Expression of surface integrin-αvβ3 in U87MG cells with the treatment of 10 nM PTX.

<p>(A) Cytometric analysis of the expression of integrin-αvβ3 was determined by specific integrin-αvβ3 antibody staining. The expression of integrin-αvβ3 in U87MG cells treated with 10 nM PTX for 12 hrs was higher than control group. (B) Immunofluorescent detection of integrin-αvβ3 and nuclei was respectively stained by anti-integrin-αvβ3 antibody (Texas red) and DAPI (blue). Integrin-αvβ3 was obviously localized in the cell periphery. (I-III) indicated the U87MG cell isotypic control group. (IV-VI) Integrin-αvβ3 of U87MG cells was induced after 12 hr 10 nM PTX. (VII–IX) U87MG cells were pre-incubated with E[c(RGDyK)]₂ peptide to antagonized PTX-induced integrin-αvβ3 expression.</p

Primers used in real-time PCR analysis.

<p>Primers used in real-time PCR analysis.</p

Quantitative expression of caspase genes in U87MG cells treated with PTX, c(RGDyK)/E[c(RGDyK)]₂ peptides or the 12 hr PTX pre-treatment following with c(RGDyK)/E[c(RGDyK)]₂ peptides exposure.

<p>U87MG cells were treated with 40 µM c(RGDyK)/E[c(RGDyK)]₂ peptides for 24 hrs with or without 10 nM PTX pre-treatment for 12 hrs. The mRNA expression of (A) caspase-3, (B) caspase-8, (C) caspase-9, (D) caspase-10, and (E) caspase-12 was determined using real-time PCR. The increased levels of caspases -3, -8 and -9 were apparent in the pre-treatment with PTX for 12 hrs and 24 hr 40 µM c(RGDyK)/E[c(RGDyK)]₂ peptides. (fold changes of caspases -3, -8 and -9; PTX+c(RGDyK) vs. PTX alone: caspase-3 = 2.4 [±0.1] vs. 1.5 [±0.02], caspase-8 = 1.5 [±0.2] vs. 1.0 [±0.1], and caspase-9 = 3.1 [±0.3] vs. 3.4 [±0.02]; PTX+E[c(RGDyK)]₂ vs. PTX alone: caspase-3 = 2.7 [±0.1] vs. 1.5 [±0.02], caspase-8 = 2.4 [±0.1] vs. 1.0 [±0.1], and caspase-9 = 5.0 [±0.4] vs. 3.4 [±0.02]). The bars are represented the mean fold change of at least three independent experiments. Asterisk indicates significant at p<0.05 compared with 10 nM PTX alone.</p

Neutralized effect of caspase-3, -8 and -9 inhibitors on the caspase mRNA expression induced by PTX pre-treatment and E[c(RGDyK)]₂ peptide.

<p>U87MG cells were respectively pre-incubated with inhibitors of caspase-3 (Z-DEVD-FMK), caspase-8 (Z-IETD-FMK) or caspase-9 (Z-LEHD-FMK) and followed by 10 nM PTX pre-treatment for 12 hr and 40 µM E[c(RGDyK)]₂ treatment for 24 hr. The mRNA expression of (A) caspase-3, (B) caspase-8, (C) caspase-9, (D) caspase-10, and (E) caspase-12 was determined using real-time PCR. Eventually, either caspase-3, -8 or -9 inhibitor effectively to suppress the expression of caspase-3, -8 and -9. Asterisk indicates significant at p<0.05 compared with 10 nM PTX alone.</p

Cytotoxicity of U87MG cells treated with RGD peptide or the combination of PTX and RGD peptide was quantified using MTT assay.

<p>(A) Cell viability of c(RGDyK) peptide or combination pre-treated by 10 nM PTX in U87MG cells. (B) Cell viability of E[c(RGDyK)]₂ peptides or combination pre-treated by 10 nM PTX in U87MG cells. Cell viability was decreased at 100 µM c(RGDyK) or E[c(RGDyK)]₂ peptide. (C) Cell viability of a serial concentration of PTX for 24 hr in U87MG cells. The results of triplicate experiments are expressed as mean ± standard deviation.</p

Middle Infrared Radiation Induces G₂/M Cell Cycle Arrest in A549 Lung Cancer Cells

<div><p>There were studies investigating the effects of broadband infrared radiation (IR) on cancer cell, while the influences of middle-infrared radiation (MIR) are still unknown. In this study, a MIR emitter with emission wavelength band in the 3–5 µm region was developed to irradiate A549 lung adenocarcinoma cells. It was found that MIR exposure inhibited cell proliferation and induced morphological changes by altering the cellular distribution of cytoskeletal components. Using quantitative PCR, we found that MIR promoted the expression levels of ATM (ataxia telangiectasia mutated), ATR (ataxia-telangiectasia and Rad3-related and Rad3-related), TP53 (tumor protein p53), p21 (CDKN1A, cyclin-dependent kinase inhibitor 1A) and GADD45 (growth arrest and DNA-damage inducible), but decreased the expression levels of cyclin B coding genes, CCNB1 and CCNB2, as well as CDK1 (Cyclin-dependent kinase 1). The reduction of protein expression levels of CDC25C, cyclin B1 and the phosphorylation of CDK1 at Thr-161 altogether suggest G₂/M arrest occurred in A549 cells by MIR. DNA repair foci formation of DNA double-strand breaks (DSB) marker γ-H2AX and sensor 53BP1 was induced by MIR treatment, it implies the MIR induced G₂/M cell cycle arrest resulted from DSB. This study illustrates a potential role for the use of MIR in lung cancer therapy by initiating DSB and blocking cell cycle progression.</p> </div

MIR exposure induced G₂/M cell cycle arrest in A549 cells.

<p>Cells were exposed to MIR for 48 h, and harvested for RNA and protein extraction. (A) Gene expression of genes involved in regulation of G₂/M transition (x-axis). The y-axis indicates the relative transcript quantities calculated using the ΔΔCt method with GAPDH as the reference gene amplified from each sample. The data are presented as mean ± S.D. (<i>n</i> = 3). * <i>P</i><0.05, ** <i>P</i><0.001. (B) Protein expression levels were examined by Western blot with actin as the internal control. All experiments were repeated three times. (C) Flow cytometric analysis of DNA content. Cells were exposed to MIR for 48 h. Cells from six independent experiments were collected for analyzing cell cycle distribution. (D) The percentage of cells in each phase was obtained by MultiCycle analysis.</p

Primers used in qPCR.

<p>Primers used in qPCR.</p

Effect of MIR exposure on the actin filaments and focal adhesions of A549 cells.

<p>Cells were seeded onto glass coverslips in 12-well plates, exposed to MIR for 48 hours, fixed for staining and visualized by fluorescence microscopy. Actin filaments were tagged with rhodamine-labeled phalloidin (red), vinculin was labeled with mouse anti-vinculin antibody and the corresponding FITC– conjugated secondary anti-mouse IgG antibody (green), and nuclei were stained with DAPI (blue). Scale bar represents 10 µm. Arrows indicate the position of vinculin.</p

Effect of MIR exposure on the microtubule networks of A549 cells.

<p>Cells were seeded onto glass coverslips in 12-well plates, exposed to MIR for 48 hours, fixed for staining and visualized by fluorescence microscopy. Microtubules were labeled with α–tubulin antibody and the corresponding FITC–conjugated secondary antibody (green), and nuclei were labeled with DAPI (blue). Scale bar represents 10 µm.</p