Extracellular Release of Annexin A2 is Enhanced upon Oxidative Stress Response via the p38 MAPK Pathway after Low-Dose X-Ray Irradiation.

The extracellular microenvironment affects cellular responses to various stressors including radiation. Annexin A2, which was initially identified as an intracellular molecule, is also released into the extracellular environment and is known to regulate diverse cell surface events, however, the molecular mechanisms underlying its release are not well known. In this study, we found that in cultured human cancer and non-cancerous cells an extracellular release of annexin A2 was greatly enhanced 1-4 h after a single 20 cGy X-ray dose, but not after exposure to ultraviolet C (UVC) radiation. Extracellular release of annexin A2 was also enhanced after H2O2 and nicotine treatments, which was suppressed by pretreatment with the antioxidant, N-acetyl cysteine. Among the oxidative stress pathway molecules examined in HeLa cells, AMP-activated protein kinase α (AMPKα) and p38 mitogen-activated protein kinase (MAPK) were mostly activated by low-dose X-ray radiation, and the p38 MAPK inhibitor, SB203580, but not compound C (an AMPKα inhibitor), suppressed the enhancement of the annexin A2 extracellular release after low-dose X irradiation. In addition, the enhancement was suppressed in the cells in which p38α MAPK was downregulated by siRNA. HeLa cells and human cultured cells preirradiated with 20 cGy or precultured in media from low-dose X-irradiated cells showed an increase in resistance to radiation-induced cell death, and the increase was suppressed by treatment of the irradiated cell-derived media with anti-annexin A2 antibodies. In addition, extracellularly added recombinant annexin A2 conferred cellular radiation resistance. These results indicate that an oxidative stress-activated pathway via p38 MAPK was involved in the extracellular release of annexin A2, and this pathway was stimulated by low-dose X-ray irradiation. Furthermore, released annexin A2 may function in low-dose ionizing radiation-induced responses, such as radioresistance.

Hochu­ekki­to (Bu­zhong­yi­qi­tang), a herbal medicine, enhances cisplatin­induced apoptosis in HeLa cells.

Hochu­ekki­to (HET), a Kampo herbal medicine composed of ten medicinal plants, is traditionally used to improve the general state of patients with malignant diseases such as cancer. Recent studies showed that HET had an anti­cancer effect against several cancer cell lines in vitro by inducing apoptosis. However, high doses of HET may have cytotoxic effects attributed to saponins or detergent­like compounds. Therefore, the present study used low doses of HET (50 µg/ml), which did not affect cell viability, to evaluate its synergistic anti­cancer effects with cisplatin. HeLa cells were cultured for 24 h with 50 µg/ml HET, followed by cisplatin treatment for 24 h at various concentrations. Subsequently, the sensitivity of the cells to cisplatin was assessed using a colony survival and a crystal violet cell viability assay. Furthermore, cisplatin­induced apoptosis was analyzed by flow cytometry. Proteins associated with cell viability and apoptosis, including phosphorylated (p­)Akt, p53, B­cell lymphoma 2 (Bcl­2), Bcl­2­associated X protein (Bax) and active caspase­3 were analyzed by immunoblotting. The present study revealed that cell survival was decreased and apoptosis was increased in HeLa cells pre­treated with HET prior to cisplatin treatment compared with HET­untreated cells. Furthermore, protein expression of p53 and active caspase­3 was increased, while the expression of p­Akt as well as the Bcl­2/Bax ratio, an index of survival activity in cells, were decreased in the HET­pre­treated cells compared with those in HET­untreated cells following incubation with cisplatin. In conclusion, the present study indicated that HET enhanced cisplatin­induced apoptosis of HeLa cells and that the administration of HET may therefore be clinically beneficial alongside apoptosis­inducing chemotherapy.