The Antitumor And Radiosenstization Effects Of Docetaxel On Human Hepatocellular Carcinoma In Vitro And In Vivo

The antitumor and radiosenstization effects of docetaxel on human hepatocellular carcinoma in vitro and in vivoPart oneThe antitumor effects of docetaxel on human hepato-cellular carcinoma in vitro and in vivoPURPOSE: To investigate the in vitro and in vivo anti-human hepatocellular carcinoma (HCC) activity of docetaxel against SMMC-7721 HCC cells and its possible mechanism. METHODS:Colony forming assay Exponential growth phase SMMC-7721 cells were seeded in Ф60 mm culture plate at a density of 200 cells/plate, after 24hrs, the media was replaced and treated with equal volume of fresh media containing different doses of docetaxel ( 0, 10-11, 10-10, 10-9, 10-8, 10-7, 10-6 M). Growing 24hrs, the media containing docetaxel was again discarded and replaced by the original media without docetaxel. The cells were cultured at the incubator for 9 days. The colonies were fixed with 95% ethanol and stained with Giemsa, and manually counted. Colonies ≥50 cells were considered survivors.Flow cytometric analysis of DNA content after docetaxel treatment During exponential growth phase, SMMC-7721 cells were treated with different doses of docetaxel(0, 10-11, 10-10, 10-9, 10-8, 10-7, 10-6 M) for 24hrs and harvested with 0.25% trypsin and resuspended in media. About 1×106 cellswere centrifuged at 300g for 5 minutes and then washed once with PBS. The cell pellets were added with 100% precooled ethanol at 4℃for 4hrs, centrifuged at 300 g for 5 minutes and then washed once, resuspended with PBS(1×106cells/ml). The cells were added 500u RNase, incubated at 37℃ for 30 minutes, washed with PBS. Cells were stained with 50 μg/ml PI for 20 minutes in the dark. The DNA content of each cell was measured using a Becton Dickinson FACSCalibur flow cytometer and analyzed with ModFit LT software.Morphological study with fluorescence microscope Exponential growth phase SMMC-7721 cells were treated with 10 nM docetaxel for 24hrs and 48hrs and harvested with 0.25% trypsin and resuspended in medium. About 1×106 cells were washed once and resuspended with 25μl of PBS. Tweenty-five μl of the cells suspension was mixed with 1μl of dye mixture containing acridine orange (AO) 100 μg/ml and ethidium bromide (EB) 100 μg/ml in PBS. The cells were visualized immediately under a fluorescence microscope, the peak excitation wavelength was 490 nm. Transmission electron microscopic observation After SMMC-7721 cells incubated in the 10 nM docetaxel for 24 hrs and 48 hrs respectively, during exponential growth phase, they were fixed in Karnovsky solution, followed by cacodylate buffer for ultrastructural examination. The cells were postfixed in 1% osmium tetroxide and dehydrated for staining with uranyl acetate and lead citrate. Thin sections were observed under electron microscope.Reactive oxygen species (ROS) measurement After exponential growth phase SMMC-7721 cells were treated with different doses of docetaxel(0.25 nM, 0.5 nM, 10 nM) for 24hrs, the cells were incubated with 2/,7/-dichlorofluorescin diacetate (DCF/DA, 5 μM ) at 37℃ for 50 minutes to estimate the ROS level. The cells were harvested and detected immediately for fluorescence intensity detection on Becton Dickinson FACSCalibur flowcytometer and data were acquired and analyzed using FACS/CELLQuest software on a Power Macintosh 7600/120 computer, and the control group was not given docetaxel but incubated with DCF/DA. Determination of intracellular glutathione (GSH) content After exponential growth phase SMMC-7721 cells (≥106) were treated with different doses of docetaxel (0.25 nM, 0.5 nM, 10 nM) for 24 hrs, harvested by trypsination, washed, resuspended in PBS and counted under phase contrast microscope. The cells suspension was centrifuged at 300 g for 5 minutes, the cell pellets were added 0.75 ml distilled water and 0.25 ml thiosalicylic acid to precipitate the protein. After centrifugation at 12000 g for 5 minutes at 4℃, the supernatant was used for GSH assay. The control group was not giv...