黃體酯酮(Progesterone)增加Epirubicin對肝癌細胞株之急性氧化壓力及減少自我吞噬作用的探討

Progesterone increased Epirubicin-induced acute oxidative stress and decreased epirubicin-induced autophagy in hepatoma HA22T/VGH cells

張文燦(Wen-Tsan Chang)

指導教授 ： 李金德

高雄醫學大學/醫學院/醫學研究所/博士(2014年)

https://doi.org/10.6832/KMU.2014.00102

摘要

研究背景：化學治療在肝癌之治療中仍有爭論存在。本研究旨在探討併用Epirubicin與黃體酯酮(progesterone)時，黃體酯酮對Epirubicin對肝癌細胞株產生之急性氧化壓力及自我吞噬作用的調節機轉的探討。
研究方法：將不同濃度的Epirubicin及黃體酯酮（progesterone）單獨或合併加入HA22T/VGH細胞之培養皿中，而用下列方法來探討其對HA22T/VGH細胞之產生之急性氧化壓力及自我吞噬作用生長抑制及細胞凋亡作用的影響。（1）使用crystal violet 及trypan blue來分析細胞之增殖情形（2）使用annexin V-FITC/propidium iodide之細胞流式儀及TUNEL來探討細胞凋亡現象（3）使用2′, 7′-Dichlorodihydrofluorescein diacetate and 4-amino-5-methylamino-2′, 7′-difluorofluorescein diacetate 流式細胞儀來探討急性氧化壓力代謝物的產生（4）使用ELISA 探討glutathione, nicotinamide adenine dinucleotide phosphate 的變化（5）使用免疫螢光染色探討FasL/Fas的變化（6）使用colorimetric 法分析caspase3、caspase8 的變化(7) 使用Western blotting 分析Fas-associated death domain(FADD)、Procaspase8、procaspase3、Bax、Bcl-xL、Beclin- 1、Light chain-3β的變化(8)以acridine orange staining 來探討 autophagy的變化(9)使用reverse RT-PCR來探討抗藥性基因multidrug resistance-related protein 1(MRP-1) 的變化。
研究結果：0.5 uMepirubicin 合併25uM的黃體酯酮能夠有意義的增加0.5ug/ml的 Epirubicin 對HA22T/VGH 細胞之急性氧化壓力，並藉由活化FasL/Fas、FADD、 caspase8、 caspase3 的途徑來增加HA22T/VGH細胞的凋亡作用。而0.3 uM epirubicin 合併30uM的黃體酯酮作用可以減少HA22T/VGH細胞的自我吞噬作用及降低抗藥性基因MRP-1 的表現。
結論： 本實驗室研究顯示併用黃體酯酮時，可以增加Epirubicin對肝癌細胞株HA22T/VGH 胞之急性氧化壓力及細胞凋亡作用並減少HA22T/VGH細胞的自我吞噬作用及降低抗藥性。本研究可作為進一步動物及臨床研究併用黃體酯酮及Epirubicin於肝癌治療的依據。

關鍵字

黃體酯酮(Progesterone) ； Epirubicin ； 氧化壓力 ； 細胞凋亡 ； 細胞自噬 ； 肝癌

並列摘要

Background: The use of chemotherapy in hepatocellular carcinoma is still controversial. The aim of this study was to investigate whether the combined use of epirubicin and progesterone has a synergistic effect on acute oxidative stress and autophagy in HA22T/VGH cells.
Materials and Methods: Different concentrations of epirubicin or progesterone were added to HA22T/VGH cells either alone or in combinations consisting of different concentrations of the two. Their effects on HA22T/VGH cells were studied by (1) crystal violet or trypan blue for cell viability assay (2) annexin V-FITC/propidium iodide flowcytometery and TUNEL for assay of cell apoptosis(3) 2′, 7′-Dichlorodihydrofluorescein diacetate and 4-amino-5-methylamino-2′, 7′-difluorofluorescein diacetate flowcytometery for assay of products of acute oxidative stress (4) ELISA assay for glutathione, nicotinamide adenine dinucleotide phosphate (5)Immunofluorescence for FasL and Fas (6)Colorimetric protease assay for caspase3、caspase8 (7) Western blotting asay for Fas-associated death domain(FADD)、Procaspase8、procaspase3、Bax、Bcl-xL、Beclin-1、Light chain-3β(8)Reverse transcription-polymerase chain reaction for expression of multidrug resistance related protein1 (MRP-1).
Results: The 25μM progesterone increased both the 0.5 μM epirubicin-induced cytotoxic and apoptotic effects in HA22T/VGH cells at 24 hr culture due to increase of acute oxidative stress (ROS and NO). This synergetic effects were the results of activation of FasL/Fas, FADD, precaspase-8, procaspase-3, caspase8, and caspase-3 pathway. And 0.3 μM epirubicin in combination with 30 μM progesterone could decrease the epirubicin-induced autopahgy in HA22T/VGH cells and also decrease expression of drug resistance gene MRP-1.
Conclusions: In vitro, progesterone can increase the acute oxidative stress and decrease the autophagy and drug resistence induced by epirubicin in HA22T/VGH. This provided the rationale of combination of use of epirubicin and progesterone in hepatocellular carcinoma

並列關鍵字

Progesterone ； epirubicin ； acute oxidative stress ； apoptosis, autophagy, multidrug resistance related protein1 (MRP-1) ； Hepatocellular carcinoma

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