Lithium chloride reduces radiation-induced intestinal injury through inhibiting apoptosis in intestinal epithelial cells

High dose radiation induces apoptosis of intestinal epithelial cells and subsequent depletion of the stem cells, resulting in lethal gastro-intestinal injury. However, effective treatment of this injury has not been established yet. Lithium chloride (LiCl) is well known to activate Wnt signal by inhibiting the activity of glycogen synthase kinase 3 (GSK3). The Wnt signal pathway has been shown to be associated with the maintenance of the stem cells of the intestinal crypt. Moreover, LiCl has been reported to inhibit neuronal apoptosis. The present study was designed to investigate effect of LiCl on intestinal injury induced by high dose radiation. Rat small intestinal epithelial cell line, IEC-6 cells and intestinal epithelial cells in primary culture obtained from 17.5-day fetal rat duodenum were used for in vitro assays. The cells were treated with LiCl 1 hr either before or after gamma-radiation of 20 Gy, and then cultured at 37˚C until 24 hr. The apoptosis was evaluated by the Hoechst33258 staining of cells. Pretreatment with 10 mM of LiCl markedly inhibited radiation-induced apoptosis in IEC-6 cells. Addition of LiCl to these cells after radiation also blocked the apoptosis. The anti-apoptotic effect of LiCl was also found in intestinal epithelial cells in primary culture. Inhibition of either phosphoinositide 3-kinase (PI3K)/Akt or mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase (MEK)/ERK pathway abrogated the anti-apoptotic effect of LiCl. In contrast, treatment with a p38 MAPK inhibitor did not affect the apoptosis. Western blot analyses showed that LiCl inhibited activation of caspase-3 and an increase of Bax level in radiated cells. Moreover, LiCl increased levels of Bcl-2 and Bcl-xL even in radiated cells. We also administered LiCl to male Balb/c mice intraperitoneally an hour prior to total-body irradiation (TBI) with 8 Gy. The numbers of crypts per circumference in jejunum were counted 3.5-day after TBI. The numbers of surviving crypts were greater in mice treated with 200 mg/kg body weight of LiCl than control mice with PBS. Thus, our results suggest that LiCl protects and rescues intestinal epithelial cells from radiation-induced apoptosis through activation of pathways involving PI3K/Akt and MEK/ERK. We also showed that LiCl reduced radiation-induced intestinal injury in vivo.AACR Annual Meeting 200

Ginsenosides Rd Prevents and Rescues Rat Intestinal Epithelial Cells From Irradiation-Induced Apoptosis

Panax ginseng has been shown to have a protective effect for irradiated animals or cells. Ginsenosides are the most active components isolated from ginseng, and ginsenoside Rd has been identified as one of the effective compounds responsible for the pharmaceutical actions of ginseng. In the present study, we studied the molecular mechanisms for the radio-protective action of ginsenoside Rd in rat intestinal epithelial IEC-6 cells. Cells were irradiated with gamma-ray, and apoptosis was examined using Hoechst staining and Western blot analysis. Treatment with ginsenoside Rd before gamma-irradiation inhibited irradiation-induced apoptosis in IEC-6 cells. Administration of Rd after irradiation also inhibited apoptosis in these cells. Irradiation of IEC-6 cells resulted in inactivation of Akt phosphorylation that was abrogated by Rd. On the other hand, irradiation activated phosphorylation of ERK1/2 but did not affect that of p38 MAPK. Inhibition of Akt phosphorylation prevented the reduction of apoptosis by Rd following irradiation. Pretreatment with an inhibitor of the MEK pathway further decreased the number of apoptotic cells. Rd decreased the ratios of Bax/Bcl-2 and Bax/Bcl-xL, the levels of cytochrome c, and the cleaved form of caspase-3 in irradiated IEC-6 cells. Our results suggest that ginsenoside Rd protects and rescues rat intestinal epithelial cells from irradiation-induced apoptosis through a pathway requiring activation of PI3K/Akt, inactivation of MEK, and also inhibition of a mitochondria/caspase pathway

Role of catalase in monocytic differentiation of U937 cells by TPA: hydrogen peroxide as a second messenger

Human promonocytic cell line U937 cells can be induced to differentiate into macrophages by treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA). TPA treatment induced the expression of the monocytic differentiation markers CD11b and CD36, with concomitant morphological changes. Moreover, TPA enhanced reactive oxygen species (ROS) generation in these cells, and phagocytic ability was also stimulated during differentiation. The antioxidant agent N-acetyl-L-cysteine inhibited the TPA-induced differentiation of U937 cells. TPA treatment decreased the expression level of catalase, which catalyzes the decomposition of hydrogen peroxide (H(2)O(2)) to H(2)O and O(2). In contrast, TPA increased the level of manganese superoxide dismutase, which catalyzes the dismutation of superoxide into H(2)O(2) and O(2) without affecting the levels of copper-zinc superoxide dismutase or glutathione peroxidase 1, which removes H(2)O(2) using glutathione as substrate. Treatment of U937 cells with catalase inhibited the enhancement of ROS generation induced by TPA, and blocked the TPA-induced differentiation of U937 cells. Human promyelocytic cell line HL60 cells were also induced to differentiate into macrophages by TPA. However, HP100-1 cells, its variant cell line overexpressing catalase, were resistant to TPA-induced differentiation. Our results suggest that catalase inhibits monocytic differentiation by TPA; the decrease in catalase level and the accumulation of H(2)O(2) are significant events for monocyte/macrophage differentiation by TPA

An FGF1:FGF2 chimeric growth factor exhibits universal FGF receptor specificity, enhanced stability and augmented activity useful for epithelial proliferation and radioprotection.

Structural instability of wild-type fibroblast growth factor (FGF)-1 and its dependence on exogenous heparin for optimal activity diminishes its potential utility as a therapeutic agent. Here we evaluated FGFC, an FGF1:FGF2 chimeric protein, for its receptor affinity, absolute heparin-dependence, stability and potential clinical applicability. Using BaF3 transfectants overexpressing each FGF receptor (FGFR) subtype, we found that, like FGF1, FGFC activates all of the FGFR subtypes (i.e., FGFR1c, FGFR1b, FGFR2c, FGFR2b, FGFR3c, FGFR3b and FGFR4) in the presence of heparin. Moreover, FGFC activates FGFRs even in the absence of heparin. FGFC stimulated keratinocytes proliferation much more strongly than FGF2, as would be expected from its ability to activate FGFR2b. FGFC showed greater structural stability, biological activity and resistance to trypsinization, and less loss in solution than FGF1 or FGF2. When FGFC was intraperitoneally administered to BALB/c mice prior to whole body gamma-irradiation, survival of small intestine crypts was significantly enhanced, as compared to control mice. These results suggest that FGFC could be useful in a variety of clinical applications, including promotion of wound healing and protection against radiation-induced damage