Abstract
Abstract Using a specific stain for phosphotidylserine (PS) – Merocyanine 540 (MC540), PS externalization (an event occurred in apoptosis) is observed. After 100, 200, 400, 800 uM SbCl3 treatment for 4 hr (protocol 1) in TK6 cells (normal p53), the results showed that PS externalization in cells treated with 800 uM SbCl3 had significant difference from that of untreated control cells, P<0.05. In SbCl3-treated WTK1 cells (mutant p53), there is a dose-dependent increasing of PS externalization. PS externalization in WTK1 cells treated with 400uM and 800uM SbCl3 had significant difference with that of untreated control cells, P<0.05. On the other hand, after same 4 hr treatment as above and another 24 hr post-incubation in drug-free medium (protocol 2) in TK6 or WTK1 cells, the results showed that PS externalization in cells treated with 200uM SbCl3 was the most. It was significantly different from that of untreated control cells, P<0.05.Using Propidium iodide (PI) to stain cells after SbCl3 treatment using protocol 2 for detecting necrosis cells, the results show that necrosis population in samples treated with □200 uM SbCl3 had significant difference from that in untreated control samples, P<0.05. Using Propidium iodide (PI) to stain cells after treatment with protocol 2 and alcohol fixation for detecting Sub-G1 cell population -- a late event in apoptosis, the results show that Sub-G1 cell population in TK6 samples treated with □100 uM SbCl3 had significant difference from that in untreated control samples. However, Sub-G1 cell population in WTK1 samples treated with □200 uM SbCl3 had significant difference from that in untreated control samples, P<0.05. And, the highest percentage of necrosis cells or sub-G1 cell population was found in WTK1 samples after 400 uM SbCl3 treatment using protocol 2. However, these highest values in WTK1 cells after above-mentioned treatment in fact were still lower than those in TK6 cells.Finally, using Trypan blue staining to observe the survival fraction of cells after SbCl3 treatment using protocol 1 or protocol 2, the result show that TK6 cells were more sensitive than WTK1 cells at same dose of treatment. All these differences between TK6 and WTK1 cells may be because of different p53 background presented in these two cells. Further, using the DAN (2,3-diaminonaphthalene) staining and DCFHDA (dichlorodihydrofluorescein diacetate) /DCFH staining, the NO content in medium released from TK6 cells and the total ROS (reactive oxygen species)/RNS (reactive nitrogen species) formed in same cells were detected. The data show that DAN-stained TK6 cells after 200 uM SbCl3 treatment using protocol 1 showed a 120% NO content of that from untreated cells. DCFHDA/DCFH fluorescence from same cells after same treatment showed 130% total ROS/RNS of that from untreated cells. ONOO- scavenger- Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid, 100 uM) and NOS (NO synthase) inhibitor- NAME (Nω-nitro-L-arginine methyl ester, 2mM) or MTC (S-methyl-L-thiocitrulline, 50 uM) could reduce NO formation in TK6 cells immediately after 200 uM SbCl3 treatment for 4 hr. Catalase (800 U) and its inhibitor 3-AT (3-amnotriazole, 40 mM) could reduce total RNS/ROS formation in TK6 cells immediately after 200 uM SbCl3 treatment.Since the NO content and total RNS/ROS increased in TK6 cells after SbCl3 treatment, the effects of RNS/ROS inhibitors and donors on SbCl3-induced apoptosis were observed. The results show that SbCl3-induced PS externalization in both TK6 and WTK1 cells could be reduced by 50-200 uM Trolox, 1-4mM NAME or 25-100 uM MTC. MTC at 25-200 uM range has the least suppressing effect among the three used chemicals. However, NO donor SNP (0.25-1.0 mM) could not further increase the SbCl3-induced PS externalization in TK6 cells. In addition, 400-1600 U Catalase and 20-80 mM 3-AT could reduce SbCl3-induced PS externalization in TK6 cells after 200 uM SbCl3 treatment using protocol 2, although the effect of catalase seemed not obvious. SOD inhibitor DEDCA (diethyldithiocarbamic acid, 50-200 uM) could increase PS externalization in normal TK6 cells, but would reduce SbCl3-induced PS externalization increasing in TK6 cells after 200 uM SbCl3 treatment using protocol 2.Finally, the effect of reduced glutathione (GSH) on SbCl3-induced apoptosis was observed. The results show that GSH content increased in TK6 cells after 200 or 400 uM SbCl3 treatment using protocol 1, though, the GSH inducer NAC (N-acetylcysteine) and GSH inhibitor BSO (L-Buthionine sulfoximine) has little effect on SbCl3-induced PS externalization increment in TK6 cells.