Synergistic effect of rapamycin (RPM) and PD98059 on cell cycle and mTOR signal transduction in human colorectal cancer cells / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To investigate the synergistic effect of rapamycin (RPM) and PD98059 on human colorectal cancer cells and its potential mechanisms.</p><p><b>METHODS</b>Three human colorectal cancer cell lines SW480, HCT116 and HT29 were treated with RPM 10 nmol/L, PD98059 (10 micromol/L, 20 micromol/L, 40 micromol/L, 50 micromol/L), or RPM plus PD98059, respectively, and the sensitivity was analyzed by MTT assay. The cell cycle progression was evaluated by flow cytometry. Western blotting analysis was performed to examine the total and phosphorylated levels of mammalian target of rapamycin (mTOR) and its downstream translational signaling intermediates, 70 kDa ribosomal protein S6 kinase (p70s6k) and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1).</p><p><b>RESULTS</b>Both RPM and PD98059 could inhibit viability of the three cell lines. The anti-proliferative effect of PD98059 exhibited a time/dose dependent manner and was strengthen by RPM. All the treatment with RPM, PD98059, and RPM + PD98059 induced arrest of cell cycle, although the arrest was confined at different cell cycle phases. In addition to their effect on proliferation and cell cycle, both inhibitors also reduced phosphorylation levels of mTOR, p70s6k, and 4E-BP1, as well as total 4E-BP1 levels in SW480 and HCT116 cells. That effect was reinforced when cells were treated with RPM plus PD98059 simultaneously, whereas total protein levels of mTOR and p70s6k remained unchanged.</p><p><b>CONCLUSION</b>RPM and PD98059 inhibit proliferation of colorectal cancer cells synergistically, and induce cell cycle arrest. The modulation of mammalian target of rapamycin signaling pathway is involved in its potential mechanisms.</p>

The relationship of mTOR signaling pathway and histone acetylation in human gastric cancer cell lines / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To evaluate the relationship between mammalian target of rapamycin (mTOR) signaling pathway and histone acetylation in cell survival, cell cycle, gene expression and protein level on human gastric cancer cells.</p><p><b>METHODS</b>Human gastric cancer cell lines, MKN45 and SGC7901 were treated with trichostatin A, rapamycin and/or LY294002, a PI3K inhibitor. Cell viability was analyzed by methylthiazolyl tetrazolium. Cell cycle distribution was evaluated by flow cytometry. The transcription level of p21(WAF1) gene was detected by using real-time polymerase chain reaction. Proteins were detected by Western blotting.</p><p><b>RESULTS</b>Cell viability remarkably reduced after treatment by more than two drugs (P< 0.01). Through flow cytometry assessment, MKN45 cells were arrested in G2 phase (P< 0.05), while SGC7901 cells were in G2 or G1 phase (P< 0.05) whether treated with single or more than two drugs. The expression of p21(WAF1) mRNA was remarkably increased in the gastric cancer cells treated with conjoined drugs (P< 0.01). Phosphorylation of Akt, p70S6K and 4E-BP1 was significantly reduced in cells treated with conjoined drugs (P< 0.01). And histone acetylation of H4/H3 was also increased in cells treated with conjoined drugs (P< 0.01).</p><p><b>CONCLUSION</b>mTOR singnaling pathway has an important relationship with histone acetylation in gastric cancer cell lines. There is a co-effect of mTOR inhibitor and histone deacetylase inhibitor on gastric cancer cells.</p>

5-Aza-2'-deoxycitydine induces demethylation and up-regulates transcription of p16INK4A gene in human gastric cancer cell lines / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>To investigate the effects of DNA methylation on the expression of tumor-associated genes and the cell cycle in human gastric cancer cells.</p><p><b>METHODS</b>Two gastric cancer cell lines (MKN-45 and HGC-27) were treated with DNA methyltransferase (DNMT) inhibitor, 5-aza-2'-deoxycytidine (5-aza-dC). The expressions of p16INK4A, p21WAF1, p53, p73, c-Ha-ras and c-myc genes mRNA were detected by using reverse transcription PCR (RT-PCR). DNA methylation status of p16INK4A gene promoter was assayed by bisulfite modification and sequencing. The cell cycle was analyzed by using flow cytometry (FCM).</p><p><b>RESULTS</b>5-aza-dC induced the demethylation of p16INK4A gene promoter. The expression of p16INK4A mRNA was obviously up-regulated by treatment with 10 micro mol/L (MKN-45 cells) or 5 micro mol/L (HGC-27 cells) of 5-aza-dC for 24 hours. However, 5-aza-dC treatment failed to regulate the expressions of p21WAF1, p53, p73, c-Ha-ras and c-myc genes in MKN-45 and HGC-27 cells. Furthermore, 5-aza-dC induced the cell cycle arrest in G1 phase in HGC-27 cell, but not in MKN-45 cell.</p><p><b>CONCLUSIONS</b>DNA methylation regulates the transcription of p16INK4A but not p21WAF1 and proto-oncogenes in human gastric cancer cell lines MKN-45 and HGC-27.</p>

Prophylactic and therapeutic effect of oxymatrine on D-galactosamine-induced rat liver fibrosis / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the prophylactic and therapeutic effect of oxymatrine on experimental liver fibrosis and to reveal its mechanism.</p><p><b>METHODS</b>By establishing D-galactosamine-induced rat liver fibrosis model, we observed the effect of oxymatrine on serum and tissue biochemical indexes, content of liver hydroxyline, expression of TGF?1 mRNA and changes of tissue pathology.</p><p><b>RESULTS</b>There was a decline of liver hydroxyline and serum AST and ALT in oxymatrine group compared to those of the D-GalN group. The hydroxyline content in oxymatrine pretreatment group was (0.50 0.11)mug/mg compared with (0.99 0.14)mug/mg in D-GalN group (t=8.366, P<0.01). The content in oxymatrine treatment group was (0.44 0.04)mug/mg compared with 0.70 0.06 in D-GalN group (t=9.839, P<0.01). The SOD activity was (149.81 15.28) NU/mg in oxymatrine pretreatment group and (95.22 16.33) NU/mg in the model group (t=7.309, P<0.01); (157.68 19.54) NU/mg in the treatment group compared with (119.88 14.94) NU/mg in the model group (t=4.348, P<0.01). MDA in the pretreatment group was (2.06 0.17) nmol/mg, lower than (4.57 0.37) nmol/mg in the model group (t=17.529, P<0.01). In the treatment group, it was (1.76 0.24)nmol/mg, lower than (3.10 0.17) nmol/mg in the model group (t=12.697, P<0.01). TGF?1 mRNA reduced in the pretreatment and treatment groups as compared with that in the model group (0.21 0.01 vs 0.50 0.01, t=48.665, P<0.01; 0.18 0.02 vs 0.38 0.01, t=22.464, P<0.01). Electron microscopy showed that oxymatrine group had milder hepatocyte degeneration and less fibrosis accumulation than did the model group. Microscopy revealed wide septa expansion from the portal area to the central venous, piecemeal and confluent necrosis and pseudo-nodular formation in part of the lobular in the model group. While in oxymatrine group these lesions were much improved.</p><p><b>CONCLUSIONS</b>Oxymatrine shows prophylactic and therapeutic effect in D-galactosamine induced rat liver fibrosis. This is partly by protecting hepatocyte and suppressing fibrosis accumulation through anti-lipoperoxidation.</p>