In vitro study on blocking mTOR signaling pathway in EGFR-TKI resistance NSCLC

OBJECTIVE@#To investigate the effect and mechanism of inhibitor everolimus on EGFR-TKI resistance NSCLC.@*METHODS@#MTT assay was used to detect proliferation of human non-small cell lung cancer cell line A549. Flow cytometry was used to detect the changes of apoptosis and cycle distribution in each group after 24 h and 48 h. RT-PCR was used to detect the changes of PTEN and 4EBP1 expression levels after 48 h of monotherapy and combination therapy.@*RESULTS@#MTT assay showed that everolimus had dose-dependent inhibition against growth of A549 cells. Flow cytometry showed when everolimus could induce apoptosis and induce G0/G1 phase cell cycle arrest, which was time-dependent (P<0.05). RT-PCR showed everolimus could increase PTEN and 4EBP1 expression.@*CONCLUSIONS@#mTOR inhibitor everolimus has an inhibitory effect on EGFR-TKI resistant NSCLC, which cannot reverse the resistance effect of EGFR-TKI resistant cell line A549. The relationship between EGFR/AKT signaling pathway and the mTOR signaling pathway and the mechanism in non-small cell lung cancer need further study.

Decreased and dysfunctional circulating endothelial progenitor cells in patients with chronic obstructive pulmonary disease / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>It has been widely demonstrated that endothelial progenitor cells are involved in several diseases and that they have therapeutic implications. In order to define the altered pulmonary vascular homeostasis in chronic obstructive pulmonary disease, we sought to observe the level and functions of circulating endothelial progenitor cells in patients with chronic obstructive pulmonary disease.</p><p><b>METHODS</b>The total study population included 20 patients with chronic obstructive pulmonary disease and 20 control subjects. The number of circulating endothelial progenitor cells (CD34(+)/CD133(+)/VEGFR-2(+) cells) was counted by flow cytometry. Circulating endothelial progenitor cells were also cultured in vitro and characterized by uptake of DiIacLDL, combining with UEA-I, and expression of von Willebrand factor and endothelial nitric oxide synthase. Adhesion, proliferation, production of nitric oxide, and expression of endothelial nitric oxide synthase and phosphorylated-endothelial nitric oxide synthase were detected to determine functions of circulating endothelial progenitor cells in patients with chronic obstructive pulmonary disease.</p><p><b>RESULTS</b>The number of circulating endothelial progenitor cells in the chronic obstructive pulmonary disease group was lower than in the control group: (0.54 ± 0.16)% vs. (1.15 ± 0.57)%, P < 0.05. About 80% of adherent peripheral blood mononuclear cells cultured in vitro were double labeled with Dil-acLDL and UEA-1. The 92% and 91% of them were positive for von Willebrand factor and endothelial nitric oxide synthase, respectively. Compared with the control, there were significantly fewer adhering endothelial progenitor cells in chronic obstructive pulmonary disease patients: 18.7 ± 4.8/field vs. 45.0 ± 5.9/field, P < 0.05. The proliferation assay showed that the proliferative capacity of circulating endothelial progenitor cells from chronic obstructive pulmonary disease patients was significantly impaired: 0.135 ± 0.038 vs. 0.224 ± 0.042, P < 0.05. Furthermore, nitric oxide synthase (112.06 ± 10.00 vs. 135.41 ± 5.38, P < 0.05), phosphorylated endothelial nitric oxide synthase protein expression (88.89 ± 4.98 vs. 117.98 ± 16.49, P < 0.05) and nitric oxide production ((25.11 ± 5.27) µmol/L vs. (37.72 ± 7.10) µmol/L, P < 0.05) were remarkably lower in endothelial cells from the chronic obstructive pulmonary disease group than the control.</p><p><b>CONCLUSION</b>Circulating endothelial progenitor cells were decreased and functionally impaired in patients with chronic obstructive pulmonary disease.</p>

Beta-arrestin2 stimulates interleukin-17 production and expression of CD4+ T lymphocytes in a murine asthma model

Allergic asthma is a complex and chronic inflammatory airway disease. Interleukin-17 is a pro-inflammatory cytokine which plays critical role in the pathogenesis of allergic asthma. It has been reported that beta-arrestin2 regulated the development of allergic asthma at a proximal step in the inflammatory cascade. In this study, the influence of beta-arrestin2 on Interleukin-17 production and expression of CD4+ T lymphocytes in a murine asthma model was investigated. Splenic CD4+ T lymphocytes from wild-type mice and those from a murine asthma model were purified. CD4+ T lymphocytes from a murine asthma model were transfected with siRNAs targeting the beta-arrestin2 or were pretreated with the ERK1/2 inhibitor, PD98059. After stimulation, the protein expression of beta-arrestin2 phosphorylated-ERK1/2 and IL-17 were detection by Western blot; the mRNA expression of IL-17 were detected by real-time PCR; the accumulation of IL-17 in supernatants were detected by ELISA. We found that beta-arrestin2 phosphorylated-ERK1/2 and IL-17 expression in CD4+ T lymphocytes from a murine asthma model was increased compared with those from wildtype mice[p<0.01]. Treatment of CD4+ T lymphocytes with siRNAs targeting the beta-arrestin2 down-regulated phosphorylated- ERK 1/2 and IL-17 expression [p < 0.01]. PD98059 decreased IL-17 production and expression in CD4+ T lymphocytes in a murine asthma model [p < 0.05]. We conclude that beta-arrestin2 stimulated IL-17 production and expression of CD4+ T lymphocytes in a murine asthma model. The effect was partly mediated by ERK 1/2 activation. Targeting beta-arrestin2 biological activity could be a valid therapeutic approach for the treatment of allergic asthma

Application of sequential noninvasive following invasive mechanical ventilation in COPD patients with severe respiratory failure by investigating the appearance of pulmonary-infection-control-window / 中南大学学报(医学版)

OBJECTIVE@#To evaluate the application of sequential noninvasive following invasive mechanical ventilation in chronic obstructive pulmonary disease (COPD) patients with severe respiratory failure by investigating the appearance of pulmonary-infection-control-window.@*METHODS@#From November 2001 to October 2004, 76 case of COPD patients with severe respiratory failure due to pulmonary infection were intubated and recruited in the study. When the pulmonary infection was significantly controlled (the time of pulmonary infection control was called PIC window) by the antibiotic and comprehensive therapy, all cases were randomized into noninvasive veatiation group (NIV) and control group. The early extubation was conducted and followed by noninvasive mechanical ventilation via facial mask with bilevel positive airway pressure mode immediately in the NIV group. Conventional invasive synchronized intermittent mandatory ventilation (SIMV) plus pressure support ventilation (PSV) was used as the weaning technique in the control group.@*RESULTS@#Thirty eight cases among 76 patients were in the NIV group, and the rest in the control group. The NIV group and the control group had similar age, sex, APACHE scores, RR, HR, MAP, PaO2 and PaCO2 at the time of commencement and PIC window (P > 0.05). The time of PIC window was (7.5 +/- 1.9) d in the NIV group, and (8.0 +/- 2.5) d in the control group (P > 0.05). In the NIV group, the durations of invasive mechanical ventilation (MV) and total MV were (7.5 +/- 1.9) d and (12.5 +/- 4.0) d respectively, while the durations were (23.5 +/- 9.5) d in the control group (P < 0.05). The durations of RICU stay and hospital stay were shorter than that in the control group. The incidence of ventilation associated pneumonia (VAP) was 18.4% (7/38) in the NIV group, 39.5% (15/38) in the control group respectively (P < 0.05). The incidence of reintubation was 13.2% (5/38) in the NIV group, 34.2% (13/38) in the control group respectively (P < 0.05). Hospital mortality was 7.9% (3/38) in the NIV group, and 28.9% (11/38) in the control group (P < 0.05).@*CONCLUSION@#In those COPD patients requiring intubation and mechanical ventilantion who have severe respiratory failure due to pulmonary infection, sequential noninvasive following invasive mechanical ventilation at the appearance of PIC window can significantly reduce the MV duration, the length of RICU stay and hospital stay, and decrease the occurrence of VAP, reintubation and hospital mortality as well. So it is an efficient strategy to be generalized.