Apoptosis Induction of Stomach Cancer Cell by TNF alpha and TGFbeta / 대한암학회지

PURPOSE: Apoptosis is a physiological mechanism for deleting cells from the body for development and homeostasis. Exogenous cytokines such as tumor necrosis factor alpha (TNFalpha) and transforming growth factor beta (TGF beta) are known to modulate apoptosis, thus can provide a new therapeutic modality for various malignancies. We studied whether TNFalpha or TGFbeta can induce apoptosis or exert antiproliferative effect on human gastric cancer cell line (AGS) and which genes are involved in the cytokine-induced apoptotic pathway. MATERIALS AND METHODS: To examine the effect of TNFalpha or TGF beta on AGS cell line (human gastric adenocarcimoma), we performed following tests; MTT test, trypan blue dye exclusion assay and colony forming efficiency. Total DNA was extracted from the TNFalpha-treated AGS cells and DNA ladder was detected as the hallmark of apoptosis, and flow cytometry analysis was performed for another apoptotic index. The effects of TNFalpha on c-myc expression was observed using RT-PCR. RESULTS: TNFalpha suppressed AGS cell growth, in a time- and dose-dependent manner, but TGFbeta had no effect on AGS cell growth. Electrophoretic analysis of total cellular DNA revealed the pattern of internucleosomal DNA cleavage, which is specific for apoptosis and the effect was observed from 24 to 72 hrs after 50 ng/ml TNFalpha treatment. Time-dependent increse of apoptotic cells by TNFalpha was detected by flow cytometry analysis. Morphological changes such as cell to cell contacts and extension of cell processes were observed in TNFalpha-treated AGS cells. RT-PCR using c-myc primers showed thatthe mRNA levels were increased 6 hrs after TNFalpha treatment and persisted for 72 hrs. CONCLUSION: It is suggested that TNFalpha, but not TGF beta, functions as an important inducer of apoptosis in AGS cell line, and c-myc may function as a critical endogenous activator of the pathway leading to cell death of AGS cells.

Rapid induction of mRNA for prostaglandin H synthase in ovine meningeal fibroblasts

We examined effects of interleukin 1 alpha (IL1 alpha) and phorbol 12, 13 dibutyrate (PDB), an activator of protein kinase C, on mRNA for Prostaglandin H synthase (PGHS) and prostanoid production in cultured ovine meningeal fibroblasts. Immuno- and morphologically-identified fibroblasts were derived from cerebral cortex and white matter from fetal lambs (approximately 120 days gestation) and grown to confluence on glass coverslips in 12 well plates. Levels of prostaglandin F2alpha and the stable hydrolysis product of prostacyclin (i.e., 6-keto-PGF1alpha) were determined using enzyme immunoassay. Relative amounts of mRNA were determined by in situ hybridization using ovine cDNA for PGHS1. IL1alpha (10 ng/ml) increased mRNA levels over baseline by 62 +/- 19% (p<0.05) at 60 min., 37 +/- 12% (NS) at 120 min., and 36 +/- 18 % (NS) at 240 min (n= 12). Levels of 6-keto-PGF1alpha were 148 +/- 18 pg/ml during baseline, 246 +/- 41 pg/ml at 60 min., 248 +/- 40 pg/ml at 120 min., and 259 +/- 62 pg/ml at 240 min (all p<0.05) (n=12). PGF2alpha was increased although it wasn't statistically significant. However, IL1alpha decreased PGE2 level significantly (all p<0.05). PDB (10-6 M) increased mRNA levels over baseline by 25+/-6% after 30 min., 40 +/- 6% after 60 min., and 20 +/- 8% after 90 min. (n=9) (all p<0.05). Levels of 6-keto-PGF1alpha were 200 +/- 43 pg/ml during baseline, 202 +/- 43 pg/ml after 30 min. (NS), 268 +/- 58 pg/ml after 60 min. (p<0.05), and 296 +/- 60 pg/ml after 90 min. (p<0.05) (n=9). Levels of PGF2alpha were 178 +/- 26 pg/ml during baseline, 300 +/- 30 pg/ml after 30 min., 299 +/- 35 pg/ml after 60 min., and 355 +/- 32 pg/ml after 90 min (all p <0.05) (n=6). Actinomycin-D (1 mg/ml) prevented increases in mRNA, 6-keto-PGF1alpha, and PGF2alpha at 60 min. for both I-L1 alpha and PDB. We conclude that cerebral fibroblasts are avid producers of prostanoids, and that enhanced production of PGHS is responsible for augmented PGF2alpha and prostacyclin production in the presence of an activator of protein kinase C and for decreased PGE2 and increased prostacyclin production in the presence of IL1alpha.