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Apelin Expression in Human Non-small Cell Lung Cancer: Role in Angiogenesis and Prognosis  Judit Berta, MS, Istvan Kenessey, MD, Judit Dobos, PhD, Jozsef.

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Presentation on theme: "Apelin Expression in Human Non-small Cell Lung Cancer: Role in Angiogenesis and Prognosis  Judit Berta, MS, Istvan Kenessey, MD, Judit Dobos, PhD, Jozsef."— Presentation transcript:

1 Apelin Expression in Human Non-small Cell Lung Cancer: Role in Angiogenesis and Prognosis 
Judit Berta, MS, Istvan Kenessey, MD, Judit Dobos, PhD, Jozsef Tovari, PhD, Walter Klepetko, MD, Hendrik Jan Ankersmit, MD, Balazs Hegedus, PhD, Ferenc Renyi-Vamos, MD, PhD, Janos Varga, MS, Zsolt Lorincz, PhD, Sandor Paku, PhD, Gyula Ostoros, MD, PhD, Anita Rozsas, MS, Jozsef Timar, MD, PhD, DSc, Balazs Dome, MD, PhD  Journal of Thoracic Oncology  Volume 5, Issue 8, Pages (August 2010) DOI: /JTO.0b013e3181e2c1ff Copyright © 2010 International Association for the Study of Lung Cancer Terms and Conditions

2 FIGURE 1 Apelin is expressed in normal human lung and non-small cell lung cancer (NSCLC) tissues and cell lines. A, Immunostaining of apelin in bronchial glands in normal human lung tissue. (B, C), Immunohistochemistry showing examples for focal (B) and diffuse (C) apelin staining patterns (corresponding to low and high apelin expression, respectively) in human NSCLC. (B) Arrows point at single apelin-expressing tumor cells within the tumor. Magnification, ×200 (A–C). D, Reverse-transcriptase polymerase chain reaction (RT-PCR) demonstrating the expression of apelin mRNA in human NSCLC cell lines and β-actin as control. E, Enzyme-linked immunosorbent assay detection of secreted apelin in conditioned medium from NSCLC cell cultures. Columns, mean for three experiments; bars, standard deviation. F, Box plots showing significant a difference between apelin mRNA levels of tumor and normal lung specimens of patients with NSCLC as depicted by median (central dots), 25 to 75% quartile ranges (boxes), and minimum/maximum levels (whiskers) (n = 46). G, Correlation between apelin mRNA and protein expressions of human NSCLC specimens as determined by quantitative real-time RT-PCR and immunohistochemistry, respectively (n = 46, R = 0.59, p < 0.01). Journal of Thoracic Oncology 2010 5, DOI: ( /JTO.0b013e3181e2c1ff) Copyright © 2010 International Association for the Study of Lung Cancer Terms and Conditions

3 FIGURE 2 Exogenous apelin does not stimulate non-small cell lung cancer (NSCLC) cell proliferation in vitro. Wild-type H1650 (A), HCC15 (B), LCLC-I03H (C), H1975 (D), H358 (E), and A549 (F) cells were cultured in serum-containing (Se+) or serum-free (Se−) medium. Apelin-36 (0 to 10−6 M) was added to the cells, and cell numbers were estimated at 72 hours by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. No significant effect of apelin on in vitro NSCLC proliferation was found. Columns, mean for three experiments; bars, standard deviation. Journal of Thoracic Oncology 2010 5, DOI: ( /JTO.0b013e3181e2c1ff) Copyright © 2010 International Association for the Study of Lung Cancer Terms and Conditions

4 FIGURE 3 Genetic manipulation increases cellular expression of apelin but does not stimulate in vitro proliferation of non-small cell lung cancer cell lines. A, Enzyme-linked immunosorbent assay demonstrating the effects of apelin overexpression on apelin levels in conditioned medium of H358 and H1975 cells stably transfected with either apelin or control vectors. Columns, mean for three experiments; bars, standard deviation; *p < 0.05 versus controls. B, No significant difference in proliferation was found by a 72-hour 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay when comparing H358 and H1975 cells stably transfected with control or apelin expression vectors. Columns, mean for three experiments; bars, standard deviation. Journal of Thoracic Oncology 2010 5, DOI: ( /JTO.0b013e3181e2c1ff) Copyright © 2010 International Association for the Study of Lung Cancer Terms and Conditions

5 FIGURE 4 Overexpression of apelin through genetic manipulation-stimulated in vivo growth of human non-small cell lung cancer cells in nude mice. Growth curves of control vector- and apelin-transfected H1975 (A) and H358 (B) cells. (▪) and (▴), means for eight mice per group; bars, standard deviation; *p < 0.05 and **p < 0.01 versus controls. Journal of Thoracic Oncology 2010 5, DOI: ( /JTO.0b013e3181e2c1ff) Copyright © 2010 International Association for the Study of Lung Cancer Terms and Conditions

6 FIGURE 5 Apelin overexpression increases non-small cell lung cancer angiogenesis in vivo. Frozen sections of 32-day-old apelin-overexpressing H358 (C) and H1975 (D) tumors or of control H358 (A) and H1975 (B) tumors were stained for the endothelial cell marker CD31 (red fluorescence). Magnification, ×200 (A–D). E, Microvessel densities (MVDs) of 32-day-old apelin-overexpressing or of control H358 and H1975 tumors. MVDs are mean blood vessel counts per square millimeter. F, Microvessel perimeters of 32-day-old apelin-overexpressing or of control H358 and H1975 tumors. Microvessel perimeters are expressed in micrometers. Columns, means for eight mice per group; bars, standard deviation; *p < 0.05 versus controls. Journal of Thoracic Oncology 2010 5, DOI: ( /JTO.0b013e3181e2c1ff) Copyright © 2010 International Association for the Study of Lung Cancer Terms and Conditions

7 FIGURE 6 Apelin expression is associated with angiogenesis and prognosis in human non-small cell lung cancer (NSCLC). A, Box plots showing median (central dots), 25 to 75% quartile ranges (boxes), and minimum/maximum levels (whiskers) of microvessel densities (MVDs), as determined by CD31 immunolabeling, in human NSCLC specimens with low versus high apelin protein expression. MVDs are mean blood vessel counts per square millimeter; n = 94. B, Box plots showing median (central dots), 25 to 75% quartile ranges (boxes), and minimum/maximum levels (whiskers) of microvessel perimeters, as determined by CD31 immunostaining, in human NSCLC specimens with low versus high apelin protein expression. Microvessel perimeters are expressed in micrometers; n = 94. C, Kaplan-Meier curves for the overall survival of the patient population with NSCLC, according to apelin expression as determined by immunohistochemistry. High apelin expression in the tumors was a significant prognostic factor for reduced overall survival; n = 94. Journal of Thoracic Oncology 2010 5, DOI: ( /JTO.0b013e3181e2c1ff) Copyright © 2010 International Association for the Study of Lung Cancer Terms and Conditions

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