Non-invasive monitoring of hypoxia-inducible factor activation by optical imaging during antiangiogenic treatment in a xenograft model of ovarian carcinoma

Open Access Article.Targeting the hypoxia response pathway and angiogenesis are two promising therapeutic strategies for cancer treatment. Their use as single strategies has important limitations. Thus, development of combined regimens has become an important step toward improving therapeutic efficacy. Also, non-invasive monitoring of the response to targeted biological therapies, as well as determination of the optimal schedule for combination regimens has become an active field of research over the last five years, with relevance for both preclinical and clinical settings. Here, we used an optical imaging method to non-invasively monitor the functional changes in HIF activity in response to antiangiogenic treatment in a xenograft model of human ovarian carcinoma. A bioluminescent reporter construct containing nine copies of the hypoxia response element upstream of the luciferase gene (9xHRE-luciferase) was characterized in vitro in a panel of tumor cell lines and in vivo in a subcutaneous xenograft model of ovarian carcinoma by means of optical imaging. We showed that in OVCAR-3 subcutaneous xenografts, the most abrupt change in the HIF functional reporter occurs before the onset of massive tumor growth. However, this system failed to detect hypoxia induced upon antiangiogenic treatment due to the compensating effects of increased hypoxia and decreased tumor cell viability caused by imbalanced neovascularization vs. tumor expansion. Therefore, the readout based on HIF functional reporter could be conditioned by the dynamics of tumor growth and angiogenesis, which is highly variable depending on the tumor type, tumor model and stage of progression.This study was supported by grants from the Ministerio de Ciencia y Tecnología/Ministerio de Ciencia e Innovación (SAF2008-03147 to LdP and SAF2010-19256 to BJ), Comunidad Autónoma de Madrid (S-SAL-0311_2006) and the 7th Research Framework Programme of the European Union (METOXIA, project ref. HEALTH-F2-2009-222741). B.M.P. and V.G. have been supported by a grant from the Comunidad Autónoma de Madrid (S-SAL-0311_2006).Peer Reviewe

Frustration induced Raman scattering in CuGeO_3

We present experimental data for the Raman intensity in the spin-Peierls compound CuGeO_3 and theoretical calculations from a one-dimensional frustrated spin model. The theory is based on (a) exact diagonalization and (b) a recently developed solitonic mean field theory. We find good agreement between the 1D-theory in the homogeneous phase and evidence for a novel dimerization of the Raman operator in the spin-Peierls state. Finally we present evidence for a coupling between the interchain exchange, the spin-Peierls order parameter and the magnetic excitations along the chains.Comment: Phys. Rev. B, Rapid Comm, in Pres

The antiferromagnetic/paramagnetic transition in mixed-spin compounds R2BaNiO5

We present an extensive Quantum Monte Carlo study of the magnetic properties ofthe mixed-spin quantum systems R2BaNiO5 (R= magnetic rare earth)which show coexistence of 3-dimensional magnetic long-range orderwith 1-dimensional quantum gap excitations.We discuss the validity of the performed simulations in thecritical region and show the excellent agreement with experimental results. We emphasize the importance of quantum fluctuations contained in ourstudy which is absent in previous mean-field-like treatments.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/43878/1/10051_2004_Article_143.pd

Hypoxia Negatively Regulates Antimetastatic PEDF in Melanoma Cells by a Hypoxia Inducible Factor-Independent, Autophagy Dependent Mechanism

Pigment epithelium-derived factor (PEDF), a member of the serine protease inhibitor (SERPIN) superfamily, displays a potent antiangiogenic and antimetastatic activity in a broad range of tumor types. Melanocytes and low aggressive melanoma cells secrete high levels of PEDF, while its expression is lost in highly aggressive melanomas. PEDF efficiently abrogates a number of functional properties critical for the acquisition of metastatic ability by melanoma cells, such as neovascularization, proliferation, migration, invasiveness and extravasation. In this study, we identify hypoxia as a relevant negative regulator of PEDF in melanocytes and low aggressive melanoma cells. PEDF was regulated at the protein level. Importantly, although downregulation of PEDF was induced by inhibition of 2-oxoglutarate-dependent dioxygenases, it was independent of the hypoxia inducible factor (HIF), a key mediator of the adaptation to hypoxia. Decreased PEDF protein was not mediated by inhibition of translation through untranslated regions (UTRs) in melanoma cells. Degradation by metalloproteinases, implicated on PEDF degradation in retinal pigment epithelial cells, or by the proteasome, was also excluded as regulatory mechanism in melanoma cells. Instead, we found that degradation by autophagy was critical for PEDF downregulation under hypoxia in human melanoma cells. Our findings show that hypoxic conditions encountered during primary melanoma growth downregulate antiangiogenic and antimetastasic PEDF by a posttranslational mechanism involving degradation by autophagy and could therefore contribute to the acquisition of highly metastatic potential characteristic of aggressive melanoma cells

UTRs are not required for PEDF downregulation by hypoxia in SBcl2 melanoma.

<p>Western blot analysis from SBcl2-pCEP4 and SBcl2-pCEP4-PEDF melanoma cell lines incubated with 1 mM DMOG or under hypoxia (1% O₂): (A) extracellular PEDF (PEDF_e) and Penta-HIS (5-HIS) protein levels in 24 h conditioned medium (CM) and (B) intracellular PEDF (PEDF_i) and HIF1α protein levels in whole-cells extract. β-tubulin was used as loading control. (C) Western blot analysis of PEDF_e protein levels in 24 h CM, PEDF_i and HIF1α protein levels in whole-cell extracts from SBcl2 melanoma cell line incubated under normoxia (21% O₂) or hypoxia (1% O₂). β-tubulin was used as loading control. (D) UTR-reporter assay in SBcl2 melanoma cell line transfected with a Renilla promoter reporter containing the 3′ UTR of PEDF (psiCHECK2-3′PEDF), the 3′UTR of GAPDH (psiCHECK2-3′GAPDH) or an empty reporter (psiCHECK2) and incubated under normoxia or hypoxia for 24 h. Renilla activity was normalized to luciferase activity, which is used as an internal control of transfection efficiency. psiCHECK2-3′GAPDH was used as a negative control. Bars represent average ± standard deviation (SD).</p

Hypoxia-induced downregulation of PEDF in melanocytes and SBcl2 melanoma cells is not mediated by metalloproteinases or proteasomal degradation.

<p>(A) Western blot analysis of extracellular PEDF (PEDF_e) protein levels in 24 h conditioned medium (CM) from M330 primary melanocytes (upper blot) and SBcl2 melanoma cell line (lower blot). Cells were treated with 1 mM DMOG for 24 h and the CM were incubated with 100 ng human recombinant PEDF (rhuPEDF) and 20 mM EDTA at 37°C for 2 h. (B) Western blot analysis of PEDF_e protein levels in CM from SBcl2 melanoma cell line treated with metalloproteinase inhibitor GM6001 (10 µM) and incubated under normoxia (21% O₂) or hypoxia (1% O₂) for 24 h. (C) Western blot analysis of PEDF_e protein levels in 16 h CM and HIF1α protein levels in whole-cell extracts from SBcl2 and WM164 melanoma cell lines after treatment with the proteasome inhibitor MG132 (5 µM and 1 µM respectively) under normoxia or hypoxia. β-tubulin was used as loading control.</p

LC3 knock-down prevents downregulation of PEDF by hypoxia in melanoma cells.

<p>(A) Transduction efficiency of SBcl2 melanoma cell line after infection with non-silencing (shNS) or shRNA^mir to LC3 (shLC3) lentivirus at multiplicity of infection of 60. Fluorescence images (20× magnification) show more than 90% GFP-positive cells. (B) Quantitative RT-PCR analysis of <i>LC3</i> mRNA levels in SBcl2-shNS and SBcl2-shLC3 melanoma cell lines. <i>LC3</i> mRNA levels are shown relative to SBcl2-shNS after normalization to <i>18s rRNA</i>. Bars represent average ± standard deviation (SD) (***<i>P</i><0.001). (C) Western blot analysis of extracellular PEDF (PEDF_e) protein levels in conditioned medium (CM), intracellular PEDF (PEDF_i), HIF1α and LC3 protein levels in whole-cell extracts from SBcl2-shNS and SBcl2-shLC3 melanoma cells incubated under normoxia (21% O₂) or hypoxia (1% O₂) for 24 h. β-tubulin was used as loading control.</p

Hypoxia-induced downregulation of PEDF is HIF-independent in melanocytes and SBcl2 melanoma.

<p>(A) Transduction efficiency of M13 primary melanocytes (left panels) and SBcl2 melanoma cell line (right panels) after infection with non-silencing (shNS) or shRNA^mir to HIF1α (shHIF1α) lentivirus at multiplicity of infection of 40 (M13) or 60 (SBcl2). Fluorescence images (40× magnification) show more than 90% GFP-positive cells. (B) Quantitative RT-PCR analysis of <i>HIF1α</i> mRNA levels in M13-shNS, M13-shHIF1α primary melanocytes and SBcl2-shNS, SBcl2-shHIF1α melanoma cell lines. <i>HIF1α</i> mRNA levels are shown relative to control shNS cells after normalization to <i>18s</i> rRNA. Bars represent average ± standard deviation (SD) (**<i>P</i><0.01; ***<i>P</i><0.001). (C) Western blot analysis of extracellular PEDF (PEDF_e) protein levels in conditioned medium (CM), intracellular PEDF (PEDF_i) and HIF1α protein levels in whole-cell extracts from M13-shNS and M13-shHIF1α primary melanocytes incubated under normoxia (21% O₂) or hypoxia (1% O₂) for 16 h and 24 h. β-tubulin was used as loading control. (D) Quantitative RT-PCR analysis of <i>VEGF</i> (left panel) and <i>BNIP3</i> (right panel) mRNA levels in M13-shNS (filled bars) and M13-shHIF1α (empty bars) primary melanocytes. <i>VEGF</i> and <i>BNIP3</i> mRNA levels are shown relative to M13-shNS under normoxia after normalization to <i>18s</i> rRNA. Bars represent average ± SD (*<i>P</i><0.05; ***<i>P</i><0.001). (E) Western blot analysis of PEDF_e protein levels in CM, PEDF_i and HIF1α protein levels in whole-cell extracts from SBcl2-shNS and SBcl2-shHIF1α melanoma cell lines incubated under normoxia or hypoxia for 16 h and 24 h. β-actin was used as loading control. (F) Quantitative RT-PCR analysis of <i>VEGF</i> (left panel) and <i>BNIP3</i> (right panel) mRNA levels in SBcl2-shNS (filled bars) and SBcl2-shHIF1α (empty bars) melanoma cell lines. <i>VEGF</i> and <i>BNIP3</i> mRNA levels are shown relative to SBcl2-shNS under normoxia after normalization to <i>18s</i> rRNA. Bars represent average ± SD (**<i>P</i><0.01; ***<i>P</i><0.001).</p

Hypoxia downregulates PEDF at the protein level in melanocytes and human melanoma cell lines.

<p>Western blot analysis of (A) extracellular PEDF (PEDF_e) protein levels in conditioned medium (CM) and (B) HIF2α protein levels in whole-cell extracts (B) from M330 primary melanocytes incubated under normoxia (21% O₂) or hypoxia (1% O₂) for 12 h or 24 h. β-tubulin was used as loading control. Quantitative RT-PCR analysis of (C) <i>PEDF</i> mRNA levels and (D) <i>VEGF</i> mRNA levels in M330 primary melanocytes incubated in normoxia or hypoxia for 12 h or 24 h. <i>PEDF</i> and <i>VEGF</i> mRNA levels are shown relative to cells in normoxia after normalization to <i>β-actin</i>. Bars represent average ± standard deviation (SD) (**<i>P</i><0.01). (E) Western blot analysis of PEDF_e protein levels in CM and HIF1α in whole-cell extracts from M438 primary melanocytes, SBcl2 and WM164 melanoma cell lines incubated in normoxia or hypoxia for 12 h or 24 h. β-actin was used as loading control. Quantitative RT-PCR analysis of (F) <i>PEDF</i> mRNA levels and (G) <i>VEGF</i> mRNA levels in M438 primary melanocytes, SBcl2 and WM164 melanoma cell lines incubated under normoxia or hypoxia for 12 h or 24 h. <i>PEDF</i> and <i>VEGF</i> mRNA levels are shown relative to normoxia after normalization to <i>β-actin</i>. Bars represent average ± SD (**<i>P</i><0.01; ***<i>P</i><0.001).</p