Emerging Roles of DDB2 in Cancer.

Damage-specific DNA-binding protein 2 (DDB2) was originally identified as a DNA damage recognition factor that facilitates global genomic nucleotide excision repair (GG-NER) in human cells. DDB2 also contributes to other essential biological processes such as chromatin remodeling, gene transcription, cell cycle regulation, and protein decay. Recently, the potential of DDB2 in the development and progression of various cancers has been described. DDB2 activity occurs at several stages of carcinogenesis including cancer cell proliferation, survival, epithelial to mesenchymal transition, migration and invasion, angiogenesis, and cancer stem cell formation. In this review, we focus on the current state of scientific knowledge regarding DDB2 biological effects in tumor development and the underlying molecular mechanisms. We also provide insights into the clinical consequences of DDB2 activity in cancers.

Newly identified tumor suppressor functions of ING proteins.

The INhibitor of Growth (ING) proteins (ING1, ING2, ING3, ING4 and ING5) are a family of epigenetic regulators. Their decreased expression in numerous cancers led to identifying the ING proteins as gatekeeper tumor suppressors as they regulate cell cycle progression, apoptosis and senescence. Subsequently, they were also described as caretaker tumor suppressors through their involvement in DNA replication and the DNA damage response (DDR). Recent studies have identified new interactions of the ING proteins with proteins or pathways implicated in cell proliferation, the maintenance of stem cells pluripotency or the DDR. Furthermore, the ING proteins have been identified as regulators of ribosomal RNA synthesis and of mRNA stability and as regulators of mitochondrial DNA transcription resulting in the regulation of metabolism. These new findings highlight new antitumorigenic activities of the ING proteins that are potential targets for cancer treatment.

PTEN expression controls cellular response to cetuximab by mediating PI3K/AKT and RAS/RAF/MAPK downstream signaling in KRAS wild-type, hormone refractory prostate cancer cells.

Overexpression of epidermal growth factor receptor (EGFR) and mutation of pten tumor suppressor gene in human cancer cells leads to activated EGFR downstream signaling including PI3-kinase/AKT (PI3K/AKT) and/or mitogen-activated protein kinases (RAS/RAF/MAPK) and have been linked to resistance to anti-EGFR targeted therapies. Cetuximab is a chimeric IgG1 monoclonal antibody that binds the EGFR with high specificity and have been developed as promising therapeutic anticancer treatments in several solid tumors, including colorectal and head and neck squamous cell carcinomas. Cetuximab activity is related to PI3K/AKT and RAS/RAF/MAPK signaling pathways functionality and its activity has been shown to be higher in wild-type KRAS tumors. To study the influence of PTEN expression on cell response to cetuximab, we used wild-type KRAS, PTEN-null, EGFR overexpressing PC3 prostate cancer cells. Reintroduction of PTEN significantly reduced the constitutive overexpression of phosphorylated-AKT (p-AKT) and downstream kinases (p-GSK3beta and p-P70S6 kinase) as well as phosphorylated-ERK1/2 (p-ERK1/2) and consequently significantly restored cetuximab-induced cell growth inhibition and apoptosis induction. Taken together, the results achieved in the present study show that PTEN controls the cellular response to cetuximab in KRAS wild-type prostate carcinoma PC3 cells through the regulation of AKT phosphorylation and restoration of the functionality of EGFR downstream signaling. Extrapolation of these findings to clinical situation, suggests that the assessment of EGFR downstream signaling functionality could be proposed as a diagnostic response predictive marker for anti-EGFR targeted therapies.

Immunoselection and characterization of a human genomic PPAR binding fragment located within POTE genes.

Peroxisome proliferator-activated receptors (PPARs) are ligand-inducible transcription factors and belong to the nuclear hormone receptor superfamily. They form heterodimers with retinoid X receptor (RXR) and bind to specific PPAR-response elements. To identify novel PPAR target genes, we developed an affinity method to isolate human genomic fragments containing binding sites for PPARs. For this, an antibody raised against all PPAR subtypes was used. Immunoselected fragments were amplified and sequenced. One of them, ISF1029, was mapped by BLAT and BLAST searches on different human chromosomes, downstream of several POTE genes. ISF1029 contained three hexamers strongly related to the AGGTCA motif organized according to a DR0/3 motif. The latter was found to bind to PPARalpha in gel mobility shift and supershift assays and to exhibit a downregulation potentiality in transfection experiments under clofibrate treatment. POTE genes were shown to be highly expressed in human Caco-2 colorectal adenocarcinoma cells and downregulated by fenofibrate and 9-cis-retinoic acid, as attested by RT-PCR assays. Microarray analysis confirmed and extended to the human T98-G glioblastoma cells, the downregulation of several POTE genes expression by Wy-14,643, a potent PPARalpha activator. Our data provide new insights about the pleiotropic action of PPARs.

[PPARs and cell-cell or cell-extracellular matrix interactions]. / PPAR et interactions des cellules entre elles ou avec la matrice extracellulaire.

The peroxisome proliferator-activated receptors (PPARs) are transcription factors and belong to the superfamily of nuclear receptors. They are encoded by three genes located on different chromosomes: PPARalpha, PPARbeta/delta and PPARgamma. PPARalpha plays a key role in the control of lipid metabolism and homeostasis. PPARbeta/delta is expressed ubiquitously and participates in skeletal muscle physiology. PPARbeta/delta and PPARgamma are important factors for placental development and function as well as for embryo implantation. In addition, PPARgamma is mainly involved in adipogenesis. PPARs also participate more or less to cell proliferation, differentiation and apoptosis. Surprisingly, the involvement of these transcription factors in cell-cell and/or cell-matrix interactions has not yet been reviewed except for their role as therapeutic agents in inflammation. Nevertheless, several pioneer reports have recently provided some new insights in this research field, by suggesting that PPARs are involved, directly or indirectly, in these interactions and that their activation by specific ligands may lead to potential therapeutic approaches.

Stimulation of mitochondrial activity by p43 overexpression induces human dermal fibroblast transformation.

Mitochondrial dysfunctions are frequently reported in cancer cells, but their direct involvement in tumorigenesis remains unclear. To understand this relation, we stimulated mitochondrial activity by overexpression of the mitochondrial triiodothyronine receptor (p43) in human dermal fibroblasts. In all clones, this stimulation induced morphologic changes and cell fusion in myotube-like structures associated with the expression of several muscle-specific genes (Myf5, desmin, connectin, myosin, AchRalpha). In addition, these clones displayed all the in vivo and in vitro features of cell transformation. This phenotype was related to an increase in c-Jun and c-Fos expression and extinction of tumor suppressor gene expression (p53, p21WAF1, Rb3). Lastly, reactive oxygen species (ROS) production was increased in positive correlation to the stimulation of mitochondrial activity. The direct involvement of mitochondrial activity in this cell behavior was studied by adding chloramphenicol, an inhibitor of mitochondrial protein synthesis, to the culture medium. This inhibition resulted in partial restoration of the normal phenotype, with the loss of the ability to fuse, a strong decrease in muscle-specific gene expression, and potent inhibition of the transformed phenotype. However, expression of tumor suppressor genes was not restored. Similar results were obtained by using N-acetylcysteine, an inhibitor of ROS production. These data indicate that stimulation of mitochondrial activity in human dermal fibroblasts induces cell transformation through events involving ROS production.

Effects of PPAR and RXR ligands in semaphorin 6B gene expression of human MCF-7 breast cancer cells.

This study tests the hypothesis that the activators of peroxisome proliferator-activated receptors (PPARs) and 9-cis-retinoic acid receptor (RXR) regulate human semaphorin 6B (Sema6B) gene expression. The human MCF-7 breast adenocarcinoma cell line was chosen because it expresses Sema6B at a high level. The Sema6B mRNA level was analyzed by RT-PCR and the semaphorin 6B protein content was determined using a polyclonal antibody that we have produced and characterized. Treatments with fenofibrate (a PPARalpha activator) and troglitazone (a PPARgamma ligand) strongly decreased the Sema6B mRNA. The drop in Sema6B mRNA level and in protein content was more important when the treatment combined the action of fenofibrate or troglitazone and 9-cis-retinoic acid. On the other hand, no significant change was observed in the Sema6B mRNA and protein levels when the cells were exposed to the combined action of GW610742 (a PPARbeta activator) and 9-cis-retinoic acid. These data suggest that PPARalpha/RXR and PPARgamma/RXR heterodimers are involved in the regulation of Sema6B gene expression and open new perspectives concerning the participation of these nuclear receptors in cell recognition and migration.

DDB2 (damaged-DNA binding 2) protein: a new modulator of nanomechanical properties and cell adhesion of breast cancer cells.

DDB2, known for its role in DNA repair, was recently shown to reduce mammary tumor invasiveness by inducing the transcription of IκBα, an inhibitor of NF-κB activity. Since cellular adhesion is a key event during the epithelial to mesenchymal transition (EMT) leading to the invasive capacities of breast tumor cells, the aim of this study was to investigate the role of DDB2 in this process. Thus, using low and high DDB2-expressing MDA-MB231 and MCF7 cells, respectively, in which DDB2 expression was modulated experimentally, we showed that DDB2 overexpression was associated with a decrease of adhesion abilities on glass and plastic areas of breast cancer cells. Then, we investigated cell nanomechanical properties by atomic force microscopy (AFM). Our results revealed significant changes in the Young's Modulus value and the adhesion force in MDA-MB231 and MCF7 cells, whether DDB2 was expressed or not. The cell stiffness decrease observed in MDA-MB231 and MCF7 expressing DDB2 was correlated with a loss of the cortical actin-cytoskeleton staining. To understand how DDB2 regulates these processes, an adhesion-related gene PCR-Array was performed. Several adhesion-related genes were differentially expressed according to DDB2 expression, indicating that important changes are occurring at the molecular level. Thus, this work demonstrates that AFM technology is an important tool to follow cellular changes during tumorigenesis. Moreover, our data revealed that DDB2 is involved in early events occurring during metastatic progression of breast cancer cells and will contribute to define this protein as a new marker of metastatic progression in this type of cancer.

Effect of acute and chronic psychostimulant drugs on redox status, AP-1 activation and pro-enkephalin mRNA in the human astrocyte-like U373 MG cells.

In order to approach the astroglial implication of addictive and neurotoxic processes associated with psychostimulant drug abuse, the effects of amphetamine or cocaine (1-100 microM) on redox status, AP-1 transcription factor and pro-enkephalin, an AP-1 target gene, were investigated in the human astrocyte-like U373 MG cells. We demonstrated an early increase in the generation of radical oxygen species and in the formation of 4-hydroxynonenal-adducts reflecting the pro-oxidant action of both substances. After 1 h or 96 h of treatment, Fos and Jun protein levels were altered and the DNA-binding activity of AP-1 was increased in response to both substances. Using supershift experiments, we observed that the composition of AP-1 dimer differed according to the substance and the duration of treatment. FRA-2 protein represented the main component of the chronic amphetamine- or cocaine-activated complexes, which suggests its relevance in the long-term effects of psychostimulant drugs. Concomitantly, the pro-enkephalin gene was differently regulated by either 6 h or 96 h of treatment. Because astrocytes interact extensively with the neurons in the brain, our data led us to conclude that oxidation-regulated AP-1 target genes may represent one of the molecular mechanisms underlying neuronal adaptation associated with psychostimulant dependence.

Comparison of cytotoxicity induced by hypolipidemic drugs via reactive oxygen species in human and rodent liver cells.

Hypolipidemic drugs (HP drugs) are xenobiotics belonging to the peroxisome proliferator family which are used as pharmaceuticals in the treatment of human hyperlipidemia and hypercholesterolemia. They cause hepatocarcinogenesis in rodents by increasing cell proliferation. One hypothesis is that this hepatocarcinogenic effect is caused by induced oxidative stress resulting from the overproduction of reactive oxygen species (ROS) and from a decreasing antioxidant defense. In addition, ROS play a role in hepatocellular proliferation by activation of NF-kappaB and AP-1, leading to an increase in mitogenic cytokines such as tumor necrosis factor-alpha. No liver cancer incidence has been noted in individuals treated with HP drugs for brief periods of time. However, the observation that old rats and mice are more susceptible than young individuals to the hepatocarcinogenic effect caused by long term exposure to HP drugs raises the question of a potential health risk for the human population. In vitro, HP drugs cause an apoptogenic effect in human hepatocytes. This effect is related to a moderate antioxidant response, dysfunction of mitochondria caused by an overproduction of ROS and release of apoptogenic factors. Finally, the apoptogenic effect of HP drugs is observed in human hepatomas, suggesting a clinical interest of these agents in antitumoral activity.

Interaction of Iron II Complexes with B-DNA. Insights from Molecular Modeling, Spectroscopy, and Cellular Biology.

We report the characterization of the interaction between B-DNA and three terpyridin iron II complexes. Relatively long time-scale molecular dynamics (MD) is used in order to characterize the stable interaction modes. By means of molecular modeling and UV-vis spectroscopy, we prove that they may lead to stable interactions with the DNA duplex. Furthermore, the presence of larger π-conjugated moieties also leads to the appearance of intercalation binding mode. Non-covalent stabilizing interactions between the iron complexes and the DNA are also characterized and evidenced by the analysis of the gradient of the electronic density. Finally, the structural deformations induced on the DNA in the different binding modes are also evidenced. The synthesis and chemical characterization of the three complexes is reported, as well as their absorption spectra in presence of DNA duplexes to prove the interaction with DNA. Finally, their effects on human cell cultures have also been evidenced to further enlighten their biological effects.

Induction of MnSOD gene by arachidonic acid is mediated by reactive oxygen species and p38 MAPK signaling pathway in human HepG2 hepatoma cells.

Metabolism of arachidonic acid (AA) is known to induce in different cell types an oxidative stress via the production of reactive oxygen species. As these latter may be scavenged by antioxidant enzymes as manganese and copper/zinc-dependent superoxide dismutase (MnSOD and Cu/ZnSOD, respectively), we investigated the effects of AA on their expression in human HepG2 hepatoma cells. RT-PCR and Western blot data revealed that AA induced an increase in the MnSOD, but not Cu/ZnSOD, expression at the mRNA and protein levels, respectively. This induction was also marked by an increase in MnSOD activity. The AA-induced MnSOD expression required de novo transcription as demonstrated by cotreatment of HepG2 cells with AA and actinomycin D. The fact that MnSOD expression was not induced when HepG2 cells were cultured with 5,8,11,14-eicosatetraynoic acid (ETYA), a nonmetabolizable analog of AA, or with different inhibitors of the AA metabolism pathways suggested that the metabolism of AA was required. Further investigations into the mechanisms by which AA induced MnSOD expression showed that superoxide anions released from AA metabolism act as second messengers via a signal-controlling pathway involving protein kinase C and p38 mitogen activated protein kinase (MAPK). These results define a novel role of p38 MAPK dependent-pathway in the regulation of MnSOD gene.

Arachidonic acid activates a functional AP-1 and an inactive NF-kappaB complex in human HepG2 hepatoma cells.

Exogenous arachidonic acid (AA) has been shown to induce the antioxidant manganese superoxide dismutase gene by reactive oxygen species (ROS) derived from AA metabolism and the participation of the p38 mitogen-activated protein kinase (MAPK) pathway in human HepG2 hepatoma cells. The goal of this study was to investigate the effect of AA on the activation of the two redox-sensitive transcription factors AP-1 and NF-kappaB in HepG2 cells. Using electrophoretic mobility shift assays, DNA-binding activities of AP-1 and NF-kappaB were markedly increased in AA-treated HepG2 cells. The c-Jun and c-Fos proteins were identified as components of the AA-induced AP-1 complex and their levels were increased. AA-activated NF-kappaB complex was constituted as a p50 homodimer resulting in a nuclear translocation for this protein only. Moreover, no degradation of IkappaBalpha was observed. These results were contrasted to the interleukin-1beta-activated p50/p65 complex used as a positive control. Using 5,8,11,14-eicosatetraynoic acid and inhibitors of AA metabolism, AP-1 and NF-kappaB activation required the lipoxygenase/cytochrome P450 monooxygenase pathways. In addition, antioxidants inhibited the AA-induced AP-1 and NF-kappaB activation, suggesting a role of ROS released from the AA metabolism. In reporter gene assays, AA induced the transcriptional activity of AP-1 but not that of NF-kappaB. Further investigations showed that the AA-induced transcriptional activity of AP-1 was regulated by protein kinase C and p38 MAPK pathways. These results suggest that the functional AP-1 activated by AA and coupled to that of p38 MAPK pathway may play an important role in response to ROS induced by AA metabolism in HepG2 cells without the involvement of the NF-kappaB pathway.

Decrease of human hepatoma cell growth by arachidonic acid is associated with an accumulation of derived products from lipid peroxidation.

We showed that the metabolism of arachidonic acid (AA) in HepG2 cells generates reactive oxygen species (ROS), which activate the p38 mitogen-activated protein kinase (MAPK) pathway and the redox-sensitive transcription factors AP-1 and NF-kappaB, leading to the induction of the antioxidant manganese superoxide dismutase gene. The present study reports that AA decreases the HepG2 cell growth by 40% and 55% after a treatment for 24 and 48 h, respectively. This effect was blocked by an inhibitor of lipoxygenase/cytochrome P450 monooxygenase pathways and by the antioxidants. In addition, AA induced an oxidative stress, as an accumulation of malondialdehyde (MDA)-modified proteins, resulting to a generation of MDA and H(2)O(2) was observed after 24 h. This AA-induced oxidative stress was associated with the lack of an increase in the H(2)O(2)-degrading enzyme level. In contrast, 5,8,11,14-eicosatetraynoic acid, a nonmetabolizable analog of AA, had not effect. The peroxisome proliferator-activated receptor gamma (PPARgamma) with AA metabolites as ligands was upregulated by the fatty acid but was not involved in the AA effect because its transcriptional activity estimated by reporter gene assays was negatively controlled by p38 MAPK pathway. These findings suggest that the effect of AA on human hepatoma cell growth by inducing an oxidative stress may present a clinical interest in the treatment of the liver cancer.

Clofibric acid down-regulation of metallothionein IIA in HepG2 human hepatoma cells.

Among the different hypotheses advanced to explain the peroxisome proliferator (PP)-induced hepatocarcinogenicity in rodents, one is based on the development of an oxidative stress due to an imbalance in the production of reactive oxygen species that leads to DNA damages and lipid peroxidation. On the other hand, human cells appear to be nonresponsive to PPs. As metallothionein proteins play an important antioxidant role, the aim of the present study was to investigate the expression of metallothionein IA (MTIA) and IIA (MTIIA) in HepG2 human hepatoma cells exposed to clofibric acid. When HepG2 cells were treated for 24 hr with 0.50 or 0.75 mM CA, a significant decrease was observed in MT protein-level determined by Western blotting and in the MTIIA mRNA content analyzed by RT-PCR and Northern blotting. No significant change was observed in the MTIA mRNA amount whatever the CA concentration and the duration of treatment. The decrease in MTIIA mRNA-level was not mediated via peroxisome proliferator-activated receptor alpha as attested by our data from gel mobility shift DNA binding assays, Dot blotting and cotransfection experiments with MTIIA promoter-driven luciferase reporter gene and PPARalpha expression vector. These results provide new insights about the pleiotropic effects of PPs on human cells.

Induction of the expression of the peroxisome proliferator-activated receptor alpha (PPARalpha) by clofibrate in jerboa tissues.

Peroxisome proliferator-activated receptor alpha (PPARalpha) is a member of the nuclear hormone receptor superfamily that can be activated by natural fatty acids and various xenobiotics, including clofibrate. This transcription factor primarily regulates genes involved in lipid metabolism and homeostasis. We present the expression pattern of the PPARalpha subtype in the adult jerboa Jaculus orientalis, determined by RT-PCR and Western blotting using specific probes and a polyclonal antibody for PPARalpha, respectively. PPARalpha is highly expressed in liver and kidney, and to a lesser extent in duodenum and colon. PPARalpha expression is increased at the mRNA and protein levels in liver and duodenum of jerboa treated for 2 weeks with the peroxisome proliferator (PP) clofibrate. The induction is tissue-specific as no significant changes are observed in kidney and colon. The present data indicate that the PP-induced PPARalpha gene expression is not dependent on the PPARalpha content in target cells.

Manganese superoxide dismutase in breast cancer: from molecular mechanisms of gene regulation to biological and clinical significance.

Breast cancer is one of the most common malignancies of all cancers in women worldwide. Many difficulties reside in the prediction of tumor metastatic progression because of the lack of sufficiently reliable predictive biological markers, and this is a permanent preoccupation for clinicians. Manganese superoxide dismutase (MnSOD) may represent a rational candidate as a predictive biomarker of breast tumor metastatic progression, because its gene expression is profoundly altered between early and advanced breast cancer, in contrast to expression in the normal mammary gland. In this review, we report the characterization of some gene polymorphisms and molecular mechanisms of SOD2 gene regulation, which allows a better understanding of how MnSOD is decreased in early breast cancer and increased in advanced breast cancer. Several studies display the biological significance of MnSOD level in proliferation as well as in invasive and angiogenic abilities of breast tumor cells by controlling superoxide anion radical (O2(•-)) and hydrogen peroxide (H2O2). Particularly, they report how these reactive oxygen species may activate some signaling pathways involved in breast tumor growth. Emerging understanding of these findings provides an interesting framework for guiding translational research and suggests a way to define precisely the clinical interest of MnSOD as a prognostic and/or predicting marker in breast cancer, by associating with some regulators involved in SOD2 gene regulation and other well-known biomarkers, in addition to the typical clinical parameters.

DDB2: a novel regulator of NF-κB and breast tumor invasion.

The DNA repair protein damaged DNA-binding 2 (DDB2) has been implicated in promoting cell-cycle progression by regulating gene expression. DDB2 is selectively overexpressed in breast tumor cells that are noninvasive, but not in those that are invasive. We found that its overexpression in invasive human breast tumor cells limited their motility and invasiveness in vitro and blocked their ability to colonize lungs in vivo, defining a new function for DDB2 in malignant progression. DDB2 overexpression attenuated the activity of NF-κB and the expression of its target matrix metalloprotease 9 (MMP9). Mechanistic investigations indicated that DDB2 decreased NF-κB activity by upregulating expression of IκBα by binding the proximal promoter of this gene. This effect was causally linked to invasive capacity. Indeed, knockdown of DDB2-induced IκBα gene expression restored NF-κB activity and MMP9 expression, along with the invasive properties of breast tumor cells overexpressing DDB2. Taken together, our findings enlighten understanding of how breast cancer cells progress to an invasive phenotype and underscore potential clinical interest in DDB2 as a prognostic marker or therapeutic target in this setting.

Regulation of the high basal expression of the manganese superoxide dismutase gene in aggressive breast cancer cells.

A high basal expression of manganese superoxide dismutase (MnSOD) has been reported in aggressive breast cancer cells, according to an unknown mechanism, and contributes to their invasive abilities. Here, we report the involvement of Sp1 and nuclear factor-κB (NF-κB) transcription factors in this high basal expression of MnSOD in aggressive breast cancer cells. Suppression or inactivation of Sp1 showed that it plays an essential role in the high MnSOD expression in aggressive breast cancer cells through a unique binding site identified by chromatin immunoprecipitation (ChIP) assay and functional analysis of the MnSOD proximal promoter. Treatment of cells with a specific NF-κB inhibitor peptide decreased significantly high basal MnSOD expression. A ChIP assay showed binding of a constitutive p50/p65 NF-κB complex to the MnSOD intronic enhancer element, associated with hyperacetylation of the H3 histone. Finally, high basal expression of MnSOD resulted in the lack of expression of Damaged DNA binding 2 (DDB2) protein in aggressive breast cancer cells. DDB2 overexpression prevented the binding of Sp1 as well as of NF-κB to their respective elements on the MnSOD gene. These results contribute to a better understanding of MnSOD up-regulation, which may be clinically important in the prediction of breast tumor progression.

Photodynamic therapy targeting neuropilin-1: Interest of pseudopeptides with improved stability properties.

The general strategy developed aims to favor the vascular effect of photodynamic therapy by targeting tumor vasculature. Since angiogenic endothelial cells represent an interesting target to potentiate this vascular effect, we previously described the conjugation of a photosensitizer to a peptide targeting neuropilins (NRPs) over-expressed specially in tumor angiogenic vessels and we recently characterized the mechanism of photosensitization-induced thrombogenic events. Nevertheless, in glioma-bearing nude mice, we demonstrated that the peptide moiety was degraded to various rates according to time after intravenous administration. In this study, new peptidases-resistant pseudopeptides were tested, demonstrating a molecular affinity for NRP-1 and NRP-2 recombinant chimeric proteins and devoid of affinity for VEGF receptor type 1 (Flt-1). To argue the involvement of NRP-1, MDA-MB-231 breast cancer cells were used, strongly over-expressing NRP-1 receptor. We evidenced a statistically significant decrease of the different peptides-conjugated photosensitizers uptake after RNA interference-mediated silencing of NRP-1. Peptides-conjugated photosensitizers allowed a selective accumulation into cells. In mice, no degradation was observed in plasma in vivo 4h after intravenous injection by MALDI-TOF mass spectrometry. This study draws attention to this potential problem with peptides, especially in the case of targeting strategies, and provides useful information for the future design of more stable molecules.