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Preliminary Study on Hydrogen Peroxide-Induced Cellular Responses in RAW 264.7 Cells as Determined by IP-HPLC
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±è¿¬¼÷ ( Kim Yeon-Sook ) - ûÁÖ´ëÇб³ º¸°ÇÀÇ·á´ëÇÐ Ä¡À§»ýÇаú
Abstract
Hydrogen peroxide (H2O2) is originally an endogenous small molecule which is reduced into water in cells. In order to know the H2O2-induced oxidative stress in RAW 264.7 cells, first of all, the optimum concentration of exogenous H2O2 which show reactive cellular responses was determined as 40 ¥ìM by MTT assay, and followed by 40 ¥ìM H2O2 application in RAW 264.7 cells for 30 min, 1, or 2 hours. The expressional changes of essential proteins for cellular proliferation, epigenetic modification, inflammation, apoptosis, survival, and protection were assessed by immunoprecipitation high performance liquid chromatography (IP-HPLC) using 51 antisera. 40 ¥ìM H2O2 treatment down-regulated proliferation-related proteins, Ki-67, PCNA, CDK4, cyclin D2, cMyc, and PLK4, induced histone methylation/ deacetylation and DNA methylation by increasing levels of HDAC10 and DMAP1 and by decreasing levels of DNMT1 and KDM4D, activated inflammatory reaction by increasing levels of MCP-1, COX-2, CD68, LTA4H, CXCR4, and lysozyme, and dramatically up-regulated cellular apoptosis-, survival-, and protection-related proteins, AIF, PARP-1, caspase 9, c-caspase 9, pAKT1/2/3, SOD-1, HO-1, NF-kB, NRF2, and GSTO1 in RAW 264.7 cells. These observations suggest exogenous 40 ¥ìM H2O2-induced oxidative stresses which resulted global cellular responses including not only antioxidant, inflammation, and apoptosis but also proliferation and epigenetic modification. Particularly, 40 ¥ìM H2O2-induced apoptosis was mainly derived from PARP-1/AIF signaling leading parthanatos, and 40 ¥ìM H2O2-induced suppression of cMyc/MAX/MAD network was relevant to reduction of RAW 264.7 cell proliferation. Accordingly, H2O2 appears to affect RAW 264.7 macrophages in several ways eliciting not only oxidative stresses but also genome-wide DNA damage.
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Hydrogen peroxide; Protein expressions; RAW 264.7 cells; IP-HPLC
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