Effect of cordycepin on apoptosis and autophagy of tongue cancer cells in vitro and the molecular mechanism / 南方医科大学学报

<p><b>OBJECTIVE</b>To study the effect of cordycepin on cell cycle, apoptosis and autophagy of human tongue cancer TCA-8113 cells and explore the mechanism of cordycepin for inhibiting the occurrence of tongue cancer.</p><p><b>METHODS</b>CCK-8 method was used to assess the inhibitory effect of cordycepin on TCA-8113 cell proliferation in vitro. The cell cycle and cell apoptosis of TCA-8113 cells treated with different concentrations of cordycepin were analyzed using flow cytometry. The expressions of apoptosis-related genes caspase-3, caspase-9, Bcl-2, and Bax were examined using quantitative real-time PCR and Western blotting, and immunohistochemistry was used to detect the expressions of autophagy-related proteins LC-3β, P62, p-mTOR, and AMPK.</p><p><b>RESULTS</b>CCK-8 assay showed that cordycepin significantly inhibited the proliferation of TCA-8113 cells in a concentration-dependent manner with an IC of 3.548 mg/mL at 24 h and an IC of 1.185 mg/mL at 48 h. Flow cytometric analysis showed that cordycepin caused cell cycle arrest at S phase and dose-dependently increased the apoptotic rate of TCA-8113 cells. Treatment of the cells with cordycepin enhanced the expressions of Bax, caspase-3 and caspase-9 at both the mRNA and protein levels and inhibited the expression of the antiapoptotic gene Bcl-2. Immunohistochemistry demonstrated that cordycepin promoted the expression of LC-3β and AMPK and inhibited the expression of P62 and p-mTOR.</p><p><b>CONCLUSION</b>Cordycepin inhibits the proliferation and induces apoptosis of HCT-116 cells through the mitochondrial pathway and induces autophagy via the AMPK/mTOR pathway.</p>

Construction of a eukaryotic expression vector for alpha-1-antitrypsin and the localization of the expression product in NIH 3T3 cells / 南方医科大学学报

<p><b>OBJECTIVE</b>To construct a eukaryotic expression vector for alpha-1-antitrypsin (AAT) and detect its expression and localization in NIH 3T3 cells.</p><p><b>METHODS</b>The total RNA was extracted from the liver tissue of BALB/c mice, and the corresponding coding sequences for mouse AAT (GenBank accession No. NM_009243) were amplified by RT-PCR and cloned into hemagglutinin (HA)-tagged vector pcDNA3-HA. The construct was then transfected into NIH 3T3 cells, which were observed under fluorescence microscope.</p><p><b>RESULTS</b>The recombinant plasmid was verified by PCR, enzyme digestion and sequence analysis, and the fusion protein was highly expressed in NIH 3T3 cells. Under fluorescence microscope, the fusion protein was found to distribute mainly in the cytoplasm.</p><p><b>CONCLUSION</b>The expression vector for AAT-HA fusion protein has been successfully constructed and effectively expressed in mammalian cells to allow future functional study of AAT in mammalian cells.</p>

High mobility group box-1 stimulates proinflammatory cytokine production in endothelial cells via MAP kinases / 南方医科大学学报

<p><b>OBJECTIVE</b>To examine the synergistic effect of recombinant human high mobility group box 1 (HMGB1) protein and lipopolysaccharides (LPS) on the release of interleukin-8 (IL-8) and monocyte chemotactic protein 1 (MCP-1) in human umbilic vein endothelial cells (HUVECs), and explore the role of mitogen-activated protein kinases (MAPK) signal transduction in cytokine release.</p><p><b>METHODS</b>HUVECs were incubated with recombinant HMGB1 (0-75 ng/ml) for 24 h and the culture medium supernatant was harvested for detection of IL-8 and MCP-1 with LiquiChip system. At 0, 1, 3, 6, 12 and 24 h after stimulation with 15 ng/ml HMGB1 or 15 ng/ml HMGB1 plus 10 ng/ml LPS, the levels of IL-8 and MCP-1 in the HUVECs were examined. To test the effect of MAPK inhibitors, HUVCs were pretreated with the inhibitors SB203580 (20 mol/L), PD98059 (20 mol/L), and JNK inhibitor II (50 nmol/L) 1 h before HMGB1 and LPS stimulation.</p><p><b>RESULTS</b>The levels of IL-8 and MCP-1 were significantly increased in the HUVECs stimulated with HMGB1 protein at the concentrations of 3, 15 and 75 ng/ml in comparison with the control levels (P<0.01). Since 3-6 h after the stimulation with HMGB1, the levels of IL-8 and MCP-1 began to increase gradually, and steadily increased at 12 and 24 h, all significantly higher than those of the control group (P<0.01). Stimulation of the HUVECs with LPS (10 ng<ml) or HMGB1 (15 ng/ml) alone resulted in significantly increased levels of IL-8 and MCP-1 (P<0.01), which were further increased after costimulation with LPS and HMGB1, suggesting a synergistic effect between HMGB1 and LPS (P<0.01). This synergistic effect was significantly inhibited by pretreatment with MAPK signaling kinases inhibitors, especially the p38 MAP kinase inhibitor SB203580, and the cocktail of MAP kinase inhibitors almost totally blocked the expression of these chemokines in HUVECs treated with HMGB1 and LPS.</p><p><b>CONCLUSION</b>HMGB1 protein can activate HUVECs to produce the chemokines IL-8 and MCP-1 in a dose- and time-dependent manner. HMGB1 also acts synergistically with LPS to induce IL-8 and MCP-1 release, which might play an important role in the development of sepsis. MAPK signal transduction plays an important role in HMGB1 and LPS-induced IL-8 and MCP-1 release.</p>