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

Abstract

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.