Acidosis-Induced Regulation of Egr1 and Ccn1 In Vitro and in Experimental Tumours In Vivo.

Extracellular acidosis is a characteristic of solid tumours, resulting from hypoxia-induced glycolytic metabolism as well as from the "Warburg effect" (aerobic glycolysis). The acidic environment has shown to affect functional tumour properties (proliferation, migration, invasion) and thus the aim of the study was to identify signalling mechanisms, mediating these pH-dependent effects. Therefore, the serum response factor (Srf) and the activation of the serum response element (SRE) by acidosis were analysed in AT-1 prostate carcinoma cells. Furthermore, the expression of downstream targets of this cascade, namely the early growth response 1 (Egr1), which seems to be involved in tumour proliferation, and the cellular communication network factor 1 (Ccn1), which both contain SRE in their promotor region were examined in two tumour cell lines. Extracellular acidification led to an upregulation of Srf and a functional activation of the SRE. Egr1 expression was increased by acidosis in AT-1 cells whereas hypoxia had a suppressive effect. In experimental tumours, in vivo Egr1 and Ccn1 were also found to be acidosis-dependent. Also, it turned out that pH regulated expression of Egr1 was followed by comparable changes of p21, which is an important regulator of the cell cycle.This study identifies the Srf-SRE signalling cascade and downstream Egr1 and Ccn1 to be acidosis-regulated in vitro and in vivo, potentially affecting tumour progression. Especially linked expression changes of Egr1 and p21 may mediate acidosis-induced effects on cell proliferation.

Evaluation of Betulinic Acid Derivatives as PET Tracers for Hypoxia-Induced Carbonic Anhydrase IX (CA IX) Expression.

Non-invasive visualisation of the expression of hypoxia-related proteins, such as carbonic anhydrase IX (CA IX), by positron emission tomography (PET) could provide important information on the oxygenation status of tumours. Since betulinic acid derivatives bind specifically to CA IX the aim of the study was the development betulinic acid-based 68Ga-labelled PET tracers and to evaluate the hypoxia detecting properties in vitro and in vivo. The binding of betulinic acid (B-DOTA) and betulinyl-3-sulfamate (BS-DOTA) was assessed in two rat tumour cell lines (AT1 prostate and Walker-256 mammary carcinomas). AT1 cells express CA IX in a hypoxia-dependent manner whereas Walker-256 cells, expressing almost no CA IX in wildtype, were transfected with the rat Car9 gene. In vivo measurements were carried out in a small animal PET/CT in AT1 tumours in rats breathing room air, 8% or 100% O2. In AT1 cells hypoxia-induced overexpression of CA IX led to a stronger binding of BS-DOTA but not of B-DOTA. The BS-DOTA binding correlated linearly with the CA IX protein expression and could be blocked by an excess of unlabelled tracer. In the transfected Walker-256 cells no specific binding of either of the tracers was seen. In vivo the intratumoral accumulation of BS-DOTA was increased in animals kept under inspiratory hypoxia and reduced by hyperoxia. Therefore, betulinyl-3-sulfamate could be used as a PET tracer of CA IX expression in tumours and to provide information about the oxygenation status. However, accumulation data indicated that binding not only depends on hypoxia-induce CA IX expression but also on the tumour-line-specific basal expression and on the initial oxygenation status of the tumour.

Role of the mTOR Signalling Pathway During Extracellular Acidosis in Tumour Cells.

The metabolic microenvironment of solid tumours is often dominated by extracellular acidosis which results from glycolytic metabolism. Acidosis can modulate gene expression and foster the malignant progression. The aim of the study was to analyse the effects of extracellular acidosis on the mTOR signalling pathway, an important regulator of anabolic and catabolic processes like cell proliferation and autophagy. The study was performed in two tumour cell lines, AT-1 prostate and Walker-256 mammary carcinoma cells. Cells were incubated at pH 7.4 or 6.6 for 3 h and 24 h. Then RNA and protein were extracted and analysed by qPCR and western blot. mTOR and P70-S6 kinase (P70-S6K), an important downstream target of mTOR, as well as the autophagic flux were studied. The effect of acidosis on P70S6K phosphorylation was compared to pharmacological mTOR inhibition with LY294002 and rapamycin. In both cell lines the total mTOR expression was not altered by acidosis, however, the mTOR phosphorylation was reduced after 3 h but not after 24 h. The P70S6K phosphorylation was reduced at both time points comparable to changes by pharmacological mTOR inhibitors. The autophagic flux, also a target of mTOR and measured by LC3-II expression, was increased in both cell lines after 24 h of acidosis. The results of this study indicate that mTOR signalling is inhibited by extracellular acidosis which then lead to a reduced activity of the P70-S6 kinase (modulating gene expression) and increased autophagy possibly mediated by ULK1/2 activity. These finding may offer new perspectives for therapeutic interventions in acidic tumours.

Impact of Acidosis-Regulated MicroRNAs on the Expression of Their Target Genes in Experimental Tumors In Vivo.

In comparison to normal tissue, solid tumors show an acidic extracellular pH, which results from hypoxia-induced glycolytic metabolism and the Warburg effect. Since acidosis modulates the expression of different microRNAs (e.g., miR-7, miR-183, miR-203, miR-215), microRNAs and their targets might be mediators between tumor acidosis and malignant behavior. The aim of this study was to investigate how modulation of these microRNAs affects the expression of their targets (Crem, cAMP-responsive element modulator; Gls2, glutaminase 2; Txnip, thioredoxin-interacting protein) in experimental tumors in vivo and whether these changes are acidosis dependent. The study was performed in two experimental tumor lines of the rat (AT-1 prostate carcinoma, Walker-256 mammary carcinoma). The results showed that all three targets were regulated by acidosis in vivo, Crem and Gls2 being downregulated and Txnip upregulated in both models. In AT-1 tumors at normal tumor pH, miR-203 overexpression increased Txnip expression by about 75%, whereas in Walker-256 tumors, miR-7 reduced protein expression. In more acidic tumors, no impact of microRNAs on Txnip expression was seen. On the other hand, Gls2 was significantly increased in acidic tumors by miR-183 or miR-7 overexpression (cell line dependent). As this increase was not present under control conditions, an acidosis-dependent effect can be assumed. These results indicate that tumor acidosis modulates the expression of targets of pH-sensitive microRNAs in experimental tumors. Especially the protein expression of Gls2 might be regulated via changes of microRNAs, which then affects the malignant progression of tumors.

Extracellular Acidosis Regulates the Expression of Inflammatory Mediators in Rat Epithelial Cells.

Acidification of the cellular microenvironment is found in different pathological states such as inflammation, ischemia and in solid tumors. It can affect cell function and phenotype, and by this aggravate the pathological process. Epithelial cells are a relevant functional part in several normal organs as well as in tumors and will thus be challenged by the acidic extracellular pH (acidosis). Therefore, the impact of acidosis on the expression of different inflammatory mediators (MCP-1, IL-6, osteopontin, iNOS, TNF-α, and COX-2), as well as the role of different signaling pathways regulating the expression, was studied in epithelial normal rat kidney cells (NRK-52E). Acidosis led to an increase in TNF-α expression but a down-regulation of MCP-1, iNOS and COX-2. Expression of IL-6 was only slightly modulated, while osteopontin was not regulated at all. Since acidosis activates ERK1/2 and p38 signaling in NRK-52E cells, the impact of MAP kinase signaling pathways on the expression of the inflammatory markers was analyzed. At normal pH, blocking ERK1/2 or p38 decreased the level of MCP-1, iNOS and partly TNF-α. However, the effect of acidosis on the expression of inflammatory mediators was not affected by inhibition of the MAP kinase pathways. In conclusion, our results show that an acidic microenvironment affects the transcriptional program of epithelial cells. Low pH mostly reduced the expression of pathological relevant genes and might thus repress inflammatory processes induced by epithelial cells.

Expression of MicroRNAs in Fibroblasts and Macrophages Is Regulated by Hypoxia-Induced Extracellular Acidosis.

Under pathological conditions like inflammation, ischemia or in solid tumors, parameters of the microenvironment like local oxygenation and extracellular pH show marked changes when compared to healthy tissue. The altered microenvironment affects cellular phenotype of omnipresent fibroblasts and immune cells. Recently, the impact of the microenvironment on the expression patterns of microRNAs, small non-coding RNAs that regulate gene expression on a post-transcriptional level, was discussed. Therefore, microRNAs might be the link between altered microenvironmental parameters and changes in cellular phenotype. In this study, the effect of hypoxia-induced extracellular acidosis (24 h pH 6.6) on microRNA expression in fibroblasts and macrophages was analyzed. MicroRNAs in rat fibroblasts (NRK-49F) were examined with the miScript miRNA PCR Array and changes in the expression validated by TaqMan qPCR. Subsequently, the identified microRNAs were analyzed in RAW 264.7 mouse macrophages. Nine out of 84 tested microRNAs were found to be acidosis-regulated in fibroblasts by miRNA PCR array, most of them up-regulated. Of those, the pH dependency could be validated by TaqMan qPCR for five of these nine microRNAs. When comparing these microRNAs in terms of their expression in macrophages, profound differences were observed. Thus, acidosis-induced alterations in the expression of microRNAs seem to be cell-type specific. Only the up-regulation of the miR-133b by low pH was seen in all normal cells, but not in tumor cells. As the identified microRNAs are involved in the regulation of proliferation, cell death and migration (amongst others), acidosis-induced changes in their expression might affect cellular behavior of fibroblasts and macrophages under pathological conditions. For instance the proto-oncogene c-Jun, which is a target of the miR-133b, was shown to be acidosis-regulated. Acidosis could regulate the biological behavior via miRNA-133b and c-Jun.

Hypoxia-Related Tumor Acidosis Affects MicroRNA Expression Pattern in Prostate and Breast Tumor Cells.

MicroRNAs (miRNAs) are small non-coding RNA sequences which are able to modulate the expression of many functional proteins. The expression level of miRNAs can be modulated by parameters of the tumor microenvironment like hypoxia, nutrient deprivation or oxidative stress. Since miRNAs can act either as oncogenes or tumor suppressors, this may affect malignant progression or therapy resistance. In the present study it was analyzed whether extracellular acidosis can impact on miRNA expression. Therefore, tumor cells (R3327-AT-1 prostate and Walker-256 mammary carcinoma cells) were incubated at pH 6.6 (acidosis) or pH 7.4 (control) for 24 h and changes in miRNA expression were analyzed by PCR array for 84 cancer-associated miRNAs and Next-Generation Sequencing (NGS) with a panel of 765 miRNAs.In the cancer-related PCR array an acidosis-induced reduction of 5 miRNAs in AT-1 and 6 miRNAs in Walker-256 cells was seen. The miR-203a was consensually down-regulated in both cell lines. Using NGS, 19 miRNAs were found to be upregulated and 14 miRNAS were downregulated in AT-1 prostate cancer cells. In Walker-256 cells the expression of 21 miRNAs was increased and decreased for 17 miRNAs. Eleven miRNAs were regulated by acidosis in both tumor cell lines in the same direction.Acidosis induced changes in the miRNA expression of prostate and breast carcinoma cells. However, miRNA profiles differed strongly between the tumor cell lines (and between the experimental methods used), indicating that cells can react individually to microenvironmental stress. However, some miRNAs were consensually regulated in both cell lines and thus might represent a general cellular response to an extracellular acidosis.

Impact of the Tumor Microenvironment on the Expression of Inflammatory Mediators in Cancer Cells.

Hypoxia and extracellular acidosis are common features of solid malignant tumors. The aim of the study was to analyze whether these pathophysiological parameters affect the expression of inflammatory mediators in tumor cells. Therefore the mRNA expression of MCP-1 (monocyte chemotactic protein 1), iNOS and osteopontin was measured under hypoxic (pO2 1 mmHg) and acidotic (pH 6.6) conditions by qPCR in AT1 R-3327 prostate cancer cells. In addition, the underlying signaling cascades were analyzed by using inhibitors of the p38 and ERK1/2 MAP kinase pathways.Hypoxia led to a significant decrease of the expression of MCP-1 and osteopontin over the complete observation period of 24 h, whereas the iNOS expression after an initial reduction slightly increased. Acidotic conditions for up to 6 h increased the iNOS expression significantly which was functional as indicated by an elevated level of nitrate/nitrite formation by 30 %. Acidosis had almost no impact on the MCP-1 expression of tumor cells, whereas the osteopontin level tended to increase leading to a significantly elevated level after 24 h at pH 6.6. Inhibiting the p38 and ERK1/2 under control conditions revealed that the MAPKs play a significant role for the regulation of the expression of inflammatory mediators. MCP-1 expression could be lowered by inhibiting ERK1/2 whereas iNOS expression was dependent on both p38 and ERK1/2 MAPK. These results indicate that the adverse tumor microenvironment affects the expression of inflammatory mediators by tumors cells and may therefore modulate the immune response within the tumor tissue.

Acidosis Promotes Metastasis Formation by Enhancing Tumor Cell Motility.

The tumor microenvironment is characterized by hypoxia, acidosis as well as other metabolic and biochemical alterations. Its role in cancer progression is increasingly appreciated especially on invasive capacity and the formation of metastasis. The effect of acidosis on metastasis formation of two rat carcinoma cell lines was studied in the animal model. In order to analyze the pH dependency of different steps of metastasis formation, invasiveness, cell adhesion and migration of AT-1 prostate cancer cells as well as possible underlying cell signaling pathways were studied in vitro. Acidosis significantly increased the formation of lung metastases of both tumor cell lines in vivo. In vitro, extracellular acidosis neither enhanced invasiveness nor affected cell adhesion to a plastic or to an endothelial layer. However, cellular motility was markedly elevated at pH 6.6 and this effect was sustained even when extracellular pH was switched back to pH 7.4. When analyzing the underlying mechanism, a prominent role of ROS in the induction of migration was observed. Signaling through the MAP kinases ERK1/2 and p38 as well as Src family kinases was not involved. Thus, cancer cells in an acidic microenvironment can acquire enhanced motility, which is sustained even if the tumor cells leave their acidic microenvironment e.g. by entering the blood stream. This increase depended on elevated ROS production and may contribute to the augmented formation of metastases of acidosis-primed tumor cells in vivo.

Acidic environment activates inflammatory programs in fibroblasts via a cAMP-MAPK pathway.

The tissue micromilieu in disorders (inflammation, ischemia, tumor) often shows pronounced metabolic acidosis that may alter signaling and transcriptional activity in resident cells which can be of special importance for omnipresent fibroblasts. In the present study we investigated the impact of metabolic acidosis on rat fibroblasts with special emphasis on their role in inflammation by regulation of TNF-α, MCP-1, COX-2 and iNOS expression and the signaling pathways involved. Extracellular acidosis led to an enhanced expression of TNF-α, COX-2 and iNOS in parallel to an activation of p38 and ERK1/2 kinases that was not observed by sole intracellular acidosis. Accordingly, the protein amounts of TNF-α and COX-2 as well as the production of nitrate and nitrite were elevated. Acidosis-induced expression of COX-2 and iNOS depended on p38 kinase, but not on ERK1/2. In contrast acidosis-induced TNF-α expression was independent of both kinases. Although GPR4, GPR68 and GPR132 are expressed in fibroblasts, the involvement of these potential candidate pH sensors could be ruled out since no acidosis-induced elevation in intracellular cAMP or free calcium content was observed. Furthermore our data show that MAPK activation by an acidic micromilieu depends on Ser/Thr phosphatase activity, but not on the production of reactive oxygen species and is sensitive to cAMP antagonism by Rp-cAMPS. In conclusion, our results show that an acidic microenvironment induces a differential transcriptional program of pathological relevant genes in fibroblasts via the cAMP-phosphatase-MAPK pathway and thereby generates a parainflammatory situation that can result in tissue remodeling.

Self-assembly and conformation of tetrapyridyl-porphyrin molecules on Ag(111).

We present a low-temperature scanning tunneling microscopy (STM) study on the supramolecular ordering of tetrapyridyl-porphyrin (TPyP) molecules on Ag(111). Vapor deposition in a wide substrate temperature range reveals that TPyP molecules easily diffuse and self-assemble into large, highly ordered chiral domains. We identify two mirror-symmetric unit cells, each containing two differently oriented molecules. From an analysis of the respective arrangement it is concluded that lateral intermolecular interactions control the packing of the layer, while its orientation is induced by the coupling to the substrate. This finding is corroborated by molecular mechanics calculations. High-resolution STM images recorded at 15 K allow a direct identification of intramolecular features. This makes it possible to determine the molecular conformation of TPyP on Ag(111). The pyridyl groups are alternately rotated out of the porphyrin plane by an angle of 60 degrees.

The incidence of radio-frequency impulsive noise within hospital buildings: initial measurements in the 450 MHz band.

This paper reports on the incidence and level of man-made electrical impulse noise capable of causing interference to low-power, ASK and FSK radio biotelemetry links. Measurements were made at two acute hospitals, one in a residential area of Belfast, Northern Ireland and the other in an industrial part of Berlin, Germany. Monitoring was effected in the 450 MHz band using a communications receiver with its AGC line disabled and AM-detector AM output fed to a PC-based logger. The RF bandwidth was 20 kHz and a rotatable folded-dipole receiving antenna was used. Seven-day impulse counts were recorded at each location, for separate RF input thresholds of -100, -110 and -120 dBm and a common trigger window of 10 micros-10 ms. Histograms depicting the average pulse count per minute are presented; values range from a maximum of 55x10(3)/min for -120 dBm sensitivity in Berlin, to a minimum of 1.2/min for -100 dBm sensitivity in Belfast, both with vertical polarisation.