The Studies On Regulating Effects Of Glyceryl Trinitrate Metabolism On Tumor Growth And Proliferation

Nitric oxide (NO) is a simple and small free radical gas which is soluble both in water and lipid, and it is freely diffusible in the environment of cells. NO react with superoxide anion radical to produce molecules with strong oxidizing activity, such as nitrogen dioxide and peroxy nitrites. These oxidants are more toxic than NO itself. In recent years many studies showed that NO could modulate proliferation of tumor cells and had potential anti-tumor effect. Glyceryl trinitrate (GTN) had been used in clinic therapy as a vasodilator for more than a century. Many studies had been carried out on the metabolic mechanism of GTN and the results showed that as an NO donor, GTN could be metabolized to NO and then yielded S-nitrosothiols (RSNO). According to the metabolic mechanism of GTN in vivo, the present study was designed to evaluate the inhibitory effects and mechanism of GTN on tumor cells proliferation. For this purpose, experiment animals and cultured tumor cells had been used in these serial studies and the results indicated that GTN had potential anti-tumor efficacy. 1. GTN administered through alimentary canal or added in cellculture media yield nitrite. Different concentration of GTN was added to RMPI 1640 medium in 96-well plate incubated at 37 C for 48 hours and the nitrite was measured by Griess regent. The results of OD values was read by Bio-Rad Model 3550 - UV Microplate Reader both in measurement wavelength 545nm and reference wavelength 595nm. As results showed that different concentration of nitrite were released from different concentration of GTN. The higher concentration of GTN was administered, the higher concentration of nitrite was detected. In the study of rabbits, blood pressures were measured by dynamic analyzer. Methemoglobin (MetHb) was measured by blood gas analyzer and Alanine transferase (ALT) was measured by HITACHI 7600 automatic analyzer. Nitrate, nitrite and GSH were measured with colorimetry method. As results blood pressure and GSH in red blood cells showed significant decreases, nitrites showed significant increases. MetHb and ALT had no significant difference before and after GTN administration. According to these results we can conclude that metabolic GTN had no injury effects on hemoglobin and liver. 2 GTN could inhibit the growth of tumor cells and modulate cell cycle of tumor cell. Hela cells in early logarithmic growth phase (3 x 104/well) were incubated with different concentrations of GTN and incubated at 37 C for 48 hours. Then Griess reagent was added to measure nitrite or 3 -(4,5-dimethylthiazole-2-yI) -2,5-diphenyl tetrazolium-bromide ( MTT) was added to measure proliferationactivity. The results indicated that high concentration of GTN could inhibit the ability of Hela cells to adhere to culture bottle. Results of MTT assay showed high concentration of GTN could inhibit the growth of Hela cells. At the same time Hela cells were treated with 1.75X10~4M GTN and incubated at 37掳C for 48 hours. After treatment, cells were fixed by cool alcohol and Cell cycle distribution was analyzed by flow cytometer analysis of Pi-stained cells. Cell cycles could be modulated by GTN which could prompte S stage into G2/M compared to the control.3 GTN deplete GSH to initiate down- expression of Bcl-2 and induce apoptosis in tumor cells. After cells were treated with 1.75X10-4M GTN and incubated at 37 C for 48 hours, 150 1 1.67% metaphosphonic acid was added to 3X106 Hela cells, centrifuged (10,000 g for 15 minutes) and collected 60 1 supernatants to 96-well plate, 200 1 0.3M Na2HPO4 and 25 1 0.04% 2-nitrobenzoic (DTNB) were added to each well. OD was read by Bio-Rad Model 3550 - UV Microplate Reader in measurement wavelength 405nm. The result of OD value showed that concentration of GSH in Hela cells decreased significantly compared to the control. GTN could deplete GSH in tumor cells (p<0.05).Cells were treated with 1.75X10-4M GTN and incubated at 37掳C for 48 hours. After treatment, cells were digested and washed 2 times with phosphate buffered saline (PBS), then dealt...