Metabolic adaptation in epithelial ovarian cancer metastasis.

Epithelial ovarian cancer (EOC) is highly lethal due to its unique metastatic characteristics. EOC spheroids enter a non-proliferative state, with hypoxic cores and reduced oncogenic signaling, all of which contribute to tumour dormancy during metastasis. We investigated the metabolomic states of EOC cells progressing through the three steps to metastasis. Metabolomes of adherent, spheroid, and re-adherent cells were validated by isotopic metabolic flux analysis and mitochondrial functional assays to identify metabolic pathways that were previously unknown to promote EOC metastasis. Although spheroids were thought to exist in a dormant state, metabolomic analysis revealed an unexpected upregulation of energy production pathways in spheroids, accompanied by increased abundance of tricarboxylic acid (TCA) cycle and electron transport chain proteins. Tracing of 13C-labelled glucose and glutamine showed increased pyruvate carboxylation and decreased glutamine anaplerosis in spheroids. Increased reductive carboxylation suggests spheroids adjust redox homeostasis by shuttling cytosolic NADPH into mitochondria via isocitrate dehydrogenase. Indeed, we observed spheroids have increased respiratory capacity and mitochondrial ATP production. Relative to adherent cells, spheroids reduced serine consumption and metabolism, processes which were reversed upon spheroid re-adherence. The data reveal a distinct metabolism in EOC spheroids that enhances energy production by the mitochondria while maintaining a dormant state with respect to growth and proliferation. The findings advance our understanding of EOC metastasis and identify the TCA cycle and mitochondrional activity as novel targets to disrupt EOC metastasis, providing new approaches to treat advanced disease.

Simulated Microgravity Influences VEGF, MAPK, and PAM Signaling in Prostate Cancer Cells.

Prostate cancer is one of the leading causes of cancer mortality in men worldwide. An unusual but unique environment for studying tumor cell processes is provided by microgravity, either in space or simulated by ground-based devices like a random positioning machine (RPM). In this study, prostate adenocarcinoma-derived PC-3 cells were cultivated on an RPM for time periods of 3 and 5 days. We investigated the genes associated with the cytoskeleton, focal adhesions, extracellular matrix, growth, survival, angiogenesis, and metastasis. The gene expression of signaling factors of the vascular endothelial growth factor (VEGF), mitogen-activated protein kinase (MAPK), and PI3K/AKT/mTOR (PAM) pathways was investigated using qPCR. We performed immunofluorescence to study the cytoskeleton, histological staining to examine the morphology, and a time-resolved immunofluorometric assay to analyze the cell culture supernatants. When PC-3 cells were exposed to simulated microgravity (s-µg), some cells remained growing as adherent cells (AD), while most cells detached from the cell culture flask bottom and formed multicellular spheroids (MCS). After 3-day RPM exposure, PC-3 cells revealed significant downregulation of the VEGF, SRC1, AKT, MTOR, and COL1A1 gene expression in MCS, whereas FLT1, RAF1, MEK1, ERK1, FAK1, RICTOR, ACTB, TUBB, and TLN1 mRNAs were not significantly changed. ERK2 and TLN1 were elevated in AD, and FLK1, LAMA3, COL4A5, FN1, VCL, CDH1, and NGAL mRNAs were significantly upregulated in AD and MCS after 3 days. After a 5-day culture in s-µg, the PC-3 cells showed significant downregulations of VEGF mRNA in AD and MCS, and FN1, CDH1, and LAMA3 in AD and SCR1 in MCS. In addition, we measured significant upregulations in FLT1, AKT, ERK1, ERK2, LCN2, COL1A1, TUBB, and VCL mRNAs in AD and MCS, and increases in FLK1, FN1, and COL4A5 in MCS as well as LAMB2, CDH1, RAF1, MEK1, SRC1, and MTOR mRNAs in AD. FAK1 and RICTOR were not altered by s-µg. In parallel, the secretion rate of VEGFA and NGAL proteins decreased. Cytoskeletal alterations (F-actin) were visible, as well as a deposition of collagen in the MCS. In conclusion, RPM-exposure of PC-3 cells induced changes in their morphology, cytoskeleton, and extracellular matrix protein synthesis, as well as in their focal adhesion complex and growth behavior. The significant upregulation of genes belonging to the PAM pathway indicated their involvement in the cellular changes occurring in microgravity.

Beneficial Effects of Low Frequency Vibration on Human Chondrocytes in Vitro.

BACKGROUND/AIMS: In articular cartilage, chondrocytes are the predominant cell type. A long-term stay in space can lead to bone loss and cartilage breakdown. Due to the poor regenerative capacity of cartilage, this may impair the crewmembers' mobility and influence mission activities. Beside microgravity other factors such as cosmic radiation and vibration might be important for cartilage degeneration. Vibration at different frequencies showed various effects on cartilage in vivo, but knowledge about its impact on chondrocytes in vitro is sparse. METHODS: Human chondrocytes were exposed to a vibration device, simulating the vibration profile occurring during parabolic flights, for 24 h (VIB) and compared to static controls. Phase-contrast microscopy, immunofluorescence, F-actin and TUNEL staining as well as quantitative real-time PCR were performed to examine effects on morphology, cell viability and shape as well as gene expression. The results were compared to earlier studies using semantic analyses. RESULTS: No morphological changes or cytoskeletal alterations were observed in VIB and no apoptotic cells were found. A reorganization and increase in fibronectin were detected in VIB samples by immunofluorescence technique. PXN, VCL, ANXA1, ANXA2, BAX, and BCL2 revealed differential regulations. CONCLUSION: Long-term VIB did not damage human chondrocytes in vitro. The reduction of ANXA2, and up-regulation of ANXA1, PXN and VCL mRNAs suggest that long-term vibration might even positively influence cultured chondrocytes.

A Special Focus on Selexipag - Treatment of Pulmonary Arterial Hypertension.

BACKGROUND: This review focuses on the treatment of pulmonary arterial hypertension (PAH) with selexipag and compares its drug-related therapeutic effects with those of prostacyclin analogues. METHODS: We searched the relevant literature and summarized the current clinical trials concerning selexipag and PAH. RESULTS: With only few cases per million, PAH is a rare disease, but when untreated it can be life-threatening. PAH is linked to elevated levels of endothelin (ET-1) and decreased levels of nitric oxide (NO) and prostacyclin (PGI2). PAH-specific therapeutic approaches concentrate on these characteristics with drugs targeting the endothelin- receptor (e.g. bosentan), phosphodiesterase-5 (e.g. sildenafil) or the prostacyclin-receptor (e.g. treprostinil). Recently, the new drug selexipag acting as a non-prostanoid IP2-receptor agonist has been approved for PAH therapy. The active form of selexipag (ACT-333679) was designed by the help of a medicinal chemistry program and it was further modified by replacing the terminal carboxyl group with an N-acylsulfonamide group to form the more stable oral drug, selexipag. Selexipag has a high selectivity for the IP2-receptor and differs from conventional prostacyclin analogues in its chemical structure. In the GRIPHON trial selexipag was demonstrated to significantly improve the primary composite endpoint of death or complications related to PAH (hazard ratio 0.6, 99% confidence interval, 0.46 to 0.78; P < 0.001) as well as exercise capacity in the form of the 6-minute walk distance (12.0 m treatment effect, 99% confidence interval, 1 to 24; P = 0.003). However, no significant reduction in all-cause mortality was achieved. Selexipag has also shown promising results in combination therapy with phosphodiesterase-5 inhibitors (PDE-5i) and/or endothelin receptor antagonists (ERA). The most common adverse effects (AEs), associated with selexipag, are headache, diarrhea, jaw pain, and nausea. Nevertheless, Selexipag was generally well tolerated during the GRIPHON trial. CONCLUSIONS: Selexipag is a valuable addition to PAH therapeutics especially by reducing the PAH-related hospitalizations and thus improving quality of life in PAH patients.