ABSTRACT
Recent reports have indicated different effects of immunostimulatory sequences containing CpG-Oligodeoxynucleotides [ODN] on various immune cells. However, the exact role of CpG-ODN in the human gut is unclear. In the present study, we assessed potential effects of CpG-ODN on non lymphoid cell [intestinal epithelial cell line HT-29] on a dose-response and time-course basis. Intestinal epithelial cell line HT-29 was treated with CpG-ODN [CpG 2006] and lipopolysaccharide [LPS] at 5, 10, 25, 50 micro g/ml and 1, 5, 10 micro g/ml concentrations, respectively. Following treatments, dose-response and time-course cytotoxicity using a colorimetric method, Metaloproteinase-2 [MMP-2] activity [using gelatin zymography] and apoptosis [using annexin-v flowcytometry method] assays were performed. Chloroquine treatment was also used for its inhibitory effect on endosomal acidification process to verify specific CpG-ODN and Toll Like Receptor 9 [TLR9] interactions. Cytotoxicity analysis of CpG-ODN showed that CpG-ODN increased significantly the proliferation of CpG-ODN treated cells, as compared to untreated cells, at concentrations of 10-25 micro g/ml [p<0.05]. Overall MMP-2 activity analysis showed significant differences between treated and untreated cells. However, minimal changes were observed when MMP-2 activity was assessed per cell. Moreover, CpG-ODN treated cells demonstrated an increasing apoptosis rate of 0.8%, 6.46% and 14.21% at concentrations of 5, 10, 25 micro g/ml, respectively. Collectively, our data indicated that intestinal epithelial cell line HT-29 is highly responsive to CpG effect in vitro and exhibits modified activities. The direct CpG-ODN and TLR-9 interactions in HT-29 cells could provide new approaches in malignant tumor therapeutic strategies
ABSTRACT
Although pyrimethamine [Tindurin[TM]] appears to be effective in the prevention and treatment of some infectious diseases, very little information exists on its unpredictable properties. We design this study to evaluate its anti-tumoral effect on a model of cell line. The cytotoxic influence of Pyrimethamine on prostate cell line was investigated using an in vitro colometric assay. The potential modulatory effects on metastasis, apoptosis, and immortality characteristics of cells were assessed with gelatin zymography, terminal deoxyribonucleotidyl transferase-mediated dUTP nick-end labeling [TUNEL] assay and telomeric repeat amplification protocol, respectively. Cytotoxicity analysis of pyrimethamine revealed a dosedependent fashion. An apoptotic influence of pyrimethamine was also confirmed by data obtained from TUNEL assay. Dose-dependent inhibitory effect on matrix metalloproteinases [MMP] was seen in pyrimethamine. A potent inhibitory effect of pyrimethamine was also established by data achieved from TRAPeze telomerase detection kit. Collectively, as induction of apoptosis together with MMP and telomerase inhibition could be indicative of cancer treatment, pyrimethamine might be considered as a chemopreventative agent in cancer
ABSTRACT
This study was conducted to examine if allergic contact dermatitis [ACD] alters the expression ofMMPs in human dermal fibroblasts. Fibroblasts are the primary source for MMP and matrix production in skin. MMPs are known to involve in a number of physiological and pathological processes. Some published data indicated a gelatinaselike activity in acute and chronic phases of allergic contact dermatitis. However, no exact source of gelatinase activity was demonstrated. Moreover, little is known about the role of MMPs in immune responses. To study and predict the pathophysiological effects of [MMP-2] in allergic contact dermatitic [ACD] patients, we established an in vitro tissue culture survey based on fibroblast explanted from ACD wounds and normal tissues respectively. We also employed a precise proliferation assay [i.e. MTT; 3-[4,5-dimethylthiazol-2-yl]-2,5- diphenyltetrazolium bromide] to analyze and compare three ACD vs. three normal cell strains Parallel to MTT assay, we assessed the activity as well as the kinetics of gelatinase [MMP-2] in conditioned media using a zymogeraphy analysis. There was a significant difference in proliferation capacity between mean ACD fibroblast strains vs. mean normal cells, particularly in days 6 to 8 post explantation, 492.5 +/- 6.6 vs. 361.75 +/- 8.25 respectively. Zymoanalyses indicated significant differences betweenACD cells and normal fibroblasts both in time-course and MMP-2 activity per cell fashions, 163.7 +/- 16.21 for meanACD fibroblasts vs. 130 +/- 9.09 for normal cells respectively. These data suggest that fibroblasts overproliferated in the process ofACD. Moreover, simultaneous overexpression of MMPs observed inACD fibroblasts vs. normal strains, is indicative of altered fibroblast functionality in the process of allergic contact dermatitis. The activity per cell analysis showed that MMP-2 expression in ACD fibroblasts is independent of cell number, suggesting that either intra- or inter-cellular control signals are also altered and that ACD fibroblasts exhibit hyper-responsiveness to mitogenic or fibrogenic stimulants. Altogether, these data address the chronocity and non-healing tendency of ACD wounds. However, more studies are required to examine possible MMPs inhibition and differential expression of mytogenic, fibrogenic and antifibrogenic cytokines in ACD wound beds. In particular, MMP-2 is postulated to be an aim for further gene therapy protocols