研究探討層流剪應力於高糖環境下對膀胱癌細胞遷移與侵襲行為之影響;Investigation of the Effects of Interstitial Fluid-Induced Laminar Shear Stress on Migration and Invasion of Bladder Cancer Cells under High Glucose Environment

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Title:	研究探討層流剪應力於高糖環境下對膀胱癌細胞遷移與侵襲行為之影響;Investigation of the Effects of Interstitial Fluid-Induced Laminar Shear Stress on Migration and Invasion of Bladder Cancer Cells under High Glucose Environment
Authors:	葉建瑄;Yeh, Chien-Hsuan
Contributors:	生醫科學與工程學系
Keywords:	高血糖;層流剪應力;細胞間隙流;膀胱癌;腫瘤遷移;腫瘤侵襲;AKT;Cav-1;MT1-MMP
Date:	2017-05-22
Issue Date:	2017-10-27 13:10:14 (UTC+8)
Publisher:	國立中央大學
Abstract:	高血糖已被廣泛證明為促進腫瘤細胞惡化如腫瘤轉移（Tumor metastasis）等的主要危險因素之一，然而，在先前的研究中較少人討論細胞間隙流（Interstitial fluid）與之共同影響腫瘤細胞生理的因素，因此腫瘤細胞在高血糖環境下受細胞間隙流所產生的層流剪應力（Laminar shear stress；LSS）影響下腫瘤轉移的機制是有待證實的。在本研究中，我們利用膀胱癌BFTC-905（Transitional cell carcinoma cells；TCCs）作為實驗細胞，施以葡萄糖做藥物處理，並結合平行板式流道系統模擬層流剪應力，進而探討細胞間隙流在葡萄糖介導的膀胱癌細胞遷移與侵襲的影響。實驗結果顯示膀胱癌在靜態環境下受葡萄糖刺激在24小時下對腫瘤遷移有顯著影響（P < 0.01），腫瘤遷移率提升2倍之多，而經層流剪應力（12 dynes/cm2）沖刷前處理後腫瘤遷移率下降了63%（P < 0.01），在結合葡萄糖與層流剪應力下腫瘤遷移率會比靜態組（含葡萄糖）下降了63%，且具有顯著差異（P < 0.01），表示葡萄糖有助於提升腫瘤遷移的能力，而層流剪應力則抑制其能力，此結果與吉薩染色實驗呈現一致；侵襲實驗方面，膀胱癌在靜態環境下受葡萄糖刺激在侵襲48小時內對腫瘤侵襲有顯著影響且腫瘤侵襲率提升3.6倍（P < 0.01），而經層流剪應力沖刷前處理後腫瘤侵襲率提升1.31倍（P = NS），在結合葡萄糖與層流剪應力下腫瘤侵襲率會比靜態組（含葡萄糖）下降了59% （P < 0.01），因侵襲實驗同時伴隨遷移能力的展現，經沖刷後腫瘤遷移能力大幅降低，以至於侵襲實驗與遷移實驗相仿，有趣的是，膀胱癌細胞僅受沖刷前處理後腫瘤侵襲率些微上升，代表層流剪應力提升腫瘤侵襲的能力。最後我們以p-AKT、p-Cav-1、MT1-MMP三種蛋白做為目標進行西方墨點法實驗分析膀胱癌在葡萄糖和層流剪應力共同處理後的表現，我們發現p-AKT、p-Cav-1的表現在單獨葡萄糖處理的情況下都有顯著提升（P < 0.01），MT1-MMP則些微下降，表示葡萄糖具有提升細胞遷移的能力，而與侵襲能力較無關聯；單獨以層流剪應力處理的情況下p-AKT的表現無顯著差異，p-Cav-1的表現下降（P < 0.05），MT1-MMP則顯著上升（P < 0.01），表示層流剪應力調控Cav-1並抑制細胞遷移能力且提升侵襲能力；在共同刺激處理的情況下p-AKT和p-Cav-1的表現皆有顯著下降（P < 0.05），顯示在葡萄糖環境下層流剪應力在調節p-AKT和p-Cav-1的表現具有抑制的作用，而MT1-MMP則同樣受到抑制作用。本研究顯示了膀胱癌在高血糖環境下受層流剪應力（12 dynes/cm2）刺激其細胞遷移程度顯著下降，且經由Cav-1路徑調控AKT，在腫瘤侵襲上同樣具有顯著下降的趨勢，但與層流剪應力較不具關聯性，我們推測MT1-MMP可能受其他路徑的影響，需要做進一步的研究。此一結果顯示層流剪應力在含有葡萄糖的環境下對於抑制膀胱癌的腫瘤轉移上扮演了一個有利的角色，可作為糖尿病患具膀胱癌其減緩腫瘤轉移的手段之一。;Hyperglycemia has been widely demonstrated as one of major risk factors for tumor deterioration such as tumor metastasis. However, the definite mechanism of how glucose affects tumor development in vivo remains unclear since the interstitial fluid; one of key physiological factors in cellular microenvironment, is usually ignored in most of prior in vitro studies. To address the above issue, in this study, we aimed to investigate the effectiveness of interstitial fluid-induced laminar shear stress (LSS) on human urinary bladder transitional cell carcinoma (BFTC-905), in respects of cellular migration and invasion, in the presence of high glucose concentration. Based on the results of Giemsa and Calcein-AM staining assays, we found that the cells with 25-mM glucose for 24 h exhibited 2.03-fold enhanced migration efficiency (P < 0.01), while the migrated cell number with 12 dynes/cm2 LSS for 4 h significantly decreased ~63% (P < 0.01) as compared with the one without glucose. On the other hand, the migrated cell number with both LSS (12 dynes/cm2) and high glucose (25 mM) is significantly decreased 63% (P < 0.01) as compared to the group treated with glucose alone, indicating that high glucose promotes cellular migration while it was inhibited by LSS. Furthermore, our data showed that the cells with 25-mM glucose for 24 h exhibited 3.6-fold (P < 0.01) enhanced invasive cell number rate, while the invasive cell number with 12 dynes/cm2 LSS for 4 h is not significant difference as compared with the one without glucose. Moreover, the invasive cell number of the BFTC-905 treated with both LSS and 25-mM glucose significantly decreased 59% as compared to the group with glucose alone. According to the Western blot analyses, we investigate the mechanism of tumor metastasis caused by synergistic effect of LSS and glucose, expressions of AKT, Cav-1 and MT1-MMP were examined. We found that expressions of both p-AKT and p-Cav-1 exhibited significantly enhanced (P < 0.01) with glucose alone, and the expressions of MT1-MMP decreased slightly. Augmented along with exposure of LSS only that the expressions of p-AKT is no significant difference, whereas LSS enabled to further decreased the p-Cav-1 (P < 0.05) but increased the MT1-MMP (P < 0.01) expression. However, the results that the expressions of p-AKT and p-Cav-1 with both LSS and glucose exhibited significant decreased, the results show that LSS has an inhibitory effect on the regulation of p-AKT and p-Cav-1 in bladder cancer cells with a high glucose environment. In addition to the expressions of MT1-MMP significant decreased with both LSS and high glucose. In our study show that cell migration of bladder cancer decreased significantly under the laminar shear stress (12 dynes/cm2) in the high glucose environment, and through the Cav-1 pathway regulate the AKT. The tumor invasion also has a decreased significantly, but it′s the less relevant with the laminar shear stress. We suppose that MT1-MMP maybe have affected by other pathways. These results showed that the LSS of tumor metastasis in bladder cancer cells suggest that mechanical microenvironment of tumor cells may play important roles, and it should be taken into account in tumor therapy and management.
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