山茱萸水萃抽物及其成份沒食子酸對人類骨肉瘤癌的抗癌作用之研究

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Title:	山茱萸水萃抽物及其成份沒食子酸對人類骨肉瘤癌的抗癌作用之研究 Anticancer effects of the water extract of Corni Fructus and the constituent gallic acid on human osteosarcoma cells
Authors:	廖慶龍;Ching-Lung Liao
Contributors:	中醫學系博士班
Keywords:	山茱萸;人類骨肉瘤癌;沒食子酸;Corni Fructus;osteosarcoma;gallic acid
Date:	2013-07-26
Issue Date:	2013-10-02 09:45:38 (UTC+8)
Publisher:	中國醫藥大學
Abstract:	自古以來山茱萸就被傳統中醫使用於治療許多疾病。而本文即是研究山茱萸水萃抽物及其成份沒食子酸對人類骨肉瘤癌的抗癌作用，並探討其作用機轉。首先在山茱萸水萃抽物方面，結果驗證山茱萸水萃抽物降低人類骨肉瘤癌細胞的存活率，造成細胞的DNA損傷、DNA condensation, 細胞週期G0/G1 arrest和細胞凋亡。山茱萸水萃抽物活化人類骨肉瘤癌細胞內caspase-8, caspase-9 和 caspase-3, 造成ROS 和 Ca2+的增加，使粒線體的膜電位差(ΔΨm)下降。山茱萸水萃抽物也上調caspase-3, caspase-9, Bax, cytochrome c, GRP78, AIF, ATF-6α, Fas, TRAIL, p21, p27, p16等的protein levels，使得細胞週期停滯而引起細胞凋亡。同時山茱萸水萃抽物也經由mitochondrial-dependent pathway，增加AIF, cytochrome c 和 Endo G, 導致細胞凋亡。由上述的結果顯示山茱萸水萃抽物引起人類骨肉瘤癌細胞凋亡，是經由ROS-modulated caspase-dependent 和 -independent pathways. 癌症的轉移是多重因素造成的，且也常使治療更複雜。本研究也探討山茱萸水萃抽物及其成份沒食子酸對人類骨肉瘤癌細胞的抑制腫瘤轉移侵入的作用與其作用機轉。結果顯示山茱萸水萃抽物可經由調控FAK, PKC, SOS1, MKK7, MEKK3, GRB2, NF-κB p65, COX-2, HIF-1α, PI3K, GRB2, Rho A, ROCK-1, IRE-1α, p-JNK1/2, p-ERK1/2, p-p38, Ras, p-PERK 和 VEGF的量，也可經由抑制MMP-2 和 -9的活性和蛋白質體表現來抑制人類骨肉瘤癌細胞腫瘤的轉移侵入。在沒食子酸方面，由 Western blotting 可得知GA下降人類骨肉瘤癌細胞內GRB2, PI3K, AKT/PKB, PKC, p38, ERK1/2, JNK, NF-κB p65的蛋白質levels.由in vitro kinase assay得知GA也抑制AKT, IKK 和 PKC的活性。GA抑制人類骨肉瘤癌細胞的migration 和 invasive能力，也降低MMP-2 and MMP-9的蛋白質、mRNA levels和secreted enzyme的活性。由上述結果顯示GA抑制人類骨肉瘤癌細胞migration 和 invasion的機轉可能經由下調PKC、抑制mitogen-activated protein kinase (MAPK) 和 PI3K/AKT，最後抑制MMP-2 and MMP-9的表現。綜上所述，山茱萸水萃抽物及其成份沒食子酸對人類骨肉瘤癌細胞有細胞毒殺作用、抑制細胞生長、抑制癌細胞轉移侵入。將來擬進一步研究考量山茱萸可做為治療人類骨肉瘤癌的新選擇方向。 Crude extract of Corni Fructus (CECF) have been used as Traditional Chinese medicine for treatment of variable dieases since long times ago. This study was to investigate the cytotoxic effects and associated mechanisms of CECF on U-2 OS human osteosarcoma cells. Our results demonstrated that CECF induced apoptosis in U-2 OS cells via ROS-modulated caspase-dependent and -independent pathways. Cancer metastasis is a multifactorial disease which complicates the treatments .In the present study, we investigated the inhibition of migration and invasion of CECF and it’s constituent - gallic acid (GA) on U-2 OS human osteosarcoma cells. We found that CECF significant inhibiting the migration and invasion of U-2 OS human osteosarcoma cells by regulating FAK, PKC, SOS1, MKK7, MEKK3, GRB2, NF-κB p65, COX-2, HIF-1α, PI3K, GRB2, Rho A, ROCK-1, IRE-1α, p-JNK1/2, p-ERK1/2, p-p38, Ras, p-PERK and VEGF expression and inhibition of MMP-2 and -9 activities and protein expression. We investigated the mechanisms for the inhibition of migration and invasion of GA on human steosarcoma U-2 OS cells. Proteins level were examined via Western blotting,and the activities of AKT, IKK and PKC were examined by in vitro kinase assay. We also examined the mRNA levels of MMP-2 and MMP-9 by real-time PCR. These results suggest that potential signaling pathways of GA-inhibited migration and invasion in U-2 OS cells may be due to down-regulation of PKC, inhibition of mitogen-activated protein kinase (MAPK) and PI3K/AKT, resulting in inhibition of MMP-2 and MMP-9 expressions. In conclusion, Crude extract of Corni Fructus (CECF) and Its constituent - gallic acid (GA) preserve the cytotoxic and anti-metastatic effects on U-2 OS human osteosarcoma cells. So we believe that the CFCE perhaps contains the capability of anti-cancer effects,and could be considered as a new strategy for anti-cancer treatment on U-2 OS human osteosarcoma cells
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