Induction of premature senescence may be a promising strategy for cancer treatment. However, biomarkers for senescent cancer cells are lacking. To identify such biomarkers, we performed comparative proteomic analysis of MCF7 human breast cancer cells undergoing cellular senescence in response to ionizing radiation (IR). IR-induced senescence was associated with up-regulation of cathepsin D (CD) and down-regulation of eukaryotic translation elongation factor 1B2 (eEF1B2), as confirmed by Western blot. The other elongation factor, eukaryotic translation elongation factor 1A1 (eEF1A1), was also down-regulated. IR-induced senescence was associated with similar changes of CD and eEF1 (eEF1A1 and eEF1B2) levels in the HCT116 colon cancer cell line and the H460 lung cancer cell line. Up-regulation of CD and down-regulation of eEF1 seemed to be specific to senescence, as they were observed during cellular senescence induced by hydrogen peroxide or anticancer drugs (camptothecin, etoposide, or 50 ng doxorubicin) but not during apoptosis induced by Taxol or 10 Mg doxorubicin or autophagy induced by tamoxifen. The same alterations in CD and eEF1A1 levels were observed during replicative senescence and Ras oncogene-induced senescence. Transient cell cycle arrest did not alter levels of eEF1 or CD. Chemical inhibition of CD (pepstatin A) and small interfering RNA-mediated knockdown of CD and eEF1 revealed that these factors participate in cell proliferation. Finally, the senescence-associated alteration in CD and eEF1 levels observed in cell lines was also observed in IR-exposed xenografted tumors. These findings show that CD and eEF1 are promising markers for the detection of cellular senescence induced by a variety of treatments. [Cancer Res 2009;69(11):4638-47]
Increased cell mass is one of the characteristics of senescent cells, but this event has not been clearly defined. When subcellular organellar mass was estimated with organelle-specific fluorescence dyes, we observed that most membranous organelles progressively increase in mass during cell senescence.
It is known that the level of interleukin-6 (IL-6) is higher in patients with active Behcet's disease (BD) than in those with inactive disease. Herpes simplex virus (HSV) type 1 inoculation of the earlobes of ICR mice resulted in the development of BD-like symptoms. To find out whether downregulation of IL-6 would affect the symptoms of BD, IL-6 small interfering RNA (siRNA) was administered to a BD mouse model. IL-6 siRNA was intraperitoneally injected into BD mice to downregulate IL-6 (n=9). IL-6 siRNA injection downregulated serum IL-6 level (118.9+/-114.4 pg ml(-1)) compared with scramble injection (439.4+/-378.0 pg ml(-1)) in BD mice (P=0.01). In seven out of nine IL-6 siRNA-injected BD mice, 77.8% improved and the severity score was decreased from 3.1+/-1.05 to 1.7+/-0.87 (P=0.005), whereas two out of six (33.3%) scramble-injected BD mice improved and the severity score changed from 2.5+/-0.84 to 2.0+/-1.41 (P=0.203). Foxp3, ROR gamma t, IL-17A, IL-17F and tumor necrosis factor-alpha were also influenced in IL-6 siRNA-injected BD mice compared with scramble-injected BD mice. Adoptive transfer of CD4+CD25+ cells to BD mice affected the decrease of IL-6 serum levels and were dependent on CD4+CD25+ cell numbers. These results showed that downregulation of IL-6 improved the inflammatory symptoms in BD mice through upregulation of regulatory T cells and inhibition of Th17 cells.
The purpose of this study was to clarify the correlation between microRNA-21 (miR-21) expression and inflammation in a herpes simplex virus (HSV)-induced Behçet’s Disease (BD) mouse model. miR-21 was compared between BD patients and healthy controls in peripheral blood mononuclear cells (PBMC). For miR-21 inhibition, miR-21 antagomir was applied to BD mice. The change of symptoms was monitored. The levels of cytokines and related molecules were determined by ELISA and real time qPCR. Treatment with colchicine or pentoxifylline down-regulated the level of miR-21 with improved symptoms in mice. miR-21 inhibition was accompanied by down-regulated serum levels of IL-17 and IL-6. The expression levels of PDCD4, RhoB, PD-1, IL-12p35, and toll-like receptor-4 were also regulated by miR-21 inhibition. miR-21 was correlated with HSV-induced BD-like inflammation in mice and BD patients. The expression of miR-21 was regulated by antagomir in mice.
Although many in vitro studies have previously been conducted to elucidate the biological effects of radio frequency (RF) radiation over the past decades, the existence and nature of any effects is still inconclusive. In an effort to further elucidate this question, we have monitored changes in protein expression profiles in RF-exposed MCF7 human breast cancer cells using two-dimensional gel electrophoresis. MCF7 cells were exposed to 849 MHz RF radiation for 1 h per day for three consecutive days at specific absorption rates (SARs) of either 2 W/Kg or 10 W/kg. During exposure, the temperature in the exposure chamber was kept in an isothermal condition. Twenty-four hours after the final RF exposure, the protein lysates from MCF cells were prepared and two-dimensional electrophoretic analyses were conducted. The protein expression profiles of the MCF cells were not significantly altered as the result of RF exposure. None of the protein spots on the two-dimensional electrophoretic gels showed reproducible changes in three independent experiments. To determine effect of RF radiation on protein expression profiles more clearly, three spots showing altered expression without reproducibility were identified using electrospray ionization tandem mass spectrometry analysis and their expressions were examined with RT-PCR and Western blot assays. There was no alteration in their mRNA and protein levels. As we were unable to observe any significant and reproducible changes in the protein expression profiles of the RF radiation-exposed MCF7 cells using high throughput and non-high throughput techniques, it seems unlikely that RF exposure modulates the protein expression profile.
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