Caffeine and other methylxanthines produce multiple physiologic effects throughout the human body, many of these effects could potentially modulate the activity of anticancer therapy. Caffeine may directly interfere with drug transport to tumor cells by formation of mixed stacking complexes with polyaromatic drugs. If formed in cells, these complexes may also prevent of intercalating drugs from DNA binding and, in this way, lower their antitumor activity. Since many of potent carcinogens are polyaromatic compounds, formation of stacking complexes with carcinogens could be associated with anti-genotoxic activity of caffeine and its use in cancer chemoprevention. Caffeine has also been reported to inhibit ATM and ATR kinases which leads to the disruption of multiple DNA damage-responsive cell cycle checkpoints and greatly sensitizes tumor cells to antitumor agents which induce genotoxic stress. Caffeine may inhibit repair of DNA lesions through a direct interference with DNA-PK activity and other repair enzymes. A number of in vitro and in vivo studies demonstrated that caffeine modulates both innate and adaptive immune responses via inhibition of cyclic adenosine monophosphate (cAMP)-phosphodiesterase. Finally, another group of effects induced by caffeine is mediated through its inhibitory action on adenosine receptors. This may modulate the stability of HIF1 alpha as well as VEGF and interleukin-8 expression in tumor cells, which could have a direct impact on neovascularization of human tumors. In this review, we present different molecular mechanisms by which caffeine and other methylxanthines may directly or indirectly modulate the effect of antitumor treatment in tumor cells and in cancer patients.

Hypoxia-inducible factors 1 and 2 (HIF1 and HIF2) are heterodimeric transcription factors consisting of alpha regulatory subunits and a constitutively expressed beta subunit. The expression of alpha regulatory subunits is promoted by hypoxia, cancer-associated mutations, and inflammatory cytokines. Thus, HIF1 and HIF2 provide a molecular link between cancer and inflammation. We have recently identified novel small molecules that selectively inhibit translation of the HIF2a message and thereby powerfully inhibit the expression of HIF2a target genes. We report here that Connectivity Map analysis links three of these compounds to the anti-inflammatory cytokine 15-deoxy-Delta(12,14)-prostaglandin J(2) (PGJ(2)). As with our identified compounds, PGJ(2) inhibits translation of the HIF2a message in a mammalian target of rapamycin-independent manner by promoting the binding of iron regulatory protein-1 (IRP1) to a noncanonical iron responsive element (IRE) embedded within the 5'-untranslated region of the HIF2a message. The IRE is necessary and sufficient for mediating the effect. Mutation of the IRE sequence, or downregulation of IRP1 expression, blocks the effect of PGJ(2) on HIF2a translation. This is the first report of an endogenous natural molecule regulating HIF2a translation, and it suggests that part of the anti-inflammatory and putative antineoplastic effects of PGJ(2) may be mediated through inhibition of HIF2a within tumor epithelial cells themselves and/or mesenchymal cells of the tumor microenvironment.

Perinatal hypoxia ischemia (HI) is a frequent cause of neonatal brain injury. This study aimed at describing molecular changes during the first 48 h after exposure of the neonatal rat brain to HI. Twelve-day-old rats were subjected to unilateral carotid artery occlusion and 90 min of 8% O2, leading to neuronal damage in the ipsilateral hemisphere only. Phosphorylated-Akt levels were decreased from 0.5 to 6 h post-HI, whereas the level of phosphorylated extracellular signal-related kinases (ERK)1/2 increased during this time frame. Hypoxia-inducible factor (HIF)-1alpha protein increased with a peak at 3 h after HI. mRNA expression for IL-beta and tumor necrosis factor-alpha and -beta started to increase at 6 h with a peak at 24 h post-HI. Expression of heat shock protein 70 was increased from 12 h after HI onwards in the ipsilateral hemisphere only. Surprisingly, HI changed the expression of cytokines, HIF1-alpha ,and P-Akt to the same extent in both the ipsi- as well as the contralateral hemisphere, although neuronal damage was unilateral. Exposure of animals to hypoxia without carotid artery occlusion induced similar changes in cytokines, HIF-1alpha, and P-Akt. We conclude that during HI, hypoxia is sufficient to regulate multiple molecular mediators that may contribute, but are not sufficient, to induce long-term neuronal damage.

HIF1 (hypoxia-inducible factor 1 alpha) is considered a central oxygen-threshold sensor in mammalian cells. In the presence of oxygen, HIF1 is marked by prolyl hydroxylases (PHDs) at the oxygen-dependent degradation (ODD) domain for ubiquitination followed by rapid proteasomal degradation. However, the actual mechanisms of oxygen sensing by HIF1 are still controversial. Thus, HIF1 expression correlates poorly with tissue oxygen levels, and PHDs are themselves target genes of HIF1 considered to readjust to new oxygen thresholds. In contrast to hypoxia chambers, we here establish an enzymatic model that allows both the rapid induction of stable hypoxia and independent control of H(2)O(2). Rapid enzymatic hypoxia only transiently induced HIF1 in various cell types and the HIF1 was completely degraded within 8-12 h despite sustained hypoxia. HIF1 degradation under sustained hypoxia could be blocked by a competitive ODD-GFP construct and PHD siRNA, but also by cobalt chloride and micromolar H(2)O(2) levels. Concomitant induction of PHDs further confirmed their role in degrading HIF1 under enzymatic hypoxia. The rapid and complete degradation of HIF1 under enzymatic hypoxia suggests that, in addition to hypoxia sensing, the HIF1/PHD loop may also compensate for fluctuations of tissue oxygen staying tuned to other, e.g., metabolic, signals. In addition to hypoxia chambers, enzymatic hypoxia provides a valuable tool for independently studying the regulatory functions of hypoxia and oxidative stress in vitro.

In Duchenne muscular dystrophy (DMD) metabolic and structural alterations of the central nervous system are described. Here, we investigated in the brain of 10 mdx mice and in five control ones, the expression of hypoxia inducible factor-1alpha (HIF-1alpha) and we correlated it with the expression of vascular endothelial growth factor (VEGF) and vascular endothelial growth factor receptor-2 (VEGFR-2) and of the endothelial tight junction proteins zonula occludens-1 (ZO-1) and claudin-1. Results showed an activation of mRNA HIF-1alpha by reverse transcription polymerase chain reaction (RT-PCR) and a strong HIF1-alpha labeling of perivascular glial cells and cortical neurons by immunohistochemistry, in mdx mouse. Moreover, overexpression of VEGF and VEGFR-2, respectively, in neurons and in endothelial cells coupled with changes to endothelial ZO-1 and claudin-1 expression in the latter were detected by immunoblotting and immunohistochemistry, in the mdx brain. Furthermore, by immunoprecipitation, an up-phosphorylation of ZO-1 was demonstrated in mdx endothelial cells in parallel with the reduction in ZO-1 protein content. These data suggest that the activation of HIF-1alpha in the brain of dystrophic mice coupled with VEGF and VEGFR-2 up-regulation and ZO-1 and claudin-1 rearrangement might contribute to both blood-brain barrier opening and increased angiogenesis.

Infections with intracellular bacteria such as chlamydiae affect the majority of the world population. Infected tissue inflammation and granuloma formation help contain the short-term expansion of the invading pathogen, leading also to local tissue damage and hypoxia. However, the effects of key aspects of damaged inflamed tissues and hypoxia on continued infection with intracellular bacteria remain unknown. We find that development of Chlamydia trachomatis is reversibly retarded by prolonged exposure of infected cells to extracellular adenosine, a hallmark of hypoxia and advanced inflammation. In epithelial cells, this effect was mediated by the A2b adenosine receptor, unique in the adenosine receptor family for having a hypoxia-inducible factor (HIF1-alpha) binding site at its promoter region, and was dependent on an increase in the intracellular cAMP levels, but was independent of cAMP-dependent protein kinase (PKA). Further study of adenosine receptor signaling during intracellular bacterial infection could lead to breakthroughs in our understanding of persistent infections with these ubiquitous pathogens.

OBJECTIVE

We aimed to characterize angiogenesis and proliferation and their correlation with clinical characteristics in a large brain metastasis (BM) series.

METHODS

Ki67 proliferation index, microvascular density (MVD) and hypoxia-inducible factor 1 alpha (HIF-1 alpha) index were determined by immunohistochemistry in BM and primary tumour specimens.

RESULTS

Six hundred thirty-nine BM specimens of 639 patients with lung cancer (344/639; 53.8%), breast cancer (105/639; 16.4%), melanoma (67/639; 10.5%), renal cell carcinoma (RCC; 52/639; 8.1%) or colorectal cancer (CRC; 71/639; 11.1%) were available. Specimens of the corresponding primary tumour were available in 113/639 (17.7%) cases. Median Ki67 index was highest in CRC BM and lowest in RCC BM (P < 0.001). MVD and HIF-1 alpha index were both highest in RCC BM and lowest in melanoma BM (P < 0.001). Significantly higher Ki67 indices, MVD and HIF-1 alpha indices in the BM than in matched primary tumours were observed for breast cancer, non-small cell lung cancer (NSCLC) and CRC. Correlation of tissue-based parameters with overall survival in individual tumour types showed a favourable and independent prognostic impact of low Ki67 index [hazard ratio (HR) 1.015; P < 0.001] in NSCLC BM and of low Ki67 index (HR 1.027; P = 0.008) and high angiogenic activity (HR 1.877; P = 0.002) in RCC.

CONCLUSIONS

Our data argue for differential pathobiological and clinical relevance of Ki67 index, HIF1-alpha index and MVD between primary tumour types in BM patients. An independent prognostic impact of tissue-based characteristics was observed in patients with BM from NSCLC and RCC, supporting the incorporation of these tissue-based parameters into diagnosis-specific prognostic scores.

Anti-PD-1 treatment increases anti-tumour immune responses in animal models of hepatocellular carcinoma (HCC). Sorafenib, the mainstay of treatment of HCC patients, however, leads to tumour hypoxia and thereby abrogates the efficacy of anti-PD-1 treatment. This served as a rationale to implement CXCR4 inhibition as adjunct to sorafenib and anti-PD-1 treatment in murine HCC models. We studied the relationship between tumour hypoxia, PD-L1 and CXCL12 expression in human HCC, aiming to test the rationale for triple therapy combining sorafenib, PD-1 immune checkpoint inhibitors and CXCR4 inhibitors. Expression of CXCL12, PD-L1 and of surrogate markers for tumour hypoxia was evaluated at messenger RNA (mRNA) level in a cohort of HCC patients from The Cancer Genome Atlas and immunohistochemically in an independent cohort from the University Hospital of Bonn. Retrospective survival analyses were conducted. CXCL12 mRNA level significantly correlated with markers indicating tumour hypoxia in HCC (HIF1-α ρ = 0.104, p = 0.047). PD-L1 expression was significantly increased in tumours with a high number of tumour-infiltrating lymphocytes (ρ = 0.533, p < 0.001). In Cox proportional hazard analyses, high PD-L1 expression and loss of nuclear CXCL12 expression showed significant prognostic value in terms of overall survival (hazard ratio (HR) = 3.35 [95%CI 1.33-8.46], p = 0.011 for PD-L1; HR = 2.64 [95%CI 1.18-5.88], p = 0.018 for CXCL12, respectively). This study supports the rationale to combine CXCR4 inhibitors and PD-1 immune checkpoint inhibitors in patients with HCC, as sorafenib-induced tumour hypoxia leads to upregulation of PD-L1 and CXCL12.

BACKGROUND

Survival upon diagnosis of brain metastases (BM) in patients with non-small cell lung cancer (NSCLC) is highly variable and established prognostic scores do not include tissue-based parameters.

METHODS

Patients who underwent neurosurgical resection as first-line therapy for newly diagnosed NSCLC BM were included. Microvascular density (MVD), Ki67 tumor cell proliferation index and hypoxia-inducible factor 1 alpha (HIF-1 alpha) index were determined by immunohistochemistry.

RESULTS

NSCLC BM specimens from 230 patients (151 male, 79 female; median age 56 years; 199 nonsquamous histology) and 53/230 (23.0%) matched primary tumor samples were available. Adjuvant whole-brain radiation therapy (WBRT) was given to 153/230 (66.5%) patients after neurosurgical resection. MVD and HIF-1 alpha indices were significantly higher in BM than in matched primary tumors. In patients treated with adjuvant WBRT, low BM HIF-1 alpha expression was associated with favorable overall survival (OS), while among patients not treated with adjuvant WBRT, BM HIF-1 alpha expression did not correlate with OS. Low diagnosis-specific graded prognostic assessment score (DS-GPA), low Ki67 index, high MVD, low HIF-1 alpha index and administration of adjuvant WBRT were independently associated with favorable OS. Incorporation of tissue-based parameters into the commonly used DS-GPA allowed refined discrimination of prognostic subgroups.

CONCLUSIONS

Ki67 index, MVD and HIF-1 alpha index have promising prognostic value in BM and should be validated in further studies.

The increasing demand in studying cellular functions in cultured cells under various levels of oxygen and hydrogen peroxide (H2O2) is only partly fulfilled by conventional approaches such as hypoxia chambers, bolus additions of H2O2 or redox-cycling drugs. This article describes the recently developed enzymatic GOX/CAT system consisting of glucose oxidase (GOX) and catalase (CAT) that allows the independent control and maintenance of both H2O2 and hypoxia in cell culture. In contrast to hypoxia chambers, the GOX/CAT system more rapidly induces hypoxia within minutes at a defined rate. The degree of hypoxia is dependent on the GOX activity and the diffusion distance of oxygen from the medium surface to the adherent cells. In contrast, H2O2 levels are solely controlled by the ratio of GOX and CAT activities. They can be adjusted at non-toxic or toxic dosages over 24 hours. Thus, the GOX/CAT system mimics a non-phosphorylating respiratory chain and allows to adjust H2O2 levels under hypoxic conditions truly simulating H2O2 release e.g. by inflammatory cells or intracellular sources. GOX/CAT can be employed to address many questions ranging from redox signaling to ischemia/reperfusion studies in transplantation medicine. Factors such as HIF1 alpha that respond both to hypoxia and H2O2 are an especially attractive target for the novel methodology. Several applications are discussed in detail to demonstrate the technical requirements and potentials. In addition, simplified protocols are presented for cell or molecular biology labs without dedicated biophysical equipment.

We report a Notch signal-induced pathway that leads to transcriptional activation of HIF1-alpha gene. HeLa/rtTAA/TRE-N1-IC cell line capable of doxycycline-induced expression of human Notch1-IC was established. The induction of Notch signaling activates HIF1-alpha and its target gene expression in HeLa/rtTAA/TRE-N1-IC cells. Notch signaling enhanced signal transducers and activators of transcription 3 (STAT3) phosphorylation required for HIF1-alpha expression. SRC kinase was found to be responsible for the enhanced STAT3 phosphorylation in response to Notch signaling. Activation of SRC/STAT3 pathway by Notch signaling was dependent on the expression of Notch effector HES1 transcription factor. The induction of HES1 enhanced STAT3 phosphorylation at Tyr 705 as well as SRC phosphorylation at Tyr 416 in inducible HeLa/rtTAA/TRE-HES1 cells, which express HES1 in response to doxycycline treatment. However, the treatment of Trichostatin A that interferes with HES1 transcriptional regulation did not affect STAT3 phosphorylation, and the expression of dominant negative HES1 failed to interfere with HES1-dependent SRC/STAT3 pathway. These observations have led us to the conclusion that HES1-dependent activation of SRC/STAT3 pathway is independent of HES1 transcription regulation. This study first reports HES1-dependent SRC/STAT3 pathway that provides a functional link between Notch signaling and hypoxia pathway.

OBJECTIVE

Exposure to a high glucose medium or diabetes has been found to protect the heart against ischaemia. The activation of antiapoptotic and proliferative factors seems to be involved in this cardioprotection. This study was designed to evaluate the role of hyperglycaemia in cardiac function, programmed cell survival, and cell death in diabetic rats after myocardial infarction (MI).

RESULTS

Male Wistar rats were divided into four groups (n = 8): control (C), diabetic (D), myocardial infarcted (MI), and diabetic myocardial infarcted (DI). The following measures were assessed in the left ventricle: size of MI, systolic and diastolic function by echocardiography, cytokines by ELISA (TNF-alpha, IL-1beta, IL-6, and IL-10), gene expression by real-time PCR (Bax, Fas, p53, Bcl-2, HIF1-alpha, VEGF, and IL8r), caspase-3 activity by spectrofluorometric assay, glucose transporter type 1 and 4 (GLUT-1 and GLUT-4) protein expression by western blotting, and capillary density and fibrosis by histological analysis. Systolic function was improved by hyperglycaemia in the DI group, and this was accompanied by no improvement in diastolic dysfunction, a reduction of 36% in MI size, reduced proinflammatory cytokines, apoptosis activation, and an increase in cell survival factors (HIF1-alpha, VEGFa and IL8r) assessed 15 days post-MI. Moreover, hyperglycaemia resulted in angiogenesis (increased capillary density) before and after MI, accompanied by a reduction in fibrosis.

CONCLUSIONS

Together, these results suggest that greater plasticity and cellular resistance to ischaemic injury result from chronic diabetic hyperglycaemia in rat hearts.

Tears in the peripheral part of the menisci have a better healing potential than tears in the central part, because the central two-thirds of the menisci are avascular. The avascular status of the meniscus is maintained by the expression of antiangiogenic factors such as endostatin. The distribution of endostatin in the menisci correlates with the degree of vascularization. Endostatin immunostaining is strong in the avascular zone and reduced in the vascularized outer one-third. Endostatin interacts with signal transduction of the vascular endothelial growth factor (VEGF) by reducing VEGF-induced kinase (Erk1/2) phosphorylation. VEGF plays an important role in angiogenesis in fetal menisci and it is down-regulated in the adult meniscus. We hypothesized that healing of meniscal tears in the avascular zone can be promoted by the local application of the angiogenic factor VEGF. To evaluate this hypothesis a tear was created in the avascular zone of the medial meniscus in 18 merino sheep. The tear was then repaired with an uncoated suture (group 1), a suture coated with PDLLA (group 2), and by a suture coated with PDLLA/VEGF (group 3). After 6 weeks we observed increased factor VIII immunostaining in the VEGF-treated group. However, in this treatment group (VEGF/PDLLA) no meniscus healed. In the uncoated suture group and in the PDLLA-coated suture group partial healing was observed in three animals and complete healing in three animals, respectively. Factor VIII expression is normally restricted to vascular endothelial cells. In this study, however, single endothelial cells could be detected in the menisci of the VEGF/PDLLA group. This finding suggests that the application of VEGF might have stimulated proliferation of vascular endothelial cells but the application of VEGF was not successful in stimulating the more complex process of vasculogenesis. Further immunohistochemical examinations of the specimen have shown that in the VEGF/PDLLA group there is strong immunostaining against matrix metalloproteinase 13 (MMP-13). In vitro studies have shown that VEGF can stimulate chondrocytes to proliferate but also to express MMP-13 via HIF1-alpha induction. Since meniscal fibrochondrocytes express the VEGF receptor 2 (KDR) the induction of MMP expression might be another factor which inhibits healing despite increased angiogenesis. In conclusion, the local application of VEGF via PDLLA-coated sutures does not promote meniscal healing. A single growth factor might not always be a promising tool for the promotion of tissue repair. Further studies have to find out if growth factor combinations (VEGF and angiopoitin) might be more effective in stimulating vasculogenesis during meniscal healing.

Hepatocellular carcinoma (HCC) is notoriously refractory to chemotherapy because of its tendency to develop multi-drug resistance (MDR), whose various underlying mechanisms make it difficult to target. The calcium signalling pathway is associated with many cellular biological activities, and is also a critical player in cancer. However, its role in modulating tumour MDR remains unclear. In this study, stimulation by doxorubicin, hypoxia and ionizing radiation was used to induce MDR in HCC cells. A sustained aggregation of intracellular calcium was observed upon these stimuli, while inhibition of calcium signalling enhanced the cells' sensitivity to various drugs by attenuating epithelial-mesenchymal transition (EMT), Hif1-α signalling and DNA damage repair. The effect of calcium signalling is mediated via transient receptor potential canonical 6 (TRPC6), a subtype of calcium-permeable channel. An in vivo xenograft model of HCC further confirmed that inhibiting TRPC6 enhanced the efficacy of doxorubicin. In addition, we deduced that STAT3 activation is a downstream signalling pathway in MDR. Collectively, this study demonstrated that the various mechanisms regulating MDR in HCC cells are calcium dependent through the TRPC6/calcium/STAT3 pathway. We propose that targeting TRPC6 in HCC may be a novel antineoplastic strategy, especially combined with chemotherapy.

Hypoxia inducible factors (HIF1, 2 and 3), consisting of alpha and beta subunits, play an essential role in various responses to hypoxia. Nuclear entry of alpha subunits is a necessary step for the formation of DNA-binding complex with beta subunit, which is constitutively localized in the nucleus. We show here that the nuclear accumulation of HIF2alpha induced by hypoxia is mediated through a novel variant of bipartite-type nuclear localization signal (NLS) in the C-terminus of the protein, which has an unusual length of spacer sequence between two adjacent basic domains. We further show that when the ubiquitin-proteasome system was deficient or inhibited, HIF2alpha accumulated in the nucleus even under normoxia, also mediated through the bipartite NLS. These findings indicate that the protein stability is critical for the nuclear localization of HIF2alpha and hypoxia is not a necessary factor for the process. Importantly, the NLS of HIF2alpha is also conserved in the other HIF family members, HIF1alpha and HIF3alpha. Mutational analyses proved that the NLS mediating the nuclear localization of HIF1alpha is indeed bipartite-, but not monopartite-type as thought before. Our results suggest that the newly identified NLS is crucial for the functional regulation of HIF family.

The concept that invasive cancer is associated with increased levels of reactive oxygen species (ROS) generated by mitochondria is consistent with an ROS-mediated signaling mechanism. As a tumor grows, it encounters adverse microenvironments, one of which is low oxygen (hypoxia), which selects tumor cells with characteristics of increased invasiveness. Hypoxic environments select for tumor cells with stabilized HIF1 apha, a transcription factor that regulates genes coding for pro-tumor cytokines that signal stromal cells such as macrophages and fibroblasts to support an invasive tumor cell phenotype. HIF1 alpha-mediated switches in the energy production of tumor cells from OXPHOS to glycolysis, as well as age-associated decreases in the metabolic rate of the host, enhance invasive qualities of tumor cells. An increase in environmental oxygen in combination with a mitochondrial targeted catalase mimetic and a metabolism booster may be of interest to investigate as a treatment strategy for invasive cancer.

Copper, a strictly regulated trace element, is essential for many physiological processes including angiogenesis. Dysregulated angiogenesis has been associated with increased copper in tumors, and thus copper chelators have been used to inhibit tumor angiogenesis. However, it remains unclear whether copper has any effect on epithelial-mesenchymal transition (EMT). Using CoCl2-induced EMT of human breast carcinoma MCF-7 cells, we found that TEPA, a copper chelator, inhibited EMT-like cell morphology and cytoskeleton arrangement triggered by CoCl2; decreased the expression of vimentin and fibronectin, markers typical of EMT; inhibited HIF-1 activation and HIF1-α accumulation in nuclear; and down-regulated the expression of hypoxia-associated transcription factors, Snail and Twist1. Moreover, knockdown copper transport protein, Ctr1, also inhibited CoCl2-induced EMT and reversed the mesenchymal phenotype. In EMT6 xenograft mouse models, TEPA administration inhibited the tumor growth and increased mice survival. Immunohistochemical analysis of the xenograft further demonstrated that TEPA administration significantly inhibited tumor angiogenesis, down-regulated hypoxia-induced transcription factors, Snail and Twist1, leading to decreased transactivation of EMT-associated marker genes, vimentin and fibronectin. These results indicate that TEPA inhibits CoCl2-induced EMT most likely via HIF1-α-Snail/Twist signaling pathway, and copper depletion may be exploited as a therapeutic for breast cancer.

Tumor radioresistance leading to local therapy failure remains a major obstacle for successful treatment of high-grade glioma. We hypothesized that distinct radiobiological features of particle therapy with carbon ions may circumvent glioma radioresistance. We demonstrate that carbon irradiation (CIR) efficiently eradicates radioresistant patient-derived glioma stem cells (GSCs), leading to growth inhibition and prolonged survival. The impact of CIR at the tumor-stroma interface was further investigated in 2 syngeneic mouse and 2 orthotopic GSC xenograft models. Intriguingly, tumor regressions and long-term local controls were observed at doses greater than or equal to 15-Gy CIR. Fractionated CIR further prolonged survival. The enhanced relative biological effectiveness of CIR in vivo was attributed to its potent antiangiogenic effects and eradication of radioresistant hypoxic tumor cells. Blockade of the HIF1-α/stromal cell-derived factor 1/CXCR4 axis by CIR reduced the recruitment of microglia and myeloid-derived suppressor cells (CD11b+Gr1+). Consequently, CIR abrogated M2-like immune polarization and enhanced the influx of CD8+ cells, generating an immunopermissive niche. We report that radiotherapy with carbon ions could surmount several central glioma resistance mechanisms by eradicating hypoxic and stem cell-like tumor cells, as well as modulating the glioma niche toward an antiangiogenic and less immunosuppressive state. Conclusively, potentially novel rationales for CIR in conquering glioma radioresistance are provided.

The interplay between p53 and RAS signaling regulates cancer chemoresistance, but the detailed mechanism is unclear. In this study, we investigated the interactive effects of p53 and RAS on ovarian cancer cisplatin resistance to explore the potential therapeutic targets. Methods: An inducible p53 and RAS mutants active in either MAPK/ERK (S35 and E38) or PI3K/AKT (C40) or both (V12) were sequentially introduced into a p53-null ovarian cancer cell line-SKOV3. Comparative microarray analysis was performed using Gene Chip Prime View Human Gene Expression arrays (Affymetrix). In vitro assays of autophagy and apoptosis and in vivo animal experiments were performed by p53 induction and/or cisplatin treatment using the established cell lines. The correlation between HDAC4 and HIF-1α or CREBZF and the association of HDAC4, HIF-1α, CREBZF, ERK, AKT, and p53 mRNA levels with patient survival in 523 serous ovarian cancer cases from TCGA was assessed. Results: We show that p53 and RAS mutants differentially control cellular apoptosis and autophagy to inhibit or to promote chemoresistance through dysregulation of Bax, Bcl2, ATG3, and ATG12. ERK and AKT active RAS mutants are mutually suppressive to confer or to deprive cisplatin resistance. Further studies demonstrate that p53 induces HIF-1α degradation and HDAC4 cytoplasmic translocation and phosphorylation. S35, E38, and V12 but not C40 promote HDAC4 phosphorylation and its cytoplasmic translocation along with HIF-1α. Wild-type p53 expression in RAS mutant cells enhances HIF-1α turnover in ovarian and lung cancer cells. Autophagy and anti-apoptotic processes can be promoted by the overexpression and cytoplasmic translocation of HDAC4 and HIF1-α. Moreover, the phosphorylation and cytoplasmic translocation of HDAC4 activate the transcription factor CREBZF to promote ATG3 transcription. High HDAC4 or CREBZF expression predicted poor overall survival (OS) and/or progression-free survival (PFS) in ovarian cancer patients, whereas high HIF-1α expression was statistically correlated with poor or good OS depending on p53 status. Conclusion: HIF-1α and HDAC4 may mediate the interaction between p53 and RAS signaling to actively control ovarian cancer cisplatin resistance through dysregulation of apoptosis and autophagy. Targeting HDAC4, HIF-1α and CREBZF may be considered in treatment of ovarian cancer with p53 and RAS mutations.

OBJECTIVE

Apoptosis and angiogenesis may be involved in the pathogenesis of atherosclerosis and plaque destabilization. In this study, we investigated if apoptosis and angiogenesis were induced in the unstable human coronary atherosclerotic plaque compared to stable atherosclerotic plaque.

METHODS

Atherosclerotic plaques from patients with stable (n = 9) and unstable angina (n = 13) were obtained by directional coronary atherectomy performed during percutaneous transluminal coronary angioplasty. Apoptosis was detected by terminal deoxynucleotidyl transferase end labelling (TUNEL), as well as by immunostaining for caspase 3, Bax and Bcl-2. Neovascularization was determined by immunostaining for the endothelial cell-specific CD31, vascular endothelial growth factor (VEGF-A), angiopoietin-1 (Ang-1), angiopoietin-2 (Ang-2), hypoxia inducible factor-1alpha (HIF-alpha), and the sections were quantified blindly.

RESULTS

The apoptotic nuclei were more frequently found in the unstable coronary atherosclerotic plaques. When the number of apoptotic cells was quantified, an increased apoptotic index was found in the unstable plaques (P = 0.04). The positive staining for caspase-3 was increased in the unstable plaques (P = 0.0008), while no difference in either Bax or Bcl-2 was found between groups. Neovascularization, as evidenced by lumens surrounded by a CD31 positive endothelial layer, was more frequently present in the plaques from patients with unstable angina (P = 0.04). The number of cells with positive staining for VEGF-A was increased in unstable plaques (P = 0.005). No difference of Ang I, Ang II, HIF1-alpha was found between groups.

CONCLUSIONS

In unstable human coronary plaques, apoptosis probably involving caspase 3 was found. The plaques had an increased neovascularization, probably induced by VEGF-A. These factors may contribute to explaining plaque destabilization and intraplaque haemorrhage.

Cellular FLIP (cFLIP) is a homologue of caspase-8 without protease activity that inhibits the apoptosis signaling initiated by death receptor ligation. We previously reported that a long form of cFLIP (cFLIP-L) inhibits ubiquitylation of beta-catenin and enhances Wnt signaling. Here we show that cFLIP-L impairs the function of the ubiquitin-proteasome system (UPS), and increases the accumulation of various short-lived proteins, such as GFP conjugated with destabilization sequence, beta-catenin and HIF1 alpha, that are subjected to rapid ubiquitylation and degradation by proteasomes. Accordingly, beta-catenin- and HIF1 alpha-mediated gene expressions are induced in the cFLIP-L-expressing cells. Exogenously expressed cFLIP-L accumulates in aggregates at the peri-nuclear region in the cells, and the cFLIP-L aggregates are refractory to solubilization. Like exogenously expressed cFLIP-L, the endogenous cFLIP in A549 lung cancer cells displays particulate distribution in the cells and more than 60% of cFLIP-L is refractory to solubilization. Down-regulation of cFLIP in A549 cells by RNA-mediated interference reduced beta-catenin- and HIF1 alpha-mediated gene expression. These results suggest that cFLIP-L is prone to aggregate and impairs UPS function, which could be involved in the pathological function of cFLIP-L expressed in certain cancer cells.

Sunitinib, a multitargeted tyrosine-kinase inhibitor, extends survival of patients with metastatic renal cell carcinoma (mRCC) and gastrointestinal stromal tumours. Between October 2005 and March 2007, we retrospectively reviewed blood glucose level variations associated with sunitinib therapy in patients treated for mRCC. Nineteen of the patients had type II diabetes. All 19 patients had a decrease in blood glucose level (mean 1.77 mmol l(-1)) after 4 weeks of treatment. This was followed by re-elevation in the 2-week rest period. After two cycles of sunitinib administration, two patients had stopped blood glucose-lowering drugs whereas five other patients had normalised their blood glucose level. On the basis of pre-clinical data, we hypothesise that several mechanisms could be involved in this process, such as capillary regression of pancreatic islets, IGF-1 modulation through HIF1-alpha or NF-kappaB activation. In addition, a decrease of glucose uptake in the context of concomitant gastrointestinal toxicity cannot be excluded. Glycaemic control should be carefully evaluated in diabetic patients treated with sunitinib, and routine monitoring is warranted.

Psoriasis is a chronic skin disease, characterized by epidermal hyperplasia, inflammation, angiogenesis and vascular remodelling. An immunohistochemical study on fifteen cryosections of psoriatic skin was performed using antibodies against VEGF, HIF1-alpha, CD34, Factor VIII, MMP-2, MMP-9, TIMP-1 and TIMP-2. Psoriatic skin showed a diffuse VEGF positive staining (13.15+/-6.6), while no expression was observed in normal epidermis. No or faint HIF-1alpha immunostaining was detected in healthy skin, while in psoriatic skin HIF-1alpha was diffusely expressed. A positive correlation between HIF-1alpha and VEGF was reported in psoriatic skin (r= 0.644; p=0.010). In psoriatic sections CD34 expression was significantly higher in respect to control skin (19.15+/-12.61 vs 3.0+/-0.23; p= 0.04), factor VIII immunostaining also demonstrated a significant increased development of the microvasculature in comparison with healthy skin (18.39+/-8.16 vs 7.4+/-0.20; p= 0.033). Total MMP-2 expression of healthy skin (30+/-2.26) was significantly lower in respect to the MMP-2 psoriatic skin (71.5+/-4.13; p= 0.0001) and a positive correlation was observed between VEGF and MMP-2 in psoriatic patients (r= 0.688; p= 0.046). In psoriatic skin MMP-9 expression was significantly increased in comparison to control skin (31+/-3.3 vs 8+/-6.1; p=0.007). All cases of psoriatic skin tissue showed that TIMP-2 and TIMP-1 expression statistically decreased in psoriatic skin (respectively 11+/-1.2 and 12+/-1.5) in comparison with healthy skin (respectively 15+/-3.2 and 53+/-3.8; p=0.0001). In conclusion, we observed that VEGF overexpression correlated with HIF-1alpha and MMP-2 expression, underlining the role of VEGF in psoriasis as a key factor in the link between inflammation and angiogenesis.

OBJECTIVE

To study the effect of a non-viral vector (carbosilane dendrimer) to efficiently deliver small interfering RNA to postmitotic neurons to study the function of hypoxia-inducible factor-1alpha (HIF1-alpha) during chemical hypoxia-mediated neurotoxicity.

METHODS

Chemical hypoxia was induced in primary rat cortical neurons by exposure to CoCl(2). HIF1-alpha levels were determined by Western Blot and toxicity was evaluated by both MTT and LDH assays. Neurons were incubated with dendriplexes containing anti-HIF1-alpha siRNA and both uptake and HIF1-alpha knockdown efficiency were evaluated.

RESULTS

We report that a non-viral vector (carbosilane dendrimer) can deliver specific siRNA to neurons and selectively block HIF1-alpha synthesis with similar efficiency to that achieved by viral vectors. Using this method, we have found that this transcription factor plays a neuroprotective role during the early phase of chemical hypoxia-mediated neurotoxicity.

CONCLUSIONS

This work represents a proof-of-concept for the use of carbosilane dendrimers to deliver specific siRNA to postmitotic neurons to block selected protein synthesis. This indicates that this type of vector is a good alternative to viral vectors to achieve very high transfection levels in neurons. This also suggests that carbosilane dendrimers might be very useful for gene therapy.