Connexin43 Modulates X-Ray-Induced Pyroptosis in Human Umbilical Vein Endothelial Cells.

OBJECTIVE: Pyroptosis is an inflammatory form of programmed cell death. This phenomenon has been recently reported to play an important role in radiation-induced normal tissue injury. Connexin43 (Cx43) is a gap junction protein that regulates cell growth and apoptosis. In this study, we investigated the effect of Cx43 on X-ray-induced pyroptosis in the human umbilical vein endothelial cells (HUVECs). METHODS: HUVECs, Cx43 overexpression, and Cx43 knockdown strains were irradiated with 10 Gy. Proteins were detected using western blot analysis. Cell pyroptosis was evaluated using the fluorescence-labeled inhibitor of caspase assay (FLICA) and propidium iodide staining through flow cytometry and confocal microscopy. Cell morphology and cytotoxicity were detected by scanning electron microscopy and lactate dehydrogenase release assay, respectively. RESULTS: Irradiation with 10 Gy X-ray induced pyroptosis in the HUVECs and reduced Cx43 expression. The pyroptosis in the HUVECs was significantly attenuated by overexpression of Cx43 as it decreased the level of active caspase-1. However, interference of Cx43 expression with siRNA significantly promoted pyroptosis by increasing the active caspase-1 level. Pannexin1 (Panx1), a gap junction protein regulates pyroptosis, and its cleaved form is used to evaluate channel opening and active state. The level of cleaved Panx1 in the HUVECs and Cx43 knockdown strains increased in the presence of X-ray, but decreased in the Cx43 overexpression strains. Furthermore, interference of Panx1 with siRNA alleviated the upregulation of pyroptosis caused by Cx43 knockdown. CONCLUSION: Results suggest that single high-dose X-ray irradiation induces pyroptosis in the HUVECs. In addition, Cx43 regulates pyroptosis directly by activating caspase-1 or indirectly by cleaving Panx1.

The protective effects of Bacillus licheniformis preparation on gastrointestinal disorders and inflammation induced by radiotherapy in pediatric patients with central nervous system tumor.

PURPOSE: we studied the effect of Bacillus licheniformis preparation (ZCS) on CNST (central nervous system tumor) patients undergoing the gastrointestinal symptoms and inflammation induced by radiotherapy. MATERIALS AND METHODS: 160 CNST patients with craniospinal irradiation (CSI) treatment were divided into experiment and control group. The experiment group patients took one capsule per time of ZCS and three times a day until the end of radiotherapy, starting one day before radiotherapy. While the patients in control group were administrated placebo without any probiotics. Serum from one day before radiotherapy and the first day after radiotherapy were collected to measure the ET, CRP, TNF-α, IL-1ß and IL-6. RESULTS: More than 70% CNST pediatric patients suffered from different degrees of gastrointestinal symptoms after radiotherapy, including mouth ulcer, nausea, vomiting, abdominal pain and diarrhea. And there was an obviously increased of serum ET, TNF-α, IL-1ß, IL-6 and CRP after RT. Importantly, a markedly decreased of ET, CRP and inflammatory cytokines were detected in the experiment group comparing to the control group after radiotherapy, as well as the relief of the gastrointestinal symptoms. However, improvement of probiotics (or ZCS) of the survival rate of CNST children and the recurrence of tumor are not observed in this study. CONCLUSIONS: Prophylactically administrated ZCS during radiotherapy for CNST patients can relieve RT-related gastrointestinal symptoms and inflammatory reaction.

Autophagy Attenuates MnCl2-induced Apoptosis in Human Bronchial Epithelial Cells.

OBJECTIVE: To investigate the role of autophagy in MnCl2-induced apoptosis in human bronchial epithelial 16HBE cells. METHODS: Cell proliferation was measured by MTT assay. Mitochondrial membrane potential (MMP) and apoptosis were measured by flow cytometry. Autophagic vacuoles were detected by fluorescence microscopy. Cellular levels of apoptosis and autophagy-related proteins were measured by western blotting. RESULTS: 16HBE cell proliferation was inhibited by MnCl2 in a dose- and time-dependent manner. MnCl2-induced 16HBE cell growth inhibition was related to MMP depolarization prior to the induction of apoptosis. Our data revealed that MnCl2-induced apoptosis in 16HBE cells was mediated by decreased expression of Bcl-2 and increased levels of cleaved caspase-3. It was observed that when we exposed 16HBE cells to MnCl2 in a dose-dependent manner, the formation of autophagic vacuoles and the levels of LC-3B-II were elevated. RNA interference of LC3B in these MnCl2-exposed cells demonstrated that MMP loss and apoptosis were enhanced. Additionally, the pan-caspase inhibitor Z-VAD-FMK increased the cellular levels of Bcl-2 and decreased apoptosis, but did not affect the cellular levels of LC3B in MnCl2-treated 16HBE cells. CONCLUSION: MnCl2 dose- and time-dependently inhibits 16HBE cell proliferation and induces MMP loss and apoptosis. Autophagy acts in a protective role against MnCl2-induced apoptosis in 16HBE cells.

Antiproliferation of berberine is mediated by epigenetic modification of constitutive androstane receptor (CAR) metabolic pathway in hepatoma cells.

Constitutive androstane receptor (CAR) regulates hepatic xenobiotic and energy metabolism, as well as promotes cell growth and hepatocarcinogenesis. Berberine is an ancient multipotent alkaloid drug which derived from Coptis chinensis plants. Here we report that berberine is able to be cellular uptake and accessible to chromatin in human hepatoma HepG2 cells. Berberine induces more apoptosis, cell cycle arrest, but less ROS production in CAR overexpressed mCAR-HepG2 cells. Moreover, berberine inhibits expressions of CAR and its target genes CYP2B6 and CYP3A4. Furthermore, berberine enhances DNA methylation level in whole genome but reduces that in promoter regions CpG sites of CYP2B6 and CYP3A4 genes under the presence of CAR condition. These results indicated that the antiproliferation of berberine might be mediated by the unique epigenetic modifying mechanism of CAR metabolic pathway, suggesting that berberine is a promising candidate in anticancer adjuvant chemotherapy, due to its distinct pharmacological properties in clinic.

S phase cell percentage normalized BrdU incorporation rate, a new parameter for determining S arrest.

In this study, a new parameter, S phase cell percentage (S fraction) normalized BrdU (SFN-BrdU) incorporation rate, was introduced to detect S arrest. The results showed a positive linear correlation between the BrdU incorporation rate and the S fraction in unperturbed 16HBE cells. Theoretical analysis indicated that only S arrest could result in a decrease in the SFN-BrdU incorporation rate. Additionally, the decrease in SFN-BrdU incorporation rate and the activation of DNA damage checkpoints further demonstrated that S arrest was induced by diethyl sulfate treatment of 16HBE cells. In conclusion, SFN-BrdU incorporation rate can be used to detecting S arrest.