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.

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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 Boyden's 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.

a b s t r a c tIt 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 « 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-1␣.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.

Redox adaptation plays an important role in cancer cells drug resistance. The antioxidant response is principally mediated by the transcription factor Nrf2, that induces the transcriptional activation of several genes involved in GSH synthesis, chemoresistance, and cytoprotection. YAP is emerging as a key mediator of chemoresistance in a variety of cancers, but its role in controlling the antioxidant status of the cells is yet elusive. Here, we show that impairing YAP protein expression reduced GSH content and Nrf2 protein and mRNA expression in bladder cancer cells. Moreover, in YAP knocked down cells the expression of FOXM1, a transcription factor involved in Nrf2 transcription, was down-regulated and the silencing of FOXM1 reduced Nrf2 expression. On the other hand, the silencing of Nrf2, as well as the depletion of GSH by BSO treatment, inhibited YAP expression, suggesting that cross-talk exists between YAP and Nrf2 proteins. Importantly, we found that silencing either YAP or Nrf2 enhanced sensitivity of bladder cancer cells to cytotoxic agents and reduced their migration. Furthermore, the inhibition of both YAP and Nrf2 expressions significantly increased cytotoxic drug sensitivity and synergistically reduced the migration of chemoresistant bladder cancer cells. These findings provide a rationale for targeting these transcriptional regulators in patients with chemoresistant bladder cancer, expressing high YAP and bearing a proficient antioxidant system.

Anaplastic thyroid carcinoma (ATC) has a rapidly fatal clinical course, being resistant to multimodal treatments. Microtubules, a/b 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 a-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.Anaplastic thyroid carcinoma (ATC), which accounts for 1% of thyroid tumors, is one of the most lethal malignancies, with a rapidly fatal clinical course.

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 Boyden's 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 Disclosure of potential conflicts of interest is found at the end of this article.