粉防己鹼經由抑制 EGFR、NF-κB 以及 MMP-2 的表現來抑制人類多型性腦神經膠質瘤 GBM8401 細胞的移行與侵襲

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Title:	粉防己鹼經由抑制 EGFR、NF-κB 以及 MMP-2 的表現來抑制人類多型性腦神經膠質瘤 GBM8401 細胞的移行與侵襲 Tetrandrine inhibited human glioblastoma multiforme GBM8401 cell migration and invasion by reducing the expression of p-EGFR(tyr1068), NF-κB p65 and MMP-2
Authors:	江以文;Yi-Wen Jiang
Contributors:	中國藥學暨中藥資源學系博士班
Keywords:	粉防己鹼;人類多型性腦神經膠質瘤;轉移;上皮生長因子受體;核轉錄因子;tetrandrine;GBM8401;metastasis;EGFR;NF-kB
Date:	2019-08-08
Issue Date:	2019-11-08 11:15:11 (UTC+8)
Publisher:	中國醫藥大學
Abstract:	人多型性腦神經膠質瘤是一種非常惡性的腦癌，且在各種腦癌當中是最致命的一種，儘管遵照標準的治療流程也很難被治癒。粉防己鹼 (tetrandirne) 由傳統中藥粉防己 (Stephania tetrandra S. Moore) 萃取而來，是一種天然微脂溶性的生物鹼，它具有許多的藥理學活性。目前，有關粉防己鹼對人類腦神經膠質瘤 GBM8401 細胞轉移與侵襲的影響機制仍然不明確。在本研究當中使用較低致死濃度的粉防己鹼來探討對人類多型性腦神經膠質瘤 GBM8401 細胞轉移的影響。本研究先利用細胞存活試驗與細胞增生試驗來檢測粉防己鹼對 GBM8401 細胞的影響。再以傷口癒合試驗與西方墨點法檢測細胞移行能力與 N-cadherin 的表現，接著以明膠電泳分析與西方墨點法檢測基質金屬蛋白? MMP-2 的表現與活性。然後利用 transwell assay 檢測 TET 對 GBM8401 細胞移行與侵襲的影響。最後以西方墨點法與電泳凝膠分析 EMSA assay 探討其抑制細胞移行與侵襲的相關機制，並利用上坪生長因子 EGFR 抑制劑探討這些蛋白在細胞轉移過程當中扮演的角色。本研究發現 GBM8401 的細胞存活率受到粉防己鹼的影響而細胞增生的部分在低濃度粉防己鹼的條件下則沒有顯著的影響。粉防己鹼藉由降低 N-cadherin 的表現來抑制細胞移行的能力，以及減少 MMP-2 表現來抑制細胞的移行與侵襲。此外，粉防己鹼也能夠經由減少 EGFR 的磷酸化以及 NF-κB p65 的表現來抑制細胞的侵襲。最後，利用蛋白質抑制劑證實了 p-EGFR 與 NF-κB p65 在人類多型性腦神經膠質瘤 GBM8401 細胞轉移的過程中扮演了相當重要的角色。由目前的觀察可以得知，在 GBM8401 細胞移行與侵襲的過程中，EGFR、NF-κB 以及 MMPs 均扮演了相當重要的角色，而粉防己鹼可以顯著地減少這些蛋白的表現。因此，本研究們認為未來粉防己鹼或許能夠做為抑制人類多型系腦神經膠質瘤 GBM8401 細胞轉移的抑制劑。 Human glioblastoma multiforme (GBM) is one of the malignant brain tumors. It is the most lethal cancer in the brain and difficult to cure even following standard treatment. Tetrandrine is one of the natural lipid alkaloid extracts from the Stephania tetrandra S. Moore which is a natural traditional herb and it exhibits widely pharmacological activities. However, the mechanism of tetrandrine affects human glioblastoma multiforme cell metastasis is still unclear. In the present study, we detected and used the lower lethal concentration of tetrandrine to investigate the anti-metastasis effects of tetrandrine on human glioblastoma multiforme GBM8401 cells in vitro. The cytotoxic effects of tetrandrine were analysis by PI staining for cell viability and MTT assay for cell proliferation. The effects of tetrandrine on cell mobility and the expression of N-cadherin were detected by wound healing assay and western blotting assay respectively; matrix metalloproteinases (MMPs) level, cell migration and invasion were detected by gelatin zymography, western blotting and trans-well assay respectively. Finally, we used Western blotting and EMSA assay to investigate other molecular mechanisms and we also used the protein inhibitors including Gefitinib and ammonium pyrrolidinedithiocarbamate (PDTC) to elucidate the role what they played in the progress of cell migration and invasion. We observed that GBM8401 cells were sensitive to tetrandrine. Tetrandrine reduced the cell mobility by decreasing the expression of N-cadherin and inhibited cell migration and invasion via reducing the expression of MMP-2. Moreover, tetrandrine also inhibited cell migration and invasion through reduced the expressions of p- epidermal growth factor receptor(tyr1068) and nuclear factor kappa-B p65. Finally, we found that epidermal growth factor receptor (EGFR), nuclear factor kappa-B (NF-κB) and MMPs played important roles in metastasis in human glioblastoma multiforme GBM8401 cells. Based on currently observations, EGFR, NF-κB p65 and MMPs played an important role in the cell migration and invasion. Tetrandrine significantly decreased the expression of p-EGFR(tyr1068), NF-κB p65 and MMP-2 in GBM8401 cells. Therefore, we suggested that it could be used as an inhibitor of migration and invasion in human glioblastoma multiforme GBM8401 cells in the future.
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