A simple method to induce hypoxiainduced vascular endothelial growth factorA (VEGF-A) expression in T24 human bladder cancer cells Jonas Magno Santos Cesário, Rodrigo Barbosa Oliveira Brito, Camila Soares Malta, Chrisna Souza Silva, Yves Silva Teles Matos, et al. In Vitro Cellular & Developmental Biology - Animal ISSN 1071-2690 In Vitro Cell.Dev.Biol.-Animal DOI 10.1007/s11626-016-0103-4
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Author's personal copy In Vitro Cell.Dev.Biol.—Animal DOI 10.1007/s11626-016-0103-4
A simple method to induce hypoxia-induced vascular endothelial growth factor-A (VEGF-A) expression in T24 human bladder cancer cells Jonas Magno Santos Cesário 1 & Rodrigo Barbosa Oliveira Brito 1 & Camila Soares Malta 1 & Chrisna Souza Silva 1 & Yves Silva Teles Matos 1 & Tânia Cristina Macedo Kunz 1 & Jessica Julioti Urbano 2 & Luis Vicente Franco Oliveira 2 & Maria Aparecida Dalboni 1 & Humberto Dellê 1
Received: 19 May 2016 / Accepted: 27 September 2016 / Editor: Tetsuji Okamoto # The Society for In Vitro Biology 2016
Abstract Angiogenesis is an essential process for the establishment, development, and dissemination of several malignant tumors including bladder cancer. The hypoxic condition promotes the stabilization of hypoxia-inducible factor 1 alpha (HIF-1α), which translocates to the nucleus to mediate angiogenic factors including the vascular endothelial growth factor A (VEGF-A). AnaeroGen system was developed for microbiology area to create a low oxygen tension required to the growth of anaerobic bacteria. Here, we hypothesized the use of AnaeroGen system to induce hypoxia in T24 human bladder carcinoma cells, in order to promote the overexpression of VEGF-A. T24 cells were cultured in six-well plates containing McCoy medium. Exposures of T24 cells to hypoxia for 1, 8, 24, and 48 h were performed using the Oxoid AnaeroGen system, while T24 cells under normoxia were used as control. The expression of VEGF-A and HIF-1α was analyzed by realtime PCR. ELISA for HIF-1α was carried out. The VEGF-A expression increased significantly by Oxoid AnaeroGeninduced hypoxia in a time-depending manner, reaching the peak in 48 h of hypoxia. Although HIF-1α mRNA was not changed, HIF-1α protein was increased in the presence of hypoxia, reaching a peak at 8 h. These results demonstrated that the Oxoid AnaeroGen system is a simple method to
* Humberto Dellê
[email protected] 1
Postgraduate Program in Medicine, Universidade Nove de Julho (UNINOVE), Rua Vergueiro, 235, 2° subsolo, São Paulo, São Paulo 01504-001, Brazil
2
Sleep Laboratory, Rehabilitation Sciences Master Degree and PhD Program, Nove de Julho University (UNINOVE), São Paulo, Brazil
expose T24 cells to hypoxia and efficiently to upregulate VEGF expression in T24 cells. Keywords Angiogenesis . Hypoxia . Bladder carcinoma . VEGF-A . HIF-1α . Oxoid AnaeroGen
Introduction Bladder cancer (BC) is the most common malignancy of the urinary system (Ritter et al. 2014). Similar to other types of cancer, the BC development depends directly on the angiogenesis process in order to supply the metabolic requirements for tumor growth. In addition, angiogenesis has a critical importance in tumor dissemination culminating in metastasis, the most cause of death in BC patients. The increase of angiogenesis markers has been implicated with poor prognosis in BC, including for the muscle-invasive form (Crew et al. 1997; Bernardini et al. 2001; Palit et al. 2005; Ajili et al. 2012). The bladder neoplastic cells are able to produce molecules that induce the angiogenesis process. One of the most potent inducers of angiogenesis is the vascular endothelial growth factor A (VEGF-A), which is activated by the hypoxia-inducible factor 1 (HIF-1), a transcription factor complex that regulates the expression of several hypoxiaresponsive genes related to angiogenesis. The HIF-1 complex is composed by two subunits: HIF-1β/ARNT (aryl hydrocarbon receptor nuclear translocator) and HIF-1α. Although both subunits are constitutively expressed, HIF1α is degraded by the ubiquitin-proteasome system under normoxic conditions (Salceda and Caro 1997). HIF-1α is hydroxylated by HIF prolyl hydroxylase to be recognized and ubiquitinated. The exposure to low oxygen tensions
Author's personal copy CESÁRIO ET AL.
inhibits the prolyl hydroxylase, since the oxygen is its cosubstrate (Maxwell et al. 1999). Inhibition of the ubiquitin-proteasome system leads to the accumulation of stable HIF-1α, which translocates from the cytoplasm to the nucleus to dimerize with HIF-1β and becomes transcriptionally active in regulating the expression of hypoxia-responsive genes (Huang et al. 1996). T24 human BC cells are widely used for in vitro studies in order to investigate the biology of the muscleinvasive BC, including angiogenesis. In general, hypoxia has been induced for T24 cells by modular incubator chambers, in which a specific atmosphere can be controlled. Another method to induce the activation of HIF1 in T24 cells is via incubation with cobalt chloride (Koga et al. 2007). This method activates HIF-1α without being necessarily deprived of oxygen; however, other effects can be triggered by cobalt chloride in addition to the HIF induction. An alternative method to generate an atmosphere with low oxygen tension has been routinely used in clinical microbiology. The Oxoid AnaeroGen systems were developed to generate an ideal atmosphere for anaerobic bacteria (Miller et al. 1995; Imhof and Heinzer 1996). The Oxoid AnaeroGen sachet containing ascorbic acid reduces the oxygen levels in the plastic pouch to below 1% within 30 min, resulting in carbon dioxide levels between 9 and 13%. Because this is simple, has low cost, and does not require special chambers with the acquisition of specific gas combination, we hypothesized the use of Oxoid AnaeroGen to promote a hypoxic condition for T24 cells in order to induce the expression of VEGF-A via HIF-1α.
Materials and Methods Cell culture Human bladder cancer T24 cells (HTB-4; American Type Culture Collection—ATCC, Manassas, VA), acquired from the cell bank of the Federal University of Rio de Janeiro, were cultured in McCoy’s 5A medium (SigmaAldrich, St. Louis, MO) supplemented with 10% fetal bovine serum and penicillin-streptomycin (Sigma-Aldrich), at 37°C with 5% CO2. To induce hypoxia, the AnaeroGen product was used (AnaeroGen Compaq AN0010C, Oxoid, Cambridge, UK). Brief, T24 cells were seeded in six-well plates and cultured until reaching 75% confluence. Each plate was placed in the plastic pouch (provided by company), together with the AnaeroGen Compact foil sachet, and sealed immediately (Fig. 1). The plates were maintained at 37°C with 5% CO2 along with the control cells (without hypoxia), during 1, 8, 24, and 48 h. In addition, T24 cells were incubated with cobalt chloride (150 μM, Sigma-Aldrich) as control for the detection of HIF-1
Fig. 1 Illustration of the Oxoid AnaeroGen system. Six-well plate with T24 cells exposed to normoxia (left) and to hypoxia (right), where the sixwell plate was incubated with the Oxoid AnaeroGen sachet to promote an atmosphere with low oxygen tension (
