Up-regulation of telomere-binding TRF1, TRF2 related to reactive oxygen species induced by As(2)O(3) in MGC-803 cells.

In this work, our study focused on As(2)O(3) action in view point of telomere. Results showed that treatment of human gastric cancer MGC-803 cells with arsenic trioxide could cause up-regulation of telomeric repeat binding factor TRF1 and TRF2 mRNA and protein levels, and induced G2/M phase arrest and cell apoptosis. At the same time, telomere length shortening and telomerase inhibitory were not obvious. Flow cytometry measurements indicated that the increase of TRF1 and TRF2 proteins is related to oxidative stress by arsenic trioxide. Results also indicate that after arsenic trioxide treatment, p53 protein levels increased significantly and also could bind directly at the telomere t-loop junction. These findings demonstrate arsenic trioxide-induced cell cycle arrest and apoptosis might involve a novel pathway related to TRF1, TRF2 protein.

Homocysteine induces cell cycle G1 arrest in endothelial cells through the PI3K/Akt/FOXO signaling pathway.

OBJECTIVE: High levels of homocysteine (Hcy) induce a sustained injury on arterial endothelial cells, which accelerates the development of thrombosis and atherosclerosis. Hcy specifically inhibits the growth of endothelial cells. The present study investigated the signaling pathways underlying this cell-cycle effect. METHODS: Human umbilical venous endothelial cells were treated with Hcy, and/or LY294002, okadaic acid, peroxovanadate (PV), antisense Akt, phosphorylation of Akt and FKHRL1 proteins. p27(kip1) protein levels were measured with Western blotting, and Akt kinase activity and cell cycle were measured with immunoprecipitation and flow cytometry, respectively. RESULTS: We demonstrate that Hcy induces dephosphorylation of Akt and FKHRL1 and upregulates the cyclin-dependent kinase inhibitors p27(kip1) in a time- and dose-dependent manner. Phosphatidylinositol-3 kinase (PI3K) activator PV and phosphatase 2A inhibitor okadaic acid could reverse it, which suggests it was dependent on PI3K activity. Moreover, Hcy induces cell cycle G1 phase arrest prevented by pretreatment with PV and okadaic acid. Transfection with specific antisense oligonucleotides to Akt further proves the observations. CONCLUSIONS: The studies implied that a novel signaling pathway, PI3K/Akt/FOXO, might play an important role in mediating cell cycle G1 arrest in endothelial cells.

A P53 target gene, PIG11, contributes to chemosensitivity of cells to arsenic trioxide.

The tumor suppressor p53 regulates the expression of various genes that promote apoptosis. PIG11 (P53-induced gene 11), also referred to as TP53I11 (tumor protein p53 inducible protein 11), is a direct p53 target gene. Recent data demonstrated that PIG11 was up-regulated markedly in arsenic trioxide induced apoptosis by DDRT-PCR, suggesting a new class of p53 target genes that sensitize cells to the effects of chemotherapeutic agents. In this study, through the construction of a recombinant GFP-PIG11 expression vector and transfection of HEK293 cells with GFP or GFP-PIG11, the role of PIG11 in apoptosis was analyzed. Results demonstrated that the percentage (11.38%) of apoptotic cells with GFP-PIG11 transfection was higher than that (7.28%) of with only GFP transfection (P<0.05). At 24 h after 1 microM of arsenic trioxide treatment, apoptotic cells exhibited a significant increase in the expression of GFP-PIG11 (36.67%+/-2.78), in contrast, 10.50%+/-2.03 only GFP and 5.25%+/-0.96 vehicle control (P<0.01). In addition, we showed that intracellular content of reactive oxygen species (ROS) was 9.66+/-0.52 in GFP-PIG11 transfection, higher than 5.21+/-0.08 in GFP only and 5.99+/-0.45 in vehicle control (P<0.01). The above results suggest that overexpression of PIG11 could induce cell apoptosis in the low levels and enhanced the apoptotic effects of arsenic trioxide. The process could be involved in intracellular generation of ROS.

Increasing sensitivity to arsenic trioxide-induced apoptosis by altered telomere state.

In this work, we investigated the synergic effects between low-dose arsenic trioxide and diethyloxadicarbocyanine (DODC), a telomerase inhibitor, on cell apoptosis. Results revealed that low-dose arsenic could block cell cycle arrest at the G2/M phase and induce apoptosis, whereas DODC could block cell cycle arrest at the G0/G1 phase but not induce apoptosis. However, cells pretreated with DODC showed greater sensitivity to arsenic than untreated cells. The percentage of apoptosis produced by combination treatment with the two agents increased and that was similar to the effect of high-dose arsenic treatment alone. Further studies showed that DODC alone could induce hairpin G-quadruplex formation and inhibit telomerase activity in a dose-dependent manner. Compared with HT1080 cells, 293 cells were more sensitive to cell growth inhibition and apoptosis and were less sensitivity to telomerase activity. These results indicate that DODC can synergistically enhance the apoptosis induced by arsenic, suggesting the increased cell senescence in response to arsenic is induced by an altered telomere state rather than by a loss of telomerase. Thus clinical application of combination treatment with arsenic and telomerase inhibitor may have potential in cancer therapy.

P53-induced gene 11 (PIG11) involved in arsenic trioxide-induced apoptosis in human gastric cancer MGC-803 cells.

Arsenic trioxide (As2O3) has been used as an effective chemotherapy agent for some human cancer, such as acute promyelocytic leukemia. We have demonstrated that low level of As2O3 relatively selectively inhibited growth of the solid tumor MGC-803 cells by triggering apoptosis. In this study, we found PIG11, a p53-induced gene, was upregulated markedly by As2O3 using the technique of differential display reverse transcriptase PCR (DDRT-PCR). Addition of anti-PIG11 phosphorothioated oligonucleotide (5'-GGC CGC CAT CTT CTC CTC-3') before As2O3 treatment, abolished the transient increase in PIG11 gene expression. Furthermore, it significantly inhibited the As2O3-induced apoptosis of MGC-803 cells, but had no effect in addition of missense (5'-GAG GAG AAG ATG GCG GCC-3') phosphorothioated oligonucleotides. These results suggest that PIG11, as a downstream target of p53, is involved in apoptosis of MGC-803 cells.

Intracellular redox status modulates monocyte chemoattractant protein-1 expression stimulated by homocysteine in endothelial cells.

Homocystinemia has been identified as an independent risk factor for atherosclerosis. Monocyte chemoattractant protein-l (MCP-l) is a potent chemokine that stimulates the migration of monocytes into the intima of the arterial wall. The authors investigated the role of intracellular redox status in the expression of MCP-l stimulated by homocysteine in endothelial cells. Homocysteine stimulated MCP-1 mRNA expression and protein production in a time-dependent and dose-dependent manner in endothelial cells, decreased intracellular glutathione (GSH) and protein thiol levels, as well as G6PDH activity and NADPH levels. Thiol reduced reagents, GSH, and dithiothreitol levels, and reversed the MCP-l mRNA expression and protein production in endothelial cells; in addition, thiol oxidized reagent, diamide, and BSO levels, and markedly potentiated homocysteine-mediated up-regulation of MCP-l mRNA expression and protein production in endothelial cells. These results demonstrate that homocysteine can trigger overexpression of the MCP-1 gene by altering the intracellular redox status, suggesting that the homocysteine-induced changes in the intracellular redox status play an important role in modulating the expression of MCP-l in endothelial cells.

Arsenite-induced reactive oxygen species and the repression of alpha-tocopherol in the MGC-803 cells.

We have investigated the action of oxidative stress in arsenite-induced apoptosis of human gastric cancer MGC-803 cells. Cells exhibited obvious characteristic of apoptosis following the treatment with 1.0 microM arsenite for 24 h. During the process, low concentration of arsenite significantly increased superoxide formation and lipid peroxidation, which was dose-dependent and was related to cell apoptosis induced by arsenite. The oxidant-dependent increase in intracellular [Ca(2+)] level and p53 gene expression were also observed at the same time. A phospholipase C inhibitor, 1-[6-([(17 beta)-3-methoxyestra-1,3,5,(10)-trien-17-yl]-amino)hexyl]-2,5-dione (U73122), could block the rapid transient increase in intracellular Ca(2+) levels, as well as the subsequent fragmentation of nuclear DNA. Addition of alpha-tocopherol before arsenite treatment abolished the transient increase in superoxide formation, lipid peroxidation, intracellular [Ca(2+)] levels and p53 gene expression, and furthermore could significantly inhibited the arsenite-induced apoptosis of MGC-803 cells. These results indicate that arsenite-induced oxidative stress, which stimulate cellular signaling systems, are involved in apoptosis of MGC-803 cells.

Opposite biological effects of arsenic trioxide and arsacetin involve a different regulation of signaling in human gastric cancer MGC-803 cells.

Opposite biological effects of arsenic trioxide (As(2)O(3)) and arsacetin on the growth of human gastric cancer MGC-803 cells have been observed. Results show that As(2)O(3) inhibited the growth of MGC-803 cells by triggering apoptosis, whereas arsacetin promoted the cell proliferation and seemed to stimulate the secretion of some growth factors at the same micromolar concentrations. Further studies showed that As(2)O(3) could regulate protein tyrosine kinase activity, protein tyrosine phosphorylation, and Bcl-2 protein and upregulate p53 protein. The ability of arsacetin to promote cell proliferation is linked with causing the opposite effects on these factors. These results indicate that the opposite biological effects of As(2)O(3) and arsacetin involve different regulations of molecular mechanisms in MGC-803 cells and that arsacetin may be a potential tumor promoter.