子的表現。我們分為三部分執行本研究，在第一部分實驗中，我們觀察乳癌病人其eNOS 的基因多型性是否與一般人有所差異？再者其相關性在停經前和停經後這兩個不同的族群中是否一致？接著在第二部分實驗中，我們探討抗氧化物葛根的補充是否能抑制乳腺腫瘤的生成，即透過同時施予化學致癌物及葛根於實驗大鼠身上，觀察其罹患乳腺腫瘤的進程，並採集血液及組織分析其生化指標、氧化壓力、和腫瘤新生相關因子的變化情形；而在第三部分的實驗中，將會分析第二部分大鼠體內eNOS 的蛋白表現，綜合以上結果，以體外細胞培養的模式，分析在MCF-7 乳癌細胞株中，葛根對於eNOS 及其相關血管新生因子的調控機轉。第一部分實驗結果顯示：不同於國外研究，eNOS 其中一個基因多型性，894G>T 與罹患乳癌的危險性有顯著性的相關性，且此危險性在停經前婦女與停經後婦女中呈現不同的結果，即帶有894G>T T allele 的停經前婦女其罹患乳癌的機率較高(P < 0.01)，但帶有894G>T T allele 的停經後婦女其罹患乳癌的機率則較低(P = 0.04)。而在第二部分實驗結果顯示：跟同樣給予化學致癌物(DMBA)的實驗大鼠相比，有給予抗氧化物葛根補充的組別其乳腺腫瘤的生成率、白血球數值、VEGF-C 和ICAM-1 含量顯著性的下降，TGF-β 及IgM 數值顯著性上升，體內氧化還原狀態也明顯的改變(P < 0.05)。而第三部分的實驗則發現高劑量的葛根會抑制MCF-7 細胞增生，伴隨著超氧陰離子及超氧陰離子歧化酶的增加，及抑制VEGF/VEGFR2、TNF-α和eNOSmRNA 的表現(P < 0.05)。由以上實驗結果我們認為，eNOS 其基因多型性與乳癌的相關性在台灣乳癌婦女族群中是存在的，且其相關性在停經前與停經後的族群中的表現呈現相反的結果。而葛根抑制DMBA 大鼠乳腺腫瘤生成和乳癌細胞株MCF-7 的增生可能包括在動物體內改善其免疫功能、增強抗氧化狀態及肝臟對於DMBA 致癌代謝物排除，和對於腫瘤生成相關因子的影響，及在乳癌細胞中抑制其血管新生相關因子mRNA的表現。期盼本研究的成果能提供臨床上，不管是個人化醫療或是透過葛根的補充，對於乳癌預防或治療上有用之參考依據。

關鍵字

乳癌；葛根；內皮細胞一氧化氮合成酶基因多型性

並列摘要

Breast cancer has a high incidence in Taiwanese women and worldwide. The risk factors for breast cancer include high caloric intake and a high-fat diet, early menophania and late menopause, obesity, high stress, and environmental pollution. In addition, studies have shown that there are significant genomic differences between patients with breast cancer in Taiwan and western countries, indicating the need for additional breast cancer studies in Taiwan. Previous studies observed increased oxidative stress in patients with breast cancer. Therefore, a few questions have been raised. First, Nitric oxide (NO), which is one of reactive oxygen species, has multiple independent roles in carcinogenesis; genetic polymorphisms in the endothelial nitric oxide synthase (eNOS) gene can modify its transcription and endogenous NO production. Is there any relationship between polymorphisms in the eNOS gene and breast cancer risk? If so, will the relationship be different with alternation of the menopausal status? Second, can the supplementation of Puerariae Radix (PR), an herb widely used in traditional Chinese medicine and as a food seasoning due to its potential anti-oxidative ability, inhibit breast tumor development? Accordingly, we have conducted three part experiments: In the first part, the effects of eNOS polymorphisms on breast cancer susceptibility were examined in terms of the menopausal status in Taiwanese women; in the second part, the effects of PR on the inhibition of breast tumors were investigated in this study using the 7,12-dimethylbenz[a]anthracene (DMBA)-treated rat model; In the final part, we investigated the eNOS protein expression in DMBA rats with/without PR supplementation, and the influence of PR on the biological effects and associated angiogenesis factors expression in the MCF-7 cells. In the first part experiment, there was a significantly higher breast cancer risk in premenopausal women carrying 894G>T T than in those with the 894G>T GG genotype (P < 0.01); however, postmenopausal women carrying 894G>T T had a lower risk of developing breast cancer (P < 0.04). In addition, based on a binary logistic regression analysis, interaction effects of these polymorphisms differed according to menopausal status. The relationship between eNOS polymorphisms and breast cancer hazard depended on menopause status, especially for the 894G>T polymorphism. In the second part experiment, we found that PR supplementation decreased tumor incidence and WBC count but increased the IgM levels, and significantly altered the redox status, elevated serum TGF-β levels, and reduced serum VEGF-C and ICAM-1 levels (P < 0.05). The mechanism by which PR decreased the incidence of DMBA-induced rat breast tumors might include ameliorating immunity, enhancing the antioxidant status, increasing the hepatic excretion of carcinogenic metabolites of DMBA, and influencing the expression of tumorigenesis-related factors. In the third part experiment, supplementation of PR at high concentration inhibited the MCF-7 cells proliferation, increased the levels of O2 •- and SOD activities, and also inhibited the mRNA expression of VEGF/VEGFR2, TNF-α, and eNOS (P < 0.05). Taken together, we thought there are associations between eNOS polymorphisms and breast cancer in Taiwanese breast cancer patients, and the contrary results were existed in pre- and post-menopausal population. The mechanism by which PR decreased the incidence of DMBA-induced rat breast tumors and proliferation of MCF-7 cells might include ameliorating immunity, enhancing the antioxidant status, increasing the hepatic excretion of carcinogenic metabolites of DMBA, influencing the expression of tumorigenesis-related factors, and inhibiting the mRNA expression of angiogenesis-related factors. We hope the results of our study can be applied in personalized medicine or serve as a reference for the prevention/treatment of breast cancer.

並列關鍵字

Breast cancer ； Puerariae Radix ； endothelial nitric oxide synthase ； polymorphism

參考文獻

Reference

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