Targeting hypoxia signaling pathways in angiogenesis.

Oxygen (O2) supply is constantly maintained by the vascular network for a proper tissue oxygenation. Hypoxia is the result of an increased O2 demand and/or decreased supply and is common in both physiological conditions and human diseases. Angiogenesis is one of the adaptive responses to hypoxia and is mainly regulated by the hypoxia-inducible factors, HIFs. These heterodimeric transcription factors are composed of one of three O2-dependent α subunits (HIF-1, HIF-2, and HIF-3) and a constitutively expressed O2-insensitive subunit (HIF-1ß). Among them HIF-1α is the most characterized and its activity is tightly controlled. Under hypoxia, its intracellular accumulation triggers the transcription of several genes, involved in cell survival/proliferation, autophagy, apoptosis, cell metabolism, and angiogenesis. HIF pathway is also modulated by specific microRNAs (miRNAs), thus resulting in the variation of several cellular responses, including alteration of the angiogenic process. The pro-angiogenic activity of HIF-1α is not restricted to endothelial cells, as it also affects the behavior of other cell types, including tumor and inflammatory/immune cells. In this context, exosomes play a crucial role in cell-cell communication by transferring bio-active cargos such as mRNAs, miRNAs, and proteins (e.g., VEGFA mRNA, miR210, HIF-1α). This minireview will provide a synopsis of the multiple factors able to modulate hypoxia-induced angiogenesis especially in the tumor microenvironment context. Targeting hypoxia signaling pathways by up-to-date approaches may be relevant in the design of therapeutic strategies in those pathologies where angiogenesis is dysregulated.

Hedgehog Pathway Inhibition by Novel Small Molecules Impairs Melanoma Cell Migration and Invasion under Hypoxia.

Melanoma is the principal cause of death in skin cancer due to its ability to invade and cause metastasis. Hypoxia, which characterises the tumour microenvironment (TME), plays an important role in melanoma development, as cancer cells can adapt and acquire a more aggressive phenotype. Carbonic anhydrases (CA) activity, involved in pH regulation, is related to melanoma cell migration and invasion. Furthermore, the Hedgehog (Hh) pathway, already known for its role in physiological processes, is a pivotal character in cancer cell growth and can represent a promising pharmacological target. In this study, we targeted Hh pathway components with cyclopamine, glabrescione B and C22 in order to observe their effect on carbonic anhydrase XII (CAXII) expression especially under hypoxia. We then performed a migration and invasion assay on two melanoma cell lines (SK-MEL-28 and A375) where Smoothened, the upstream protein involved in Hh regulation, and GLI1, the main transcription factor that determines Hh pathway activation, were chemically inhibited. Data suggest the existence of a relationship between CAXII, hypoxia and the Hedgehog pathway demonstrating that the chemical inhibition of the Hh pathway and CAXII reduction resulted in melanoma migration and invasion impairment especially under hypoxia. As in recent years drug resistance to small molecules has arisen, the development of new chemical compounds is crucial. The multitarget Hh inhibitor C22 proved to be effective without signs of cytotoxicity and, for this reason, it can represent a promising compound for future studies, with the aim to reach a better melanoma disease management.

SQSTM1/p62 inhibition impairs pro-survival signaling in hypoxic human dendritic cells.

The sequestosome 1 (SQSTM1)/p62 is an adaptor protein which plays multiple roles in several cell functions, including cell survival and autophagy. Dendritic cells (DCs) are the most prominent antigen presenting cells and during their lifespan they are exposed to different oxygen tensions, including hypoxia. By using a siRNA approach we found out that p62 was implicated in the maintenance of Erk1/2 phosphorylation and preservation of hypoxic DC survival, as well as in the reduction of AMPK activation. Thus, p62 expression in DCs in hypoxic microenvironments, such as in the lymphoid organs, may extend their lifespan to ensure their functions.

Role of the Hedgehog Pathway and CAXII in Controlling Melanoma Cell Migration and Invasion in Hypoxia.

BACKGROUND: Malignant melanoma is the leading cause of death among skin cancer patients due to its tendency to metastasize. Alterations at the molecular level are often evident, which is why melanoma biology has garnered increasing interest. The hedgehog (Hh) pathway, which is essential for embryonic development, is aberrantly re-activated in melanoma and may represent a promising therapeutic target. In addition, carbonic anhydrase XII (CAXII) represents a poor prognostic target for hypoxic tumors, such as melanoma, and is involved in cell migration. Thus, we decided to investigate whether and how the Hh pathway and CAXII may control melanoma cell migration and invasiveness. METHODS: The migratory and invasive capabilities of SK-MEL-28 and A375 cell lines, either un-transfected or transiently transfected with Smoothened (SMO), GLI1, or CAXII siRNA, were studied under normoxic or hypoxic conditions. RESULTS: For the first time, we showed that SMO and GLI1 silencing resulted in the downregulation of CAXII expression in both moderately and highly invasive melanoma cells under hypoxia. The Hh pathway as well as CAXII inhibition by siRNA resulted in impaired malignant melanoma migration and invasion. CONCLUSION: Our results suggest that CAXII and the Hh pathway are relevant in melanoma invasion and may be novel and promising therapeutical targets for melanoma clinical management.

Mouse dendritic cells in the steady state: Hypoxia, autophagy, and stem cell factor.

Dendritic cells (DCs) are innate immune cells with a central role in immunity and tolerance. Under steady-state, DCs are scattered in tissues as resting cells. Upon infection or injury, DCs get activated and acquire the full capacity to prime antigen-specific CD4+ and CD8+ T cells, thus bridging innate and adaptive immunity. By secreting different sets of cytokines and chemokines, DCs orchestrate diverse types of immune responses, from a classical proinflammatory to an alternative pro-repair one. DCs are highly heterogeneous, and physiological differences in tissue microenvironments greatly contribute to variations in DC phenotype. Oxygen tension is normally low in some lymphoid areas, including bone marrow (BM) hematopoietic niches; nevertheless, the possible impact of tissue hypoxia on DC physiology has been poorly investigated. We assessed whether DCs are hypoxic in BM and spleen, by staining for hypoxia-inducible-factor-1α subunit (HIF-1α), the master regulator of hypoxia-induced response, and pimonidazole (PIM), a hypoxic marker, and by flow cytometric analysis. Indeed, we observed that mouse DCs have a hypoxic phenotype in spleen and BM, and showed some remarkable differences between DC subsets. Notably, DCs expressing membrane c-kit, the receptor for stem cell factor (SCF), had a higher PIM median fluorescence intensity (MFI) than c-kit- DCs, both in the spleen and in the BM. To determine whether SCF (a.k.a. kit ligand) has a role in DC hypoxia, we evaluated molecular pathways activated by SCF in c-kit+ BM-derived DCs cultured in hypoxic conditions. Gene expression microarrays and gene set enrichment analysis supported the hypothesis that SCF had an impact on hypoxia response and inhibited autophagy-related gene sets. Our results suggest that hypoxic response and autophagy, and their modulation by SCF, can play a role in DC homeostasis at the steady state, in agreement with our previous findings on SCF's role in DC survival.

Hypoxia Induces Autophagy in Human Dendritic Cells: Involvement of Class III PI3K/Vps34.

Hypoxia is a component of both physiological and pathological conditions, including inflammation, solid tumors, and lymphoid tissues, where O2 demand is not balanced by O2 supply. During their lifespan, dendritic cells (DCs) are exposed to different pO2 and activate different adaptive responses, including autophagy, to preserve their viability and functions. Autophagy plays multiple roles in DC physiology. Very recently, we demonstrated that hypoxia shapes autophagy in DCs upon their differentiation state. Here, we proposed a role for PI3Ks, and especially class III PI3K/Vps34, that could be relevant in hypoxia-induced autophagy, in either immature or mature DCs. Hypoxia inhibited mTOR phosphorylation and activated a pro-autophagic program. By using different pharmacological inhibitors, we demonstrated that hypoxia-induced autophagy was mediated by PI3Ks, especially by Vps34. Furthermore, Vps34 expression was enhanced by LPS, a TLR4 ligand, along with the promotion of autophagy under hypoxia. The Vps34 inhibitor, SAR405, abolished hypoxia-induced autophagy, inhibited pro-survival signaling and viability, and increased the expression of proinflammatory cytokines. Our results underlined the impact of autophagy in the maintenance of DC homeostasis at both cell survival and inflammatory response levels, therefore, contributing to a better understanding of the significance of autophagy in DC physiology and pathology.

Hypoxia Shapes Autophagy in LPS-Activated Dendritic Cells.

During their lifespan, dendritic cells (DCs) are exposed to different pO2 levels that affect their differentiation and functions. Autophagy is one of the adaptive responses to hypoxia with important implications for cell survival. While the autophagic machinery in DCs was shown to impact signaling of TLRs, its regulation by the MD-2/TLR4 ligand LPS is still unclear. The aim of this study was to evaluate whether LPS can induce autophagy in DCs exposed to either aerobic or hypoxic conditions. Using human monocyte-derived DCs and the combination of immunofluorescence confocal analysis, measure of mitochondrial membrane potential, Western blotting, and RT-qPCR, we showed that the ability of LPS to modulate autophagy was strictly dependent upon pO2 levels. Indeed, LPS inhibited autophagy in aerobic conditions whereas the autophagic process was induced in a hypoxic environment. Under hypoxia, LPS treatment caused a significant increase of functional lysosomes, LC3B and Atg protein upregulation, and reduction of SQSTM1/p62 protein levels. This selective regulation was accompanied by activation of signalling pathways and expression of cytokines typically associated with DC survival. Bafilomycin A1 and chloroquine, which are recognized as autophagic inhibitors, confirmed the induction of autophagy by LPS under hypoxia and its impact on DC survival. In conclusion, our results show that autophagy represents one of the mechanisms by which the activation of the MD-2/TLR4 ligand LPS promotes DC survival under hypoxic conditions.

The Shc protein Rai enhances T-cell survival under hypoxia.

Hypoxia occurs in physiological and pathological conditions. T cells experience hypoxia in pathological and physiological conditions as well as in lymphoid organs. Indeed, hypoxia-inducible factor 1α (HIF-1α) affects T cell survival and functions. Rai, an Shc family protein member, exerts pro-survival effects in hypoxic neuroblastoma cells. Since Rai is also expressed in T cells, we here investigated its role in hypoxic T cells. In this work, hypoxia differently affected cell survival, proapoptotic, and metabolic programs in T cells, depending upon Rai expression. By using Jurkat cells stably expressing Rai and splenocytes from Rai-/- mice, we demonstrated that Rai promotes T cell survival and affects cell metabolism under hypoxia. Upon exposure to hypoxia, Jurkat T cells expressing Rai show (a) higher HIF-1α protein levels; (b) a decreased cell death and increased Akt/extracellular-signal-regulated kinase phosphorylation; (c) a decreased expression of proapoptotic markers, including caspase activities and poly(ADP-ribose) polymerase cleavage; (d) an increased glucose and lactate metabolism; (e) an increased activation of nuclear factor-kB pathway. The opposite effects were observed in hypoxic splenocytes from Rai-/- mice. Thus, Rai plays an important role in hypoxic signaling and may be relevant in the protection of T cells against hypoxia.

Rhinovirus 2A is the key protease responsible for instigating the early block to gene expression in infected cells.

Human rhinoviruses (HRVs) express 2 cysteine proteases, 2A and 3C, that are responsible for viral polyprotein processing. Both proteases also suppress host gene expression by inhibiting mRNA transcription, nuclear export and cap-dependent translation. However, the relative contribution that each makes in achieving this goal remains unclear. In this study, we have compared both the combined and individual ability of the two proteases to shut down cellular gene expression using a novel dynamic reporter system. Our findings show that 2A inhibits host gene expression much more rapidly than 3C. By comparing the activities of a representative set of proteases from the three different HRV species, we also find variation in the speed at which host gene expression is suppressed. Our work highlights the key role that 2A plays in early suppression of the infected host cell response and shows that this can be influenced by natural variation in the activity of this enzyme.

Inhibition of smoothened in breast cancer cells reduces CAXII expression and cell migration.

Breast cancer (BC) relapse and metastasis are the leading cause of death and, together with drug resistance, keep mortality still high. The Hedgehog (Hh) pathway is expressed during embryogenesis, organogenesis and in adult tissue homeostasis and its aberrant activation is often associated with cancer. Carbonic anhydrase (CA) enzymes are important during development; they play a key role in controlling several cellular mechanisms, such as pH regulation, survival, and migration, and they are aberrantly expressed in cancer. The goal of this study was to investigate the interplay between the Hh pathway and CAXII in terms of BC cell migration. We here demonstrated that smoothened (SMO) silencing resulted in a reduction of CAXII expression at mRNA and protein level. This led to a decrease in cell migration, which was restored when cells were treated with an SMO agonist, Sag dihydrochloride (SAG), but not when cells were cotreated with SAG and the CAs inhibitor Acetazolamide. This suggested that the ability of SAG to promote cell migration was impaired when CAXII was inhibited. The reduction was also confirmed within hypoxic and inflammatory microenvironment, typical of BC, indicating a key role of the Hh pathway in controlling CAXII expression. Our results may contribute to further understand the physiology of BC cells and indicate that the Hh pathway controls BC cell migration and cell invasion also through CAXII, with important implications in identifying novel therapeutic targets.

Different Adaptive Responses to Hypoxia in Normal and Multiple Myeloma Endothelial Cells.

BACKGROUND/AIMS: Hypoxia is a powerful stimulator of angiogenesis under physiological as well as pathological conditions. Normal endothelial cells (EC), such as human umbilical vein EC (HUVEC), are relatively affected by hypoxic insult in terms of cell survival. In contrast, EC from tumors are particularly resistant to hypoxia-induced cell death. Previous reports have shown that EC in bone marrow from multiple myeloma (MM) patients had a hypoxic phenotype, even under normoxic conditions. The aim of this study was to evaluate whether HUVEC and MMEC adapt differently to hypoxia. METHODS: Cell proliferation was assessed by the CyQUANT assay. Cdc25A, p21, Bax, Bcl-xl, BNIP3, glucose transporter (GLUT)-1, monocarboxylate transporter (MCT)-4 and carbonic anhydrase (CA)IX mRNA expression was determined by qRT-PCR. HIF-1α, BNIP3, Beclin-1, LC3B, livin, Bax, Bcl-xl, p21, p62 and ß-actin protein expression was analyzed by western blot. Apoptosis was determined by TUNEL assay. Silencing of BNIP3 was achieved by stealth RNA system technology. RESULTS: While HUVEC survival was reduced after prolonged hypoxic exposure, MMEC were completely unaffected. This difference was also significant in terms of livin, cdc25A and p21 expression. Hypoxia induced apoptosis and inhibited autophagy in HUVEC, but not in MMEC, where hypoxic treatment resulted in a more sustained adaptive response. In fact, MMEC showed a more significant increase in the expression of genes regulated transcriptionally by hypoxia-inducible factor (HIF)-1α. Interestingly, they showed higher expression of BNIP3 than did HUVEC, indicating a more pronounced autophagic (and pro-survival) phenotype. The potential role of BNIP3 in EC survival was confirmed by BNIP3 siRNA experiments in HUVEC, where BNIP3 inhibition resulted in reduced cell survival and increased apoptosis. CONCLUSION: These findings provide further information on how hypoxia may affect EC survival and could be important for a better understanding of EC physiology under normal and pathological conditions, such as in multiple myeloma.

The Shc protein RAI promotes an adaptive cell survival program in hypoxic neuroblastoma cells.

Neuroblastoma (NB) is a highly malignant pediatric solid tumor where a hypoxic signature correlates with unfavorable patient outcome. The hypoxia-inducible factor (HIF)-1α plays an important role in NB progression, contributing to cell proliferation and invasiveness. RAI belongs to the Shc family proteins, it is mainly neuron specific and protects against cerebral ischemia. RAI is also expressed in several NB cell lines, where it promotes cell survival. In this work, hypoxia differently affected cell survival and pro-apoptotic program in two NB cell lines, either expressing RAI (SKNBE) or not (SKNMC). RAI expression appeared to promote NB cell survival and to reduce some pro-apoptotic markers under hypoxia. Accordingly, the RAI silencing in SKNBE cells resulted in a reduction of cell survival and HIF-1α expression. Furthermore, using SKNMC cells stably expressing RAI, we defined a role of RAI in NB cell responses to hypoxia. Of interest, in hypoxic SKNMC cells expressing RAI HIF-1α protein levels were higher than in control cells. This was associated with a) an increased cell survival; b) an increased expression of anti-apoptotic markers; c) a pro-autophagic and not pro-apoptotic phenotype; and d) an increased metabolic activity. We may conclude that RAI plays an important role in hypoxic signaling in NB cells and the interplay between RAI and HIF-1α may be relevant in the protection of NB cells against hypoxia. Our results may contribute to a further understanding the physiology of NB cells and the molecular mechanisms involved in their survival, with important implications in NB progression.

GM-CSF Inhibits c-Kit and SCF Expression by Bone Marrow-Derived Dendritic Cells.

Stem cell factor (SCF), the ligand of c-kit, is a key cytokine for hematopoiesis. Hematopoietic precursors express c-kit, whereas differentiated cells of hematopoietic lineage are negative for this receptor, with the exception of NK cells, mast cells, and a few others. While it has long been recognized that dendritic cells (DCs) can express c-kit, several questions remain concerning the SCF/c-kit axis in DCs. This is particularly relevant for DCs found in those organs wherein SCF is highly expressed, including the bone marrow (BM). We characterized c-kit expression by conventional DCs (cDCs) from BM and demonstrated a higher proportion of c-kit+ cells among type 1 cDC subsets (cDC1s) than type 2 cDC subsets (cDC2s) in both humans and mice, whereas similar levels of c-kit expression were observed in cDC1s and cDC2s from mouse spleen. To further study c-kit regulation, DCs were generated with granulocyte-macrophage colony-stimulating factor (GM-CSF) from mouse BM, a widely used protocol. CD11c+ cells were purified from pooled non-adherent and slightly adherent cells collected after 7 days of culture, thus obtaining highly purified BM-derived DCs (BMdDCs). BMdDCs contained a small fraction of c-kit+ cells, and by replating them for 2 days with GM-CSF, we obtained a homogeneous population of c-kit+ CD40hi MHCIIhi cells. Not only did BMdDCs express c-kit but they also produced SCF, and both were striking upregulated if GM-CSF was omitted after replating. Furthermore, a small but significant reduction in BMdDC survival was observed upon SCF silencing. Incubation of BMdDCs with SCF did not modulate antigen presentation ability of these cells, nor it did regulate their membrane expression of the chemokine receptor CXCR4. We conclude that the SCF/c-kit-mediated prosurvival circuit may have been overlooked because of the prominent use of GM-CSF in DC cultures in vitro, including those human DC cultures destined for the clinics. We speculate that DCs more prominently rely on SCF in vivo in some microenvironments, with potential implications for graft-versus-host disease and antitumor immunity.

Different Expression of Hypoxic and Angiogenic Factors in Human Endometriotic Lesions.

Endometriosis is associated with local angiogenic and hypoxic mechanisms. Indeed, peritoneal fluid of women with endometriosis generates a specific microenvironment to support the growth and development of ectopic endometrial tissues. The association between proangiogenic markers and hypoxic processes in different endometriosis phenotypes was investigated in the present study, analyzing the expression of several genes, related to hypoxic signaling pathway and involved in angiogenic processes, in nonpregnant women with different forms of endometriosis. Samples of ovarian endometrioma (OMA; n = 16) or deep infiltrating endometriosis (DIE; n = 11) were collected, and in addition, control endometrium was collected from healthy women by hysteroscopy. The gene expression of the hypoxia-inducible factors (HIF) 1/2α, protease-activated receptors (PARs) », and vascular endothelial growth factor (VEGF) A was evaluated by quantitative reverse-transcription polymerase chain reaction. Ovarian endometrioma expresses high levels of HIF-1/2α, PAR-1/4, and VEGF-A, while DIE did not show significantly different gene expression compared to endometrium from unaffected women. A positive correlation between the expression of HIF-1/2α and VEGF-A mRNA was observed in OMA. The overall data point out that the heterogeneity of the disease reflects differences in expression levels of genes associated with hypoxia and angiogenesis, suggesting that such conditions may have an active role in the development of the disease.

Interleukin-1ß Affects MDAMB231 Breast Cancer Cell Migration under Hypoxia: Role of HIF-1α and NFκB Transcription Factors.

Inflammation and tumor hypoxia are intimately linked and breast cancer provides a typical example of an inflammation-linked malignant disease. Indeed, breast cancer progression is actively supported by inflammatory components, including IL-1ß, and by the hypoxia-inducible factor- (HIF-) 1α. In spite of many attempts where the role of either IL-1ß or HIF-1α was evaluated, detailed mechanisms for their effects on breast cancer cell migration under hypoxia are still unclear. We here report that IL-1ß increased MDAMB231 cell migration under hypoxic conditions along with HIF-1α accumulation and upregulation of CXCR1, which is transcriptionally regulated by HIF-1α, as well as an increased expression of CXCL8 and NFκB. In addition, IL-1ß-induced cell migration in hypoxia was not affected when HIF-1α was inhibited by either siRNA or Topotecan, well known for its inhibitory effect on HIF-1α. Of interest, HIF-1α inhibition did not reduce NFκB and CXCL8 expression and the reduction of IL-1ß-induced cell migration under hypoxia was achieved only by pharmacological inhibition of NFκB. Our findings indicate that inhibition of HIF-1α does not prevent the migratory program activated by IL-1ß in hypoxic MDAMB231 cells. They also suggest a potential compensatory role of NFκB/CXCL8 pathway in IL-1ß-induced MDAMB231 cell migration in a hypoxic microenvironment.

Short-term hypoxia enhances the migratory capability of dendritic cell through HIF-1α and PI3K/Akt pathway.

Hypoxia represents an inadequate oxygen supply to tissues, which can modulate cell functions, primarily through the hypoxia-inducible transcription factor HIF-1α. Dendritic cells (DC) are professional antigen-presenting cells and their migration maybe affected by hypoxia, since the local microenvironment in lymphoid organs, as well as in inflamed and tumor tissues, is characterized by low oxygen tensions. In this study we observed an enhanced migratory capability of human monocyte-derived DC, using in vitro migration assays performed under hypoxic conditions. Such enhancement was independent on either the chemoattractants involved or the maturation level of DC. However, HIF-1α appeared to be crucial for the migration only of immature DC and not for mature DC under hypoxia, as indicated by HIF-1α siRNA approaches. Furthermore, we observed that while Akt phosphorylation was enhanced in both immature and mature DC exposed to hypoxia, other signaling pathways, such as p38 and p42/p44 MAPK, were differently affected during hypoxic treatment. More interestingly, aspecific and specific inhibition of PI3K/Akt indicated that such pathway was relevant for the migration of both immature and matured DC under hypoxia, even when DC were transfected with HIF-1α siRNA. Our results indicate that, besides HIF-1α, several other pathways, including PI3K/Akt, may be involved in the response to hypoxia of immature and, more specifically, of mature DC to sustain their trafficking and functions within hypoxic microenvironments.

Design, synthesis, and biological evaluation of pyrazolo[3,4-d]pyrimidines active in vivo on the Bcr-Abl T315I mutant.

Starting from our in-house library of pyrazolo[3,4-d]pyrimidines, a cross-docking simulation was conducted on Bcr-Abl T315I mutant. Among the selected compounds (2a-e), the 4-bromo derivative 2b showed the best activity against the Bcr-Abl T315I mutant. Deeper computational studies highlighted the importance of the bromine atom in the para position of the N1 side chain phenyl ring for the interaction with the T315I mutant. A series of 4-bromo derivatives was thus synthesized and biologically evaluated. Compound 2j showed a good balance of different ADME properties, high activity in cell-free assays, and a submicromolar potency against T315I Bcr-Abl expressing cells. In addition, it was converted into a water-soluble formulation by liposome encapsulation, preserving a good activity on leukemic T315I cells and avoiding the use of DMSO as solubilizing agent. In vivo studies on mice inoculated with 32D-T315I cells and treated with 2j showed a more than 50% reduction in tumor volumes.

Downregulation of hypoxia-related responses by novel antitumor histone deacetylase inhibitors in MDAMB231 breast cancer cells.

The tumor microenvironment is characterized by a poor circulation which results in the selection of neoplastic cells that can grow or survive under hypoxic conditions. The relationship between hypoxia and histone deacetylase (HDAC) inhibitors has been previously established. In this work we evaluated the effects of novel HDAC inhibitors (the natural peptide FR235222 and three tetrapeptide analogs) in the human breast cancer cell line MDAMB231, cultured under hypoxia (2% O2 ≉ 14 mmHg) or normoxia (20% O2 ≉ 140 mmHg). First, we found that the novel HDAC inhibitors reduced cell proliferation in MDAMB231 cells at an extent which was similar or even higher than that exerted by the classic HDAC inhibitors trichostatin-A and suberoylanilide hydroxamic acid. More interestingly, the antiproliferative effects of the novel HDAC inhibitors were, in general, significantly higher in hypoxic cells than in normoxic controls. Hypoxic MDAMB231 cells expressed high levels of the hypoxia-inducible factor (HIF)-1α and HIF-1α-related genes, such as vascular endothelial growth factor, Bcl-2/E1B 19 kDa interacting protein-3, glucose transporter-1, carbonic anhydrase IX, as determined by Western blot analysis and qRT-PCR. Finally, we found that HIF-1α and HIF-1α-related genes were significantly downregulated by FR235222 and analogs. In conclusion, the identification of novel effects exerted by the HDAC inhibitors, characterized by a strong efficacy in inhibiting the expression of HIF-1α and its related genes, may have important implications in the pharmacological control of several tumors, including breast cancer, characterized by the presence of hypoxia, angiogenesis and metabolic derangements.

N-[2-Methyl-5-(triazol-1-yl)phenyl]pyrimidin-2-amine as a scaffold for the synthesis of inhibitors of Bcr-Abl.

N-[2-Methyl-5-(triazol-1-yl)phenyl]pyrimidin-2-amine derivatives were synthesized and evaluated in vitro for their potential use as inhibitors of Bcr-Abl. The design is based on the bioisosterism between the 1,2,3-triazole ring and the amide group. The synthesis involves a copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) as the key step, with the exclusive production of anti-(1,4)-triazole derivatives. One of the compounds obtained shows general activity similar to that of imatinib; in particular, it was observed to be more effective in decreasing the fundamental function of cdc25A phosphatases in the K-562 cell line.

Design, synthesis, biological activity, and ADME properties of pyrazolo[3,4-d]pyrimidines active in hypoxic human leukemia cells: a lead optimization study.

A family of dual Src/Abl inhibitors characterized by a substituted pyrazolo[3,4-d]pyrimidine scaffold was previously reported by us and proved to be active against several tumor cell lines. Among these compounds, a promising antileukemia lead (1) has been recently identified, but, unfortunately, it suffers from substandard pharmaceutical properties. Accordingly, an approach for the optimization of the lead 1 is described in the present work. A series of more soluble pyrazolo[3,4-d]pyrimidine derivatives were rationally designed and proved to maintain the dual Src/Abl activity of the lead. Selected compounds showed an interesting activity profile against three different leukemic cells also in hypoxic conditions, which are usually characterized by imatinib-resistance. Finally, in vitro ADME properties (PAMPA permeation, water solubility, microsomal stability) for the most promising inhibitors were also evaluated, thus allowing the identification of a few optimized analogues of lead 1 as promising antileukemia agents.