Effect of insulin on the proliferation of colon cancer cells and the underlying mechanism, and the intervention effect of the ginsenoside compound K

The progression of multiple cancers, including liver, pancreatic, breast, colon/rectum and bladder cancer may exhibit a direct correlation between combination of hyperglycemia and hyperinsulinemia in diabetes mellitus (DM) patients. The survival rate of the patients with DM and colorectal cancer (CRC) is much lower than that of patients with CRC only, and hyperglycemia may be a mediator to induce hyperinsulinemia and increase cancer risk. Compound K (CK) is a metabolite of panaxndiol ginsenosides in the intestinal and possesses anti-cancer properties such as anti-proliferation, cell cycle arrest and pro-apoptotic effect on CRC cancer. CK has been considered to exhibit anti-cancer and improving diabetes. Therefore, the human colon cancer HCT 116 cell line was used to investigate the effect of insulin on cell proliferation and its underlying molecular mechanism, as well as the effect of CK on insulin-induced cell proliferation and metastasis. The results showed that insulin can promote the cell proliferation, migration and anchorage-independent growth of HCT116 cells. Insulin enhanced the expression of mRNA and activity of matrix metalloproteinases-2 (MMP-2) and MMP-9, leading to a rise of the metastatic ability in HCT 116 cells. The results also showed that insulin combined with its insulin receptor (IR) to activate insulin receptor substrate 1 (IRS-1), which altered its downstream signals of PI3K-Akt and MAPKs to cause a rise of cell proliferation-associated transcription factor, HSP27. To further verify the effects of the major signals on insulin-promoted cell proliferation, the protein expressions of PI3K and MAPKs were inhibited. It was found that the suppressions of PI3K and ERK signals can attenuate insulin-induced proliferation. Furthermore, insulin activated by PI3K-Akt pathway caused an increase of cell proliferation-related protein expression (e.g. phosphorylation of GSK3β and mTOR) to increase the level of cell cycle-related protein cyclin D1 and to decrease c-Myc expression. However, insulin also stimulated the expression of p53 protein, suggesting that p53 has a feedback regulation to stimulate a dramatic increase, leading to inhibition of cell proliferation. The results indicated that CK can significantly reduce the migration ability of the insulin-induced HCT 116 cells, and it slightly reduced the effect of cell proliferation and anchorage-independent growth. In conclusion, insulin can activate the downstream molecules through IRS-1 signal and increase the expression of PI3K-Akt and MAPKs, which eventually result in the effects of cell proliferation and cell cycle-related protein and enhance cell proliferation and metastasis of CRC. Therefore, it is suggested that ginsenoside CK has the potential to reduce the effects of insulin-induced cancer progression.糖尿病患者因體內合併高血糖及高胰島素血症之症狀，可能與多種癌症 (肝癌、胰臟癌、乳癌、大腸癌及膽囊癌等) 之進展有直接之關聯性。研究指出，罹患糖尿病之大腸癌患者存活率，遠低於未罹患糖尿病之大腸癌病患，而高血糖可能為高胰島素血症等誘發因子之媒介，因而增加癌細胞增生及侵入轉移能力。Compound K (CK) 為二醇型人參皂苷於腸道中之降解產物。文獻指出，CK 可抑制大腸癌之細胞增生及細胞週期進展且促細胞凋亡。許多研究指出，人參皂苷CK 具抗癌及改善糖尿病之功效。因此本研究分為兩部分進行探討：(一) 胰島素對大腸癌細胞增生之影響及其機轉探討；(二) CompoundK 抑制胰島素誘導大腸癌細胞增生及侵入轉移之影響。 研究結果顯示，胰島素會加速 HCT 116 大腸癌細胞之增生、移行及非依附性生長 (anchorage-independent growth) 能力 同時促基質，金屬蛋白 2/9 (matrix metalloproteinase-2/9, MMP-2/9) 之 mRNA 及活性表現，而使癌細胞之侵入轉移能力上升。深入了解相關分子機制後發現胰島素可與其專一性細胞接受器 (insulin receptor, IR) 結合，因而活化胰島素接受器受質蛋白 1 (insulin receptor substrate 1, IRS-1)而影響其下游途徑 PI3K-Akt 及 MAPKs 等相關訊息傳遞路徑，導致與細胞增生相關之轉錄因子 HSP27 之轉錄活性上升。為了解其中影響胰島素促進癌細胞增生之主要訊息因子，實驗進一步抑制 PI3K及 MAPKs 等相關訊息蛋白之表現。結果發現，抑制 PI3K 及 ERK能顯著減弱胰島素促癌之細胞增生作用。另外，本研究亦發現，胰島素可能透過活化 PI3K-Akt 訊息傳遞路徑導致細胞增生相關之蛋白GSK3β 及 mTOR 磷酸化表現上升，並促使與細胞週期相關之蛋白cyclin D1 表現上升及 c-Myc 表現量下降；然而，胰島素亦會提升抑癌蛋白 p53 之表現量，推測此現象為 p53 蛋白本身回饋調控機制促使其表現大量增加並反制細胞大量增生之結果。人參皂苷相關功效評估指出，CK 能顯著降低胰島素誘導之大腸癌細胞爬行能力，而對於細胞增生及非依附性生長則可些微降緩。 綜合上述，胰島素可透過 HCT 116 細胞其專一接受器活化下游相關訊息蛋白，並可活化 PI3K-Akt 及 MAPKs 及增加其表現量，進而影響與細胞增生及週期相關之訊息傳遞，促進大腸癌細胞增生並且影響其侵入轉移能力。而人參皂苷 CK 可能具有減緩胰島素促癌現象之潛力。全文摘要.............i Abstract .............iii 目次.............v 圖次v ix 附圖.............xi 表次.............xii 縮寫表.............xiii 前言.............1 第一章：文獻回顧 .............3 壹、 糖尿病 .............4 一、 背景 .............4 二、 定義 .............4 貳、 糖尿病與癌症之相關性.............7 一、 高糖與癌症之相關性.............10 二、 胰島素與癌症之相關性.............12 參、 大腸癌 .............18 一、 癌症 .............18 二、 大腸癌 .............18 三、 大腸癌之分期階段.............21 四、 大腸癌之流行病學.............21 肆、 高血糖及高胰島素血症與大腸癌之關係 .............23 伍、 癌症之細胞週期及增生.............26 一、 細胞增生.............26 二、 細胞週期 .............26 陸、 癌症之侵入轉移.............31 一、 轉移之前提條件 .............31 二、 轉移之起始與擴散 .............31 三、 轉移性定殖 (Metastatic colonization) .............32 捌、 樣品介紹.............34 一、 人參之簡介 .............34 二、 人參皂苷 - Compound K .............35 捌、 研究目的 .............38 玖、 研究架構.............40 第二章、胰島素對大腸癌細胞增生之影響及其機轉探討 .............41 摘要 .............42 Abstract .............43 前言.............45 實驗架構 .............47 材料與方法 .............48 結果.............55 一、 胰島素對人類大腸癌 HCT 116 細胞存活率之影響 ..........55 二、 胰島素對 HCT 116 細胞增生之影響.............55 三、 胰島素對 HCT 116 細胞長期生長之影響 (7 天) .............55 四、 胰島素對 HCT 116 細胞中 IR 途徑相關蛋白表現之影響...56 五、 胰島素對 HCT 116 細胞中 PI3K-Akt 途徑下游相關蛋白表現之影響 ..... 57 六、 胰島素對 HCT 116 細胞中 MAPKs 途徑相關蛋白表現之影響 ........57 七、 胰島素對 HCT 116 細胞中細胞週期相關之 c-Myc 及cyclin D1 蛋白表現之影響...... 57 八、 胰島素對 HCT 116 細胞中 c-Myc 及 HSP27 轉錄因子轉位活性之影響 ...... 58 九、 胰島素促進 HCT 116 細胞增生之主要分子機轉分析 ......58 討論 .............60 第三章、Compound K 抑制胰島素誘導大腸癌細胞增生及侵入轉移之影響.............79 摘要 .............80 Abstract .............81 前言 .............82 實驗架構 .............83 材料與方法.............84 結果 .............93 一、 胰島素對 HCT 116 細胞增生之影響 .............93 二、 胰島素對 HCT 116 細胞移行能力之影響.............93 三、 胰島素對 HCT 116 細胞基質金屬蛋白酶 2 及 9 mRNA表現之影響.......93 四、 胰島素對 HCT 116 細胞基質金屬蛋白酶 2 及 9 活性之影響..........94 五、 Compound K 對 HCT 116 細胞存活率之影響 .............94 六、 Compound K 對胰島素誘導 HCT 116 細胞增生之影響 ..94 七、 Compound K 對胰島素誘導 HCT 116 細胞移行之影響 ..95 八、 Compound K 對胰島素誘導 HCT 116 細胞基質金屬蛋白酶 2 與 9 活性之影響 ............95 討論 .............97 總結論.............111 參考文獻.............11