Andrea Bandino

Redox mechanisms switch on hypoxia-dependent epithelial–mesenchymal transition in cancer cells

Epithelial-mesenchymal transition (EMT) and hypoxia are considered as crucial events favouring invasion and metastasis of many cancer cells. In this study, different human neoplastic cell lines of epithelial origin were exposed to hypoxic conditions in order to investigate whether hypoxia per se may trigger EMT programme as well as to mechanistically elucidate signal transduction mechanisms involved. The following human cancer cell lines were used: HepG2 (from human hepatoblastoma), PANC-1 (from pancreatic carcinoma), HT-29 (from colon carcinoma) and MCF-7 (from breast carcinoma). Cancer cells were exposed to carefully controlled hypoxic conditions and investigated for EMT changes and signal transduction by using morphological, cell and molecular biology techniques. All cancer cells responded to hypoxia within 72 h by classic EMT changes (fibroblastoid phenotype, SNAIL and beta-catenin nuclear translocation and changes in E-cadherin) and by increased migration and invasiveness. This was involving very early inhibition of glycogen synthase kinase-3beta (GSK-3beta), early SNAIL translocation as well as later and long-lasting activation of Wnt/beta-catenin-signalling machinery. Experimental manipulation, including silencing of hypoxia-inducible factor (HIF)-1alpha and the specific inhibition of mitochondrial generation of reactive oxygen species (ROS), revealed that early EMT-related events induced by hypoxia (GSK-3beta inhibition and SNAIL translocation) were dependent on transient intracellular increased generation of ROS whereas late migration and invasiveness were sustained by HIF-1alpha- and vascular endothelial growth factor (VEGF)-dependent mechanisms. These findings indicate that in cancer cells, early redox mechanisms can switch on hypoxia-dependent EMT programme whereas increased invasiveness is sustained by late and HIF-1alpha-dependent release of VEGF.

The biphasic nature of hypoxia-induced directional migration of activated human hepatic stellate cells.

Liver fibrogenesis is sustained by pro‐fibrogenic myofibroblast‐like cells (MFs), mainly originating from activated hepatic stellate cells (HSC/MFs) or portal (myo)fibroblasts, and is favoured by hypoxia‐dependent angiogenesis. Human HSC/MFs were reported to express vascular‐endothelial growth factor (VEGF) and VEGF‐receptor type 2 and to migrate under hypoxic conditions. This study was designed to investigate early and delayed signalling mechanisms involved in hypoxia‐induced migration of human HSC/MFs. Signal transduction pathways and intracellular generation of reactive oxygen species (ROS) were evaluated by integrating morphological, cell, and molecular biology techniques. Non‐oriented and oriented migration were evaluated by using wound healing assay and the modified Boydens chamber assay, respectively. The data indicate that hypoxia‐induced migration of HSC/MFs is a biphasic process characterized by the following sequence of events: (a) an early (15 min) and mitochondria‐related increased generation of intracellular ROS which (b) was sufficient to switch on activation of ERK1/2 and JNK1/2 that were responsible for the early phase of oriented migration; (c) a delayed and HIF‐1α‐dependent increase in VEGF expression (facilitated by ROS) and its progressive, time‐dependent release in the extracellular medium that (d) was mainly responsible for sustained migration of HSC/MFs. Finally, immunohistochemistry performed on HCV‐related fibrotic/cirrhotic livers revealed HIF‐2α and haem‐oxygenase‐1 positivity in hepatocytes and α‐SMA‐positive MFs, indicating that MFs were likely to be exposed in vivo to both hypoxia and oxidative stress. In conclusion, hypoxia‐induced migration of HSC/MFs involves an early, mitochondrial‐dependent ROS‐mediated activation of ERK and JNK, followed by a delayed‐ and HIF‐1α‐dependent up‐regulation and release of VEGF. Copyright

Dissection of the Biphasic Nature of Hypoxia-Induced Motogenic Action in Bone Marrow-Derived Human Mesenchymal Stem Cells†‡§

Hypoxic conditions have been reported to facilitate preservation of undifferentiated mesenchymal stem cell (MSC) phenotype and positively affect their colony‐forming potential, proliferation, and migration/mobilization. In this study, designed to dissect mechanisms underlying hypoxia‐dependent migration of bone marrow‐derived human MSC (hMSC), signal transduction, and molecular mechanisms were evaluated by integrating morphological, molecular, and cell biology techniques, including the wound healing assay (WHA) and modified Boydens chamber assay (BCA) to monitor migration. Exposure of hMSCs to moderate hypoxia resulted in a significant increase of migration of hMSCs in both WHA (from 6 to 20 hours) and BCA (within 6 hours). Mechanistic experiments outlined the following sequence of hypoxia‐dependent events: (a) very early (15 minutes) increased generation of intracellular reactive oxygen species (ROS), which (b) was sufficient to switch on activation of extracellular regulated kinase 1/2 and c‐Jun N‐terminal protein kinase 1/2, found to be relevant for the early phase of hMSC migration; (c) hypoxia inducible factor‐1 (HIF‐1)–dependent increased expression of vascular endothelial growth factor (VEGF) (facilitated by ROS) and its progressive release that was responsible for (d) a delayed and sustained migration of hMSCs. These results suggest that hypoxia‐dependent migration relies on a previously unrecognized biphasic scenario involving an early phase, requiring generation of ROS, and a delayed phase sustained by HIF‐1‐dependent expression and release of VEGF. STEM CELLS 2011;29:952–963

Cobalt induces oxidative stress in isolated liver mitochondria responsible for permeability transition and intrinsic apoptosis in hepatocyte primary cultures

It is well established that cobalt mediates the occurrence of oxidative stress which contributes to cell toxicity and death. However, the mechanisms of these effects are not fully understood. This investigation aimed at establishing if cobalt acts as an inducer of mitochondrial-mediated apoptosis and at clarifying the mechanism of this process. Cobalt, in the ionized species Co(2+), is able to induce the phenomenon of mitochondrial permeability transition (MPT) in rat liver mitochondria (RLM) with the opening of the transition pore. In fact, Co(2+) induces mitochondrial swelling, which is prevented by cyclosporin A and other typical MPT inhibitors such as Ca(2+) transport inhibitors and bongkrekic acid, as well as anti-oxidant agents. In parallel with mitochondrial swelling, Co(2+) also induces the collapse of electrical membrane potential. However in this case, cyclosporine A and the other MPT inhibitors (except ruthenium red and EGTA) only partially prevent DeltaPsi drop, suggesting that Co(2+) also has a proton leakage effect on the inner mitochondrial membrane. MPT induction is due to oxidative stress, as a result of generation by Co(2+) of the highly damaging hydroxyl radical, with the oxidation of sulfhydryl groups, glutathione and pyridine nucleotides. Co(2+) also induces the release of the pro-apoptotic factors, cytochrome c and AIF. Incubation of rat hepatocyte primary cultures with Co(2+) results in apoptosis induction with caspase activation and increased level of expression of HIF-1alpha. All these observations allow us to state that, in the presence of calcium, Co(2+) is an inducer of apoptosis triggered by mitochondrial oxidative stress.

Cytotoxic activity of the histone deacetylase inhibitor panobinostat (LBH589) in anaplastic thyroid cancer in vitro and in vivo.

Anaplastic thyroid carcinoma (ATC) has a rapidly fatal clinical course, being resistant to multimodal treatments. Microtubules, α/β tubulin heterodimers, are crucial in cell signaling, division and mitosis and are among the most successful targets for anticancer therapy. Panobinostat (LBH589) is a potent deacetylase inhibitor acting both on histones and nonhistonic proteins, including α‐tubulin. In vitro LBH589, evaluated in three ATC cell lines (BHT‐101, CAL‐62 and 8305C), resulted in impairment of cell viability, inhibition of colony formation, cell cycle arrest and apoptosis induction. Mechanistically, we showed that LBH589 not only affected the expression of p21 and cyclin D1, but markedly determined microtubule stabilization as evidenced by tubulin acetylation and increased tubulin polymerization. In a SCID xenograft model implanted with CAL‐62 cells, the cytotoxic properties of LBH589 were confirmed. The drug at the dose of 20 mg/kg significantly impaired tumor growth (final tumor volume 2.5‐fold smaller than in untreated animals); at this dose, no relevant side effects were observed. In tumors of treated animals, a significant reduction of Ki67, which was negatively correlated with tubulin acetylation, was observed. Moreover, acetyl‐tubulin levels negatively correlated with tumor volume at sacrifice, reinforcing the opinion that tubulin acetylation has a role in the inhibition of tumor growth. In conclusion, LBH589, acting on both histones and nonhistonic proteins in anaplastic thyroid cancer, appears to be a promising therapeutic agent for the treatment of this kind of cancer which is known not to respond to conventional therapy.

Histone Deacetylase Inhibition Modulates E-Cadherin Expression and Suppresses Migration and Invasion of Anaplastic Thyroid Cancer Cells

CONTEXT Anaplastic thyroid cancer cells are characterized by a mesenchymal phenotype, as revealed by spindle-shaped cells and absent or reduced levels of E-cadherin. Epigenetic silencing is considered one of the leading mechanisms of E-cadherin impairment, which causes the acquisition of the invasive and metastatic phenotype of anaplastic thyroid cancer. OBJECTIVES In this study we investigated the effects of histone deacetylase inhibition on E-cadherin expression, cell motility, and invasion in anaplastic thyroid cancer cell cultures. DESIGN Three stabilized cell lines and primary cultures of anaplastic thyroid cancer were treated with various histone deacetylase inhibitors. After treatment, we evaluated histone acetylation by Western blotting and E-cadherin expression by RT-real time PCR. The proper localization of E-cadherin/β-catenin complex was assessed by immunofluorescence and Western blot. Transcription activity of β-catenin was measured by luciferase reporter gene and cyclin D1 expression. The effect on cell motility and invasion was studied both in vitro using scratch-wound and transwell invasion assays and in anaplastic thyroid carcinomas tumor xenografts in mice in vivo. RESULTS Histone deacetylase inhibition induced the E-cadherin expression and the proper membrane localization of the E-cadherin/β-catenin complex, leading to reduced cancer cell migration and invasion. CONCLUSIONS We here demonstrate an additional molecular mechanism for the anticancer effect of histone deacetylase inhibition. The antiinvasive effect in addition to the cytotoxic activity of histone deacetylase inhibitors opens up therapeutic perspectives for the anaplastic thyroid tumor that does not respond to conventional therapy.

Evidence for an Elevated Aspartate pKa in the Active Site of Human Aromatase

Background: Crystallography and mutagenesis indicate that Asp309 is required for substrate binding and catalysis. Results: Substrate binding in aromatase is pH-dependent. Such a dependence is missing in D309N mutant. Conclusion: The apparent pKa for Asp309 is 8.2, and the residue is protonated at physiological pH. Significance: The assigned pKa indicates the role of Asp309 in proton delivery for aromatization reaction. Aromatase (CYP19A1), the enzyme that converts androgens to estrogens, is of significant mechanistic and therapeutic interest. Crystal structures and computational studies of this enzyme shed light on the critical role of Asp309 in substrate binding and catalysis. These studies predicted an elevated pKa for Asp309 and proposed that protonation of this residue was required for function. In this study, UV-visible absorption, circular dichroism, resonance Raman spectroscopy, and enzyme kinetics were used to study the impact of pH on aromatase structure and androstenedione binding. Spectroscopic studies demonstrate that androstenedione binding is pH-dependent, whereas, in contrast, the D309N mutant retains its ability to bind to androstenedione across the entire pH range studied. Neither pH nor mutation perturbed the secondary structure or heme environment. The origin of the observed pH dependence was further narrowed to the protonation equilibria of Asp309 with a parallel set of spectroscopic studies using exemestane and anastrozole. Because exemestane interacts with Asp309 based on its co-crystal structure with the enzyme, its binding is pH-dependent. Aromatase binding to anastrozole is pH-independent, consistent with the hypothesis that this ligand exploits a distinct set of interactions in the active site. In summary, we assign the apparent pKa of 8.2 observed for androstenedione binding to the side chain of Asp309. To our knowledge, this work represents the first experimental assignment of a pKa value to a residue in a cytochrome P450. This value is in agreement with theoretical calculations (7.7–8.1) despite the reliance of the computational methods on the conformational snapshots provided by crystal structures.

Endocellular polyamine availability modulates epithelial-to-mesenchymal transition and unfolded protein response in MDCK cells

Epithelial-to-mesenchymal transition (EMT) is involved in embryonic development as well as in several pathological conditions. Literature indicates that polyamine availability may affect transcription of c-myc, matrix metalloproteinase (MMP)1, MMP2, TGFβ1, and collagen type I mRNA. The aim of this study was to elucidate polyamines role in EMT in vitro. Madin-Darby canine kidney (MDCK) cells were subjected to experimental manipulation of intracellular levels of polyamines. Acquisition of mesenchymal phenotype was evaluated by means of immunofluorescence, western blots, and zymograms. MDCK cells were then subjected to 2D gel proteomic study and incorporation of a biotinilated polyamine (BPA). Polyamine endocellular availability modulated EMT process. Polyamine-depleted cells treated with TGFβ1 showed enhanced EMT with a marked decrease of E-cadherin expression at plasma membrane level and an increased expression of mesenchymal markers such as fibronectin and α-smooth muscle actin. Polyamine-depleted cells showed a twofold increased expression of the rough endoplasmic reticulum (ER)-stress proteins GRP78, GRP94, and HSP90 α/β in 2D gels. The latter data were confirmed by western blot analysis. Administration of BPA showed that polyamines are covalently linked, within the cell, to ER-stress proteins. Intracellular polyamine availability affects EMT in MDCK cells possibly through the modulation of ER-stress protein homeostasis.

The pan-histone deacetylase inhibitor LBH589 (panobinostat) alters the invasive breast cancer cell phenotype

Triple-negative breast cancer (TNBC) is a very aggressive type of tumour and its aggressiveness is linked to E-cadherin downregulation. In estrogen-sensitive breast cancer, high levels of E-cadherin fit with high levels of ERα and MTA3 (a component of the transcription Mi-2/NuRD complex with intrinsic DAC activity). In TNBC the E-cadherin downregulation could be due to epigenetic silencing of the CDH1 gene as well as to the lack of a fully functioning ERα-activated pathway. We report that the pan-histone deacetylase inhibitor LBH589, a potent anti-proliferative agent, induced E-cadherin expression on cell membranes of MDA-MB-231 cells (TNBC), determining a reduction of cell invasion and migration. Even though E-cadherin expression in breast cancer is also regulated by estradiol and the ERα/MTA3/Snail/Slug pathway, LBH589 is able to increase E-cadherin without affecting the estrogen pathway. In fact, no expression of ERα, PR and FoxA1 was observed in MDA-MB-231 cells before and after LBH589 treatment; furthermore, the drug caused an increase in Snail and Slug expression with a concomitant reduction of MTA3 levels. Taking into consideration its anti-proliferative and anti-invasive properties, we suggest the use of LBH589 in aggressive breast cancer refractory to hormonal therapy.

Impact of R264C and R264H polymorphisms in human aromatase function

The cytochrome P450 aromatase is involved in the last step of sex hormones biosynthesis by converting androgens into estrogens. The human enzyme is highly polymorphic and literature data correlate aromatase single nucleotide polymorphisms to the onset of pathologies such as breast cancer and neurodegenerative diseases. The aims of this study were i) to study the influence of the mutations R264C and R264H on the structure-function of the enzyme also upon phosphorylation by selected kinases and ii) to compare the activity of the variants to that of aromatase wild type in two different cell lines. Far-UV circular dichroism spectroscopy, thermal denaturation experiments and CO-binding assay showed that the two polymorphic variants are correctly folded. Steady-state kinetics experiments showed that rArom R264C and R264H exhibit a 1.5 and 3.4 folds lower catalytic efficiency, respectively, when compared to the wild type protein. Since R264 is part of the consensus motif of PKA and PKG1, phosphorylation experiments were performed to study the effect on aromatase function. Phosphorylation by PKA caused a decrease in activity by 36.2%, 49.3% and 27.9% in the wild type, R264C and R264H proteins respectively. Phosphorylation by PKG1 was also found to decrease the activity by 30.3%, 30.5% and 15.4% in the wild type, R264C and R264H proteins respectively. Experiments performed on the three full-length proteins expressed in human MCF-7 breast cancer cells and rat ST14A neuronal cells showed that, depending on the cell line used, the activity of the proteins is different, implicating different cellular mechanisms modulating aromatase activity. This work demonstrate that R264 polymorphism causes an intrinsic alteration of aromatase activity together with a different consensus for phosphorylation by different kinases, indicating that estrogen production can be different when such mutations are present. These findings are significant in understanding the onset and treatment of pathologies in which aromatase has been shown to be involved.