Acidic Environment Leads to ROS-Induced MAPK Signaling in Cancer Cells

Tumor micromilieu often shows pronounced acidosis forcing cells to adapt their phenotype towards enhanced tumorigenesis induced by altered cellular signalling and transcriptional regulation. In the presents study mechanisms and potential consequences of the crosstalk between extra- and intracellular pH (pHe, pHi) and mitogen-activated-protein-kinases (ERK1/2, p38) was analyzed. Data were obtained mainly in AT1 R-3327 prostate carcinoma cells, but the principle importance was confirmed in 5 other cell types. Extracellular acidosis leads to a rapid and sustained decrease of pHi in parallel to p38 phosphorylation in all cell types and to ERK1/2 phosphorylation in 3 of 6 cell types. Furthermore, p38 phosphorylation was elicited by sole intracellular lactacidosis at normal pHe. Inhibition of ERK1/2 phosphorylation during acidosis led to necrotic cell death. No evidence for the involvement of the kinases c-SRC, PKC, PKA, PI3K or EGFR nor changes in cell volume in acidosis-induced MAPK activation was obtained. However, our data reveal that acidosis enhances the formation of reactive oxygen species (ROS), probably originating from mitochondria, which subsequently trigger MAPK phosphorylation. Scavenging of ROS prevented acidosis-induced MAPK phosphorylation whereas addition of H2O2 enhanced it. Finally, acidosis increased phosphorylation of the transcription factor CREB via p38, leading to increased transcriptional activity of a CRE-reporter even 24 h after switching the cells back to a normal environmental milieu. Thus, an acidic tumor microenvironment can induce a longer lasting p38-CREB-medited change in the transcriptional program, which may maintain the altered phenotype even when the cells leave the tumor environment

Preliminary assessment of the imaging capability of the YAP-(S)PET small animal scanner in neuroscience

The new and fully engineered version of the YAP–(S)PET small animal scanner has been tested at the University of Mainz for preliminary assessment of its imaging capability for studies related to neuropharmacology and psychiatry. The main feature of the scanner is the capability to combine PET and SPECT techniques. It allows the development of new and interesting protocols for the investigation of many biological phenomena, more effectively than with PET or SPECT modalities alone. The scanner is made up of four detector heads, each one composed of a 4 � 4c m 2 of YAlO3:Ce (or YAP:Ce) matrix, and has a field of view (FOV) of 4 cm axially � 4c m + transaxially. In PET mode, the volume resolution is less than 8 mm 3 and is nearly constant over the whole FOV, while the sensitivity is about 2%. The SPECT performance is not so good, due to the presence of the multi-hole lead collimator in front of each head. Nevertheless, the YAP–PET scanner offers excellent resolution and sensitivity for performing on the availability of D2-like dopamine receptors on mice and rats in both PET and SPECT modalities

Causes and Consequences of A Glutamine Induced Normoxic HIF1 Activity for the Tumor Metabolism

The transcription factor hypoxia-inducible factor 1 (HIF1) is the crucial regulator of genes that are involved in metabolism under hypoxic conditions, but information regarding the transcriptional activity of HIF1 in normoxic metabolism is limited. Different tumor cells were treated under normoxic and hypoxic conditions with various drugs that affect cellular metabolism. HIF1ff was silenced by siRNA in normoxic/hypoxic tumor cells, before RNA sequencing and bioinformatics analyses were performed while using the breast cancer cell line MDA-MB-231 as a model. Differentially expressed genes were further analyzed and validated by qPCR, while the activity of the metabolites was determined by enzyme assays. Under normoxic conditions, HIF1 activity was significantly increased by (i) glutamine metabolism, which was associated with the release of ammonium, and it was decreased by (ii) acetylation via acetyl CoA synthetase (ACSS2) or ATP citrate lyase (ACLY), respectively, and (iii) the presence of L-ascorbic acid, citrate, or acetyl-CoA. Interestingly, acetylsalicylic acid, ibuprofen, L-ascorbic acid, and citrate each significantly destabilized HIF1ff only under normoxia. The results from the deep sequence analyses indicated that, in HIF1-siRNA silenced MDA-MB-231 cells, 231 genes under normoxia and 1384 genes under hypoxia were transcriptionally significant deregulated in a HIF1-dependent manner. Focusing on glycolysis genes, it was confirmed that HIF1 significantly regulated six normoxic and 16 hypoxic glycolysis-associated gene transcripts. However, the results from the targeted metabolome analyses revealed that HIF1 activity affected neither the consumption of glucose nor the release of ammonium or lactate; however, it significantly inhibited the release of the amino acid alanine. This study comprehensively investigated, for the first time, how normoxic HIF1 is stabilized, and it analyzed the possible function of normoxic HIF1 in the transcriptome and metabolic processes of tumor cells in a breast cancer cell model. Furthermore, these data imply that HIF1 compensates for the metabolic outcomes of glutaminolysis and, subsequently, theWarburg effect might be a direct consequence of the altered amino acid metabolism in tumor cells

Tumor Acidosis and Hypoxia Differently Modulate the Inflammatory Program: Measurements In Vitro and In Vivo

Inflammatory mediators produced by the tumor cells are of importance for immune response but also for malignant progression. The aim of the study was to analyze the expression of monocyte chemoattractant protein-1, interleukin-6 (IL-6), tumor necrosis factor-α, inducible isoform of nitric oxide synthase (iNOS), cyclooxygenase-2, and osteopontin in vitro in two different tumor cell lines under hypoxia (pO2 ≈ 1.5 mmHg) and/or acidosis (pH = 6.6) for up to 24 hours since hypoxia and acidosis are common characteristics of solid tumors. Additionally, the same tumor cell lines implanted in vivo were made hypoxic and acidotic artificially for 24 hours, after which the cytokine expression was measured. Finally, the activation of ERK1/2 and p38 by acidosis/hypoxia and their impact on cytokine expression were studied. The results indicate that acidosis and hypoxia have fundamentally different (often opposing) effects on cytokine expression. In addition, these effects were tumor cell line specific. When combining hypoxia and acidosis, the overall changes reflect an additive effect of both conditions alone, indicating that hypoxia and acidosis act by independent mechanisms. The in vivo changes corresponded well with the results obtained in the isolated tumor cells. Only iNOS expression was downregulated in vivo but increased in cell culture. For IL-6 expression, the acidosis-induced changes were dependent on ERK1/2 activation. In conclusion, it was demonstrated that the environmental pO2 and pH strongly affect the expression of inflammatory mediators in tumor cells. In vivo, most of the inflammatory mediators were downregulated, which could limit the activation of immune cells and by this foster the immune escape of tumors

Impact of Extracellular Acidity on the Activity of P-glycoprotein and the Cytotoxicity of Chemotherapeutic Drugs

The expression and activity of P-glycoprotein (pGP) play a role in the multidrug resistance of tumors. Because solid-growing tumors often show pronounced hypoxia or extracellular acidosis, this study attempted to analyze the impact of an acidic environment on the expression and activity of pGP and on the cytotoxicity of chemotherapeutic agents. For this, prostate carcinoma cells were exposed to an acidic extracellular environment (pH 6.6) for up to 24 hours. pGP activity was more than doubled after 3 to 6 hours of incubation in acidic medium, whereas cellular pGP expression remained constant, indicating that increased transport rate is the result of functional modulation. In parallel, the cytotoxic efficacy of daunorubicin showed pronounced reduction at low pH, an effect that was reversible on coincubation with a pGP inhibitor. A reduction of intracellular Ca(2+) concentration by 35% under acidic conditions induced a higher transport rate of pGP, an effect comparable to that found on inhibition of protein kinase C (PKC). These data indicate that pGP activity is increased by acidic pH presumably as a result of lowered intracellular calcium levels and inhibition of PKC. These findings may explain the reduced cytotoxicity of chemotherapeutic agents in hypoxic/acidic tumors

Supplementary Material: Publication in "Advances in Experimental Medicine and Biology"

Supplementary Material for the publication "Impact of acute or chronic acidosis and hypoxia on gene expression patterns in tumor cells: potential functional implications" in Advances in Experimental Medicine and Biology</p

Extracellular Acidosis Modulates the Expression of Epithelial-Mesenchymal Transition (EMT) Markers and Adhesion of Epithelial and Tumor Cells

Epithelial-to-mesenchymal transition (EMT) is an important process of tumor progression associated with increased metastatic potential. EMT can be activated by external triggers such as cytokines or metabolic parameters (e.g. hypoxia). Since extracellular acidosis is a common finding in tumors, the aim of the study is to analyze its impact on the expression of EMT markers in vitro and in vivo as well as the functional impact on cell adhesion. Therefore, three tumor and two normal epithelial cell lines were incubated for 24 h at pH 6.6 and the expression of EMT markers was studied. In addition, mRNA expression of transcription and metabolic factors related to EMT was measured as well as the functional impact on cell adhesion, either during acidic incubation or after priming cells in an acidic milieu. E-cadherin and N-cadherin were down-regulated in all tumor and normal cell lines studied, whereas vimentin expression increased in only two tumor and one normal cell line. Down-regulation of the cadherins was seen in total protein and to a lesser extent in surface protein. In vivo an increase in N-cadherin and vimentin expression was found. Acidosis up-regulated Twist1 and Acsl1 but down-regulated fumarate hydratase (Fh). Cell adhesion during acidic incubation decreased in AT1 prostate carcinoma cells whereas preceding acidic priming increased their subsequent adhesion. Low tumor pH is able to modulate the expression EMT-related proteins and by this may affect the stability of the tissue structure

Hyperbaric oxygenation and glucose/amino acids substitution in human severe placental insufficiency

In the first case, the AA and glucose were infused through a perinatal port system into the umbilical vein at 30 weeks' gestation due to severe IUGR. The patient received daily hyperbaric oxygenation (HBO, 100% O2) with 1.4 atmospheres absolute for 50 min for 7 days. At 31+4 weeks' gestation, the patient gave birth spontaneously to a newborn weighing 1378 g, pH 7.33, APGAR score 4/6/intubation. In follow‐up examinations at 5 years of age, the boy was doing well without any neurological disturbance or developmental delay. In the second case, the patient presented at 25/5 weeks' gestation suffering from severe IUGR received HBO and maternal AA infusions. The cardiotocography was monitored continuously during HBO treatment. The short‐time variations improved during HBO from 2.9 to 9 msec. The patient developed pathologic CTG and uterine contractions 1 day later and gave birth to a hypotrophic newborn weighing 420 g. After initial adequate stabilization, the extremely preterm newborn unfortunately died 6 days later. Fetal nutrition combined with HBO is technically possible and may allow the prolongation of the pregnancy. Fetal‐specific amino‐acid composition would facilitate the treatment options of IUGR fetuses and extremely preterm newborn