The Effect of Hepatoma-Derived Growth Factor, Bcl-2, and Glutathione on Radiation-Induced Apoptosis in Hepatocellular Carcinoma Cell Lines

肝癌細胞生長激素、glutathione及Bcl-2對肝癌細胞株之凋亡之關係

10.6316/TRO/200714(3)179

溫明達(Ming-Dar Woon)；王毓慧(Yu-Huei Wang)；羅海韻(Hoi-Wan Lo)；鍾昌宏(David Chung)；劉燦榮(Tsan-Zon Liu)；邱仲峰(Jeng-Fong Chiou)

Bcl-2 ； GSH ； HDGF ； 肝癌細胞株 ； Bcl-2 ； GSH ； HDGF ； Hepatoma cell line

放射治療與腫瘤學

14卷3期（2007 / 09 / 01）

179 - 188

英文

目的：為了能更了解細胞內肝癌細胞生長激素(HDGF)、glutathione(GSH)及Bcl-2量等因子對肝癌細胞之放射敏感性的影響。 材料與方法：這四種不同分化之肝癌細胞株，經20 Gy劑量放射處理後，用TUNEL螢光染色、接著用共焦顯微鏡來計算細胞凋亡之百分比。而未放射處理癌細胞株內Bcl-2蛋白及細胞內GSH的活性量是用以ELISA來定量。而肝癌細胞生長激素的測定是以每種細胞內HDGF/β-tubulin蛋白在Western blot上的相對定量比值計算得到，再將上個三參數與細胞凋亡之百分比作比對，Pearson線性關係被用來作其關連性分析。 結果：經放射處理之Mahlavu、G2、SK、及3B四種肝癌細胞株中，引起不同程度之細胞凋亡，這些未放射處理細胞株中Bcl-2蛋白表現的量分別是419, 204, 58及22units/mg；GSH的量則是95, 247, 70及177umol/mg；而HDGF/β-tubulin蛋白比是0.827, 0.152, 0.987與0.520。GSH及HDGF/β-tubulin蛋白比之間有負關係(R^2=0.994)，而Bcl-2並沒有與GSH及HDGF有Pearson線性、二次方、或其它關係。除了Mahlavu外，細胞凋亡程度與HDGF有6正向關係(R^2=0.98)、與GSH有逆向關係(R^2=0.993)。 結論：肝癌細胞生長激素、Bcl-2、GSH的量在四種不同分化之肝癌細胞株內有不同表現，肝癌細胞株內肝癌細胞生長激素的量及GSH活性有很強關係，而肝癌細胞之放射細胞凋亡反應，除Mahlavu細胞外與細胞株內肝癌細胞生長激素的量及GSH活性有很強關係。在Mahlavu細胞中Bcl-2是所有細胞株中，有最較高的表現且細胞較不易凋亡，其相互關係仍需進一步求證，以進一步了解肝癌細胞生長激素、GSH、及Bcl-2對腫瘤細胞經放射處理後細胞凋亡之關係，甚至改變細胞內這些蛋白的量來改善我們治療肝癌的效果。

Purpose: To determine the factors affecting the apoptosis of hepatoma cells by irradiation. Methods and Materials: The mean percentages of apoptotic cells were determined using confocal microscope after hepatoma cells were irradiated with a single dose of 20 Gy and stained using TUNEL stain. The level of Bcl-2 protein and glutathione (GSH) content in the naive cell lysate was measured using ELISA. The ratio of hepatoma derived growth factor (HDGF)/β-tubulin of the untreated cells was calculated from the protein band in Western blot gel after densitometer quantization. The Pearson correlation between each paired parameters and the apoptosis were compared and correlation coefficiency was measured for their relationship. Results: The expressed factors were listed as 419, 204, 58, and 22 units/mg for Bcl-2 in the tested Mahlavu, Hep G2, Hep SK, and Hep 3B cell lysates. The GSH content were 95, 247, 70, and 177 umol/mg and the HDGF/ beta-tubulin ratio were 0.827, 0.152, 0.987, and 0.520 in those 4 cell lines. The level of HDGF/β-tubulin was inversely correlated with GSH (R^2=0.994, p=0.0032). However, Bcl-2 amounts did not correlate with GSH or HDGF/β-tubulin. Linear correlation was also observed between apoptosis and GSH except Mahlavu cell with R2 value of 0.98 (p=0.05). Mahlavu cell, with highest Bcl-2 level, exhibited relatively lower level of apoptosis. Conclusions: The HDGF, GSH, and Bcl-2 were expressed differently among these four hepatoma cell lines with different level of radiation-induced apoptosis. The HDGF/beta-tubulin level correlated well with GSH activity. HDGF levels in the 4 hepatoma cells correlated with the degree of the radiation-induced apoptosis treated with 20 Gy. However, this differential apoptotic effect among the four different Hepatoma cell lines can not be solely attributed to the amount of GSH or HDGF contents. Highest Bcl-2 concentration was observed in the Mahlavu cell which might rescue the apoptotic effect due to irradiation. Further investigation for the Bcl-2 effect on GSH is necessary to elucidate this relationship to explore the possibility of using the combined pattern of these biomarkers as the prognostic factors.