植物化合物於乳癌化學預防之研究

Abstract

HER2 是一種已知的致癌基因，屬於表皮生長因子接受器家族 (epidermal growth factor receptor family; EGF receptor family) 的一員。臨床的研究發現，在乳癌的病人中，約有 25％ 到 30％ 其癌細胞有 HER2 基因放大 (amplification) 或過度表現 (overexpression) 的現象，這些 HER2 過度表現的癌症患者往往伴隨著較差的癒後，對荷爾蒙治療或化學治療的效果也較差；此外，相較於無 HER2 過度表現患者，HER2 過度表現的患者在短時間內復發的機率相對較高，甚至病人存活時間也較短。目前已知 HER2 可以藉由多個訊息傳導途徑來提高癌細胞的生長、分裂與轉移能力。由這些觀察中可以推測 HER2 過度表現與癌症有明顯的關聯性，並且導致治療上的困難，所以 HER2 被認為是預防或治療乳癌的重要標靶之一。 首先，在本論文的第二章，類黃酮化合物中的 luteolin 可以抑制 HER2 的表現，進一步的研究顯示 luteolin 可以促進 HER2 蛋白質的降解，其分子機制可能與 luteolin 可減少 HER2 與 Hsp90 的交互作用有關。此外，在 HER2 不同表現量的癌細胞中，luteolin 對於 HER2 過度表現的癌細胞具有較佳的抑制生長效果。這可能與 luteolin 會造成 HER2 過度表現的癌細胞走向細胞凋亡有關。 再者，低濃度的 luteolin 會增加 p21 蛋白質的表現，而高濃度的 luteolin 則會降低 p21 蛋白質的表現。近年來研究發現 HER2 過度表現的細胞中，p21 的高表現與細胞的存活有密切的關係；因此，推測 p21 的增加可以保護 HER2 過度表現的癌細胞不走向細胞凋亡。在過度表現 HER2 的癌細胞中，已知一個包含 Akt/mTOR 的訊息傳導途徑對於抑制細胞凋亡有重要的地位；由此引發探討此訊息傳導途徑在 p21 的增加與細胞的存活中是否扮演扮演一個重要的角色。研究結果顯示低濃度的 luteolin 只能暫時抑制 Akt/mTOR 的信號傳遞，這也暗示著若結合此訊息傳導途徑抑制劑可能可以提升 luteolin 造成細胞凋亡的效果。 同時處理 mTOR 的抑制劑 rapamycin 可以抑制由低濃度 luteolin 所引發 p21 蛋白質的增加。此外，rapamycin 也可以提升 luteolin 抑制癌細胞生長的效果，增加 luteolin 所造成的細胞凋亡。進一步利用 p21 siRNA 也能得到類似的結果。在以 SKOV3.ip1 所進行的動物實驗中，對於 HER2 過度表現的癌細胞，luteolin 不但能抑制 HER2 的表現並且抑制其生長；此外，和細胞實驗的結果類似，低濃度 luteolin 會增加 p21 的表現，而 rapamycin 不但可以抑制 p21 的表現也能提升 luteolin 抑制癌細胞的生長的效果。 由這些結果可以推測在 HER2 過度表現的癌細胞中，藥物如果不能長時間抑制 Akt/mTOR 的信號傳遞，會影響其作用的效果，這可能與 p21 的增加有關。此外，這裡的結果也暗示結合 mTOR 的抑制劑可能可以提高藥物對於 HER2 過度表現細胞的抑制效果。 脂肪酸合成酶 (Fatty acid synthase, FAS) 可將乙醯輔酶A (acetyl-CoA) 和丙二醯輔酶A (malonyl-CoA) 轉換為為棕櫚酸 (palmitate)，是能量代謝過程中一個重要的酵素複合體，在肝臟和脂肪組織的脂質合成扮演著重要的角色。其表現量異常與肥胖、心血管疾病和癌症有密切關聯。

近年來的研究發現，在 HER2 過度表現的癌細胞中，FAS的表現有顯著提高的情形。抑制 FAS 的表現可以很明顯地減低 HER2 過度表現癌細胞的致癌能力，顯示 FAS 可作為預防或治療 HER2 過度表現癌細胞的重要標靶之一。在本論文的第三章，一種類似固醇類荷爾蒙化學結構式的皂素生物鹼 (diosgenin) 被發現可以抑制 HER2 過度表現癌細胞中 FAS 的表現。Diosgenin 不但對於 HER2 過度表現的癌細胞具有較佳的抑制生長效果，亦會造成 HER2 過度表現的癌細胞走向細胞凋亡。此外，HER2 過度表現的癌細胞中，diosgenin 除了可以抑制 Akt 和 mTOR 的磷酸化，還可以促進 JNK 的磷酸化。利用抑制劑進一步確認 diosgenin 可能藉由調控Akt, mTOR 以及 JNK 的磷酸化來抑制 HER2 過度表現癌細胞中 FAS 的表現。再者，diosgenin 可以專一性的針對 HER2 過度表現的癌細胞提升 paclitaxel 抑制細胞生長的能力。這些結果顯示 diosgenin 具有預防或是治療 HER2 過度表現的癌細胞的效果。 最近很多研究指出茶多酚具有改善高血脂症、糖尿病及肥胖的現象，然而其分子機制仍有待探討。在本實驗室初步的動物試驗中發現，餵以 SD rats 茶葉可以有效降低其血清中膽固醇及三酸甘油酯含量。在綠茶、紅茶、烏龍茶及普洱茶中，以普洱茶的效果最好。有趣的是，飲食攝取量並沒有顯著的改變。考量到 FAS 是能量平衡中一個重要的酵素，推測茶多酚可能藉由抑制脂肪酸合成酶的表現來達到降血脂的效果。 在本論文的第四章，首先針對 FAS 在肝臟之表現進行分析。結果顯示餵食大白鼠普洱茶可以有效降低其肝臟 FAS 的表現。為了釐清其可能的作用機制，使用 HepG2 肝臟細胞株來做進一步的探討。研究結果顯示，普洱茶的萃取物可以降低 HepG2 細胞中 FAS 的表現。在訊息傳遞途徑方面的影響，發現包括 Akt 及 JNK 的訊息傳導途徑都有受到普洱茶萃取物的抑制。利用 PI3K 及 JNK 的抑制劑也可以降低 HepG2 細胞中 FAS 的表現。這些結果顯示普洱茶可能藉由抑制 Akt 以及 JNK 的訊息傳導途徑來調控肝臟中 FAS 的表現。這些結果顯示對於普洱茶有效成分之分離與鑑定值得進一步地研究。

The human epidermal growth factor 2 gene (HER2; also called ErbB2 or neu) is a proto-oncogene that belongs to the EGF receptor family. Amplification of the HER2 gene or overexpression of HER2 protein was found in up to 25-30% of breast carcinoma. Breast cancer patients whose tumor cells overexpress HER2 has a poor clinical outcome, such as shorter survival or earlier relapse. HER2 overexpression has been shown to enhance proliferative, prosurvival, and metastatic signals in breast cancer cell lines. Therefore, HER2 is an important target in the prevention or treatment of breast cancer. In chapter II, luteolin, a naturally occurring flavonoid, was found to be a potent stimulator of HER2 degradation, and Hsp90 molecular chaperone is suggested to be involved in luteolin-induced HER2 depletion. Luteolin preferentially inhibited the growth of HER2-overexpressing cancer cells compared with cells expressing a basal level of HER2. Furthermore, the induction of apoptosis was also observed in the luteolin-treated HER2-overexpressing cancer cells as measured by flow cytometry and PARP cleavage. Low doses of luteolin upregulated the p21 expression and high doses of luteolin downregulated its expression. Examination of the Akt/mTOR signaling revealed that this signaling was only transiently inhibited by low doses of luteolin, which suggested that the inability to cause sustained Akt/mTOR inhibition may contribute to p21 induction and provide a survival advantage to HER2-overexpressing cancer cells. To test this hypothesis, the combined use of luteolin and mTOR inhibitor rapamycin prevented low doses of luteolin from inducing p21 expression, and HER2-overexpressing cancer cells would be sensitized towards luteolin-induced apoptosis. In addition, p21 siRNA also increased the luteolin-induced cell death. In nude mice with xenografted SKOV3.ip1-induced tumors, luteolin significantly inhibited HER2 expression and tumor growth in a dose-dependent manner, and rapamycin further enhanced the effect of luteolin with a concomitant p21 inhibition. These results reveal an intriguing finding that suppressing p21 expression might have therapeutic implications and further suggest that combination of mTOR inhibitors may be a promising strategy to help increase the efficacy of preventive or therapeutic compounds against HER2-overexpressing tumors. Fatty acid synthase (FAS, EC 2.3.1.85), which catalyzes the reductive synthesis of palmitate from acetyl-CoA and malonyl-CoA, plays a central role in the anabolic conversion of dietary calories into storage form of energy in mammals. The expression of FAS reflects the regulation of lipogenesis. FAS is an important enzyme participating in the energy metabolism and is related to various human diseases including obesity, cardiovascular disease and cancer. FAS expression is markedly elevated in HER2-overexpressing breast cancer cells that are associated with poor prognosis. Pharmacological inhibition of FAS has led to a significant antitumor effect in HER2-overexpressing breast cancer cells. In chapter III, diosgenin (3B-hydroxy-5-spirostene), a plant-derived steroid, was found to be effective in suppressing FAS expression in HER2-overexpressing breast cancer cells. Diosgenin preferentially inhibited proliferation and induced apoptosis in HER2-overexpressing cancer cells. Furthermore, diosgenin significantly inhibited the phosphorylation of Akt and mTOR, and up-regulated JNK phosphorylation. The use of pharmacological inhibitors revealed that the modulating Akt, mTOR and JNK phosphorylation was potentially required for diosgenin-induced FAS suppression. Finally, diosgenin could specifically sensitize paclitaxel-induced cytotoxicity in HER2-overexpressing cancer cells. These results indicate that diosgenin could down-regulate FAS expression and induce apoptosis in HER2-overexpressing cancer cells through modulating Akt, mTOR and JNK phosphorylation, and further suggest that diosgenin alone or in combination with chemotherapeutic agents may provide a new approach for prevention or treatment of cancers that overexpress HER2. The significant hypolipidemic and growth suppressive effects of pu-erh, oolong, black and green tea leaves in Sprague-Dawley rats has been demonstrated by our laboratories. Pu-erh tea could lower the levels of cholesterol and triacylglycerol more significantly than other teas. No significant difference in food intake were observed among these tested and control groups. This striking phenomenon caused me to consider that the hypolipidemic and anti-obesity effects of tea treated groups may have come from the suppression of the key lipogenic enzymes. Considering the facts that FAS is an important enzyme participating in the energy metabolism, and the bulk of physiological lipogenesis occurs in liver, this promoted me to investigate the levels of FAS in the hepatic tissues of both control and tea-treated rats. In chapter IV, the expression of FAS in the livers of rats fed pu-erh tea leaves was significantly suppressed. FAS expression in the hepatoma HepG2 cells were also suppressed by the extracts of pu-erh tea at both the protein and mRNA levels. FAS expression in HepG2 cells was strongly inhibited by PI3K inhibitor LY294002 and JNK inhibitor II and slightly inhibited by p38 inhibitor SB203580 and MEK inhibitor PD98059, separately. Based on these findings, it suggests that the suppression of FAS in the livers of rats fed pu-erh tea leaves may occur through down-regulation of the Akt and JNK signaling pathways. The active principles and molecular mechanisms that exerted these biological effects in pu-erh tea deserve for future exploration.