Loss of glucocorticoid receptor expression by DNA methylation prevents glucocorticoid induced apoptosis in human small cell lung cancer cells

Human small cell lung cancer (SCLC) is highly aggressive, and quickly develops resistance to therapy. SCLC cells are typically insensitive to glucocorticoids due to impaired glucocorticoid receptor (GR) expression. This is important as we have previously shown that expression of a GR transgene induces cell death in-vitro, and inhibits tumor growth in-vivo. However, the underlying mechanism for loss of GR expression is unknown. The SCLC cell line, DMS79, has low GR expression, compared to non-SCLC cell lines and normal bronchial epithelial cells. Retroviral GR expression in DMS79 cells caused activation of the apoptotic pathway as evidenced by marked induction of caspase-3 activity. Methylation analysis of the GR promoter revealed some methylation in the 1D, and 1E promoters of the GR gene, however the ubiquitous constitutively active 1C promoter was heavily methylated. In the 1C promoter there was a highly significant increase in DNA methylation in a panel of 14 human SCLC cell lines compared to a mixed panel of GR expressing, and non-expressing cell lines, and to peripheral blood mononuclear cells. Furthermore, within the panel of SCLC cell lines there was a significant negative correlation seen between methylation of the 1C promoter, and GR protein expression. Reversal of GR gene methylation with DNA methyltransferase inhibition caused increased GR mRNA and protein expression in SCLC but not non-SCLC cells. This resulted in increased Gc sensitivity, decreased Bcl-2 expression and increased caspase-3 activity in SCLC cells. These data suggest that DNA methylation decreases GR gene expression in human SCLC cells, in a similar manner to that for conventional tumor suppressor genes.     

De-phosphorylation of GR at Ser203 in nuclei associates with GR nuclear translocation and GLUT5 gene expression in Caco-2 cells

Glucocorticoid hormones and p44/42 mitogen-activated protein kinase (MAPK) inactivation are considered to be important in small-intestinal differentiation/maturation. In this study, we found that co-treatment with glucocorticoid hormone agonist dexamethasone and p44/42 MAPK inhibitor PD98059 in intestinal cell line Caco-2 strongly induced GLUT5 gene expression. Glucocorticoid hormone receptor (GR) was translocated from the cytoplasm to the nucleus and de-phosphorylated at serine residue 203 in the nucleus, by combined treatment with dexamethasone and PD98059. The binding of GR, as well as acetylated histones H3 and H4, to the promoter/enhancer region of GLUT5 gene was enhanced by combined treatment with dexamethasone and PD98059. These results suggest that the inactivation of p44/42 MAP kinase enhances glucocorticoid hormone-induced GLUT5 gene expression, probably through controlling the phosphorylation at serine 203 and nuclear transport of GR, as well as histone acetylation on the promoter/enhancer region of GLUT5 gene.     

Redox capacity of cells affects inactivation of glutathione reductase by nitrosative stress

Glutathione reductase (GR) plays a pivotal role in maintaining glutathione (GSH) in its reduced form. We have isolated a cDNA for rat GR and constructed a baculovirus system to produce recombinant GR on a large scale. This protein was purified by simple, two-step chromatographic procedure using DE52 and 2',5'-ADP Sepharose. Tissue distributions of GR were examined by Northern and Western blotting with a rabbit antibody to purified GR. GR was expressed in the order of reactivity; kidney, colon, liver, stomach, etc. Western blot analysis showed that both the cytosolic and the mitochondrial fractions of liver homogenate gave immunoreactive bands of similar size. This indicates that the same gene products exist in these fractions. Since nitric oxide (NO) produced under inflammatory conditions causes nitrosative stress and affects the redox states of surrounding tissues, we investigated the effects of NO donors on the enzymatic activities of purified GR. S-nitrosoglutathione (GSNO), 3-morpholinosydnonimine N-ethylcarbamide (SIN-1), and S-nitroso-N-acetyl-D,L-penicillamine (SNAP) at 1 mM gave 39, 15, and 12% inhibitions, respectively. In RAW 264.7 cells the GR activity was reported to be inhibited by GSNO. In A549 cells, however, no such change in the activity, protein levels and mRNA of GR was noted. Since these cells have a much higher redox capacity than RAW 264.7 cells as judged by GR activity and thioredoxin reductase activity it wound minimize cellular damage, including inactivation of GR caused by nitrosative stress.     

Effect of nitrosative stress on Schizosaccharomyces pombe: inactivation of glutathione reductase by peroxynitrite

Oxidative stress has been shown to alter cellular redox status in various cell types. Changes in expressions of several antioxidative and antistress-responsive genes along with activation or inactivation of various proteins were also reported during oxidative insult as well as during nitrosative stress. In the present study, we show the effect of nitrosative stress on cellular redox status of fission yeast Schizosaccharomyces pombe. This is the first report of S-nitrosoglutathione (GSNO) reductase activity in S. pombe and its inactivation by GSNO. We also show the inactivation of glutathione reductase (GR) and glutathione peroxidase in the presence of various reactive nitrogen species in vivo. In addition, we first observe the inactivation of GR by peroxynitrite in vivo using S. pombe cells and also similar observations under in vitro conditions. An immunoreactive band against monoclonal anti-3-nitrotyrosine antibody confirms the modification of GR under in vitro conditions. We also show the effect of nitrosative stress on Deltapap1 cells of S. pombe, which are more sensitive to nitrosative stress, indicating the involvement of Pap1 in the protection against nitrosative stress. Finally, exposure of S. pombe cells to reactive nitrogen species reveals an important role of cellular thiol pool in protection against nitrosative stress.     

AUY922 Effectively Overcomes MET- and AXL-Mediated Resistance to EGFR-TKI in Lung Cancer Cells

The activation of bypass signals, such as MET and AXL, has been identified as a possible mechanism of EGFR-TKI resistance. Because various oncoproteins depend on HSP90 for maturation and stability, we investigated the effects of AUY922, a newly developed non-geldanamycin class HSP90 inhibitor, in lung cancer cells with MET- and AXL-mediated resistance. We established resistant cell lines with HCC827 cells harboring an exon 19-deletion mutation in of the EGFR gene via long-term exposure to increasing concentrations of gefitinib and erlotinib (HCC827/GR and HCC827/ER, respectively). HCC827/GR resistance was mediated by MET activation, whereas AXL activation caused resistance in HCC827/ER cells. AUY922 treatment effectively suppressed proliferation and induced cell death in both resistant cell lines. Accordingly, the downregulation of EGFR, MET, and AXL led to decreased Akt activation. The inhibitory effects of AUY922 on each receptor were confirmed in gene-transfected LK2 cells. AUY922 also effectively controlled tumor growth in xenograft mouse models containing HCC827/GR and HCC827/ER cells. In addition, AUY922 reduced invasion and migration by both types of resistant cells. Our study findings thus show that AUY922 is a promising therapeutic option for MET- and AXL-mediated resistance to EGFR-TKI in lung cancer.     

自噬与非小细胞肺癌对吉非替尼耐药关系的实验研究

目的:探讨细胞自噬与非小细胞肺癌对Gefitinib耐药的相关性,寻找逆转非小细胞肺癌对Gefitinib耐药的新靶点。方法:以体外培养的人非小细胞肺癌Gefitinib敏感细胞PC-9与Gefitinib耐药细胞PC-9/GR为研究对象,通过MTT法检测Gefitinib对PC-9及PC-9/GR细胞存活率的影响;Western blot检测Gefitinib对PC-9及PC-9/GR细胞中自噬相关蛋白LC3的表达的影响;流式细胞术检测自噬诱导剂雷帕霉素和Gefitinib对PC-9/GR细胞凋亡率的影响。结果:PC-9/GR细胞Gefitinib IC50为PC-9细胞的200倍以上,具有非常明显的耐药性。PC-9/GR细胞中LC3II的表达显著低于PC-9/GR细胞(P0.05)。Rapamycin联合Gefitinib作用于PC-9/GR细胞可以明显提高其细胞凋亡率(P0.05)。结论:细胞自噬减弱与非小细胞肺癌对Gefitinib耐药有关,诱导细胞自噬可能逆转非小细胞肺癌对Gefitinib耐药。     

Negative cross-talk between p53 and the glucocorticoid receptor and its role in neuroblastoma cells

The tumour suppressor p53 and the glucocorticoid receptor (GR) respond to different types of stress. We found that dexamethasone-activated endogenous and exogenous GR inhibit p53-dependent functions, including transactivation, up- (Bax and p21(WAF1/CIP1)) and down- (Bcl2) regulation of endogenous genes, cell cycle arrest and apoptosis. GR forms a complex with p53 in vivo, resulting in cytoplasmic sequestration of both p53 and GR. In neuroblastoma (NB) cells, cytoplasmic retention and inactivation of wild-type p53 involves GR. p53 and GR form a complex that is dissociated by GR antagonists, resulting in accumulation of p53 in the nucleus, activation of p53-responsive genes, growth arrest and apoptosis. These results suggest that molecules that efficiently disrupt GR-p53 interactions would have a therapeutic potential for the treatment of neuroblastoma and perhaps other diseases in which p53 is sequestered by GR.     

Normal lung development and function after Sox9 inactivation in the respiratory epithelium

Heterozygous mutations in the human SOX9 gene cause campomelic dysplasia (CD), a skeletal malformation syndrome with various other organ defects. Severely affected CD patients usually die in the neonatal period due to respiratory distress. We analyzed the dynamic expression pattern of Sox9 in the developing mouse lung throughout morphogenesis. To determine a role of Sox9 in lung development and function, Sox9 was specifically inactivated in respiratory epithelial cells of the mouse lung using a doxycycline-inducible Cre/loxP system. Immunohistochemical and RNA analysis demonstrated extensive inactivation of Sox9 in the embryonic stage of lung development as early as embryonic day (E) 12.5. Lung morphogenesis and lung function after birth were not altered. Compensatory upregulation of Sox2, Sox4, Sox8, Sox10, Sox11, and Sox17 was not detected. Although Sox9 is expressed at high levels throughout lung morphogenesis, inactivation of Sox9 from the respiratory epithelial cells does not alter lung structure, postnatal survival, or repair following oxygen injury.