The bacterial metabolite, lithocholic acid, has antineoplastic effects in pancreatic adenocarcinoma.

Lithocholic acid (LCA) is a secondary bile acid. LCA enters the circulation after bacterial synthesis in the gastrointestinal tract, reaches distantly located cancer cells, and influences their behavior. LCA was considered carcinogenic, but recent studies demonstrated that LCA has antitumor effects. We assessed the possible role of LCA in pancreatic adenocarcinoma. At the serum reference concentration, LCA induced a multi-pronged antineoplastic program in pancreatic adenocarcinoma cells. LCA inhibited cancer cell proliferation and induced mesenchymal-to-epithelial (MET) transition that reduced cell invasion capacity. LCA induced oxidative/nitrosative stress by decreasing the expression of nuclear factor, erythroid 2-like 2 (NRF2) and inducing inducible nitric oxide synthase (iNOS). The oxidative/nitrosative stress increased protein nitration and lipid peroxidation. Suppression of oxidative stress by glutathione (GSH) or pegylated catalase (pegCAT) blunted LCA-induced MET. Antioxidant genes were overexpressed in pancreatic adenocarcinoma and decreased antioxidant levels correlated with better survival of pancreatic adenocarcinoma patients. Furthermore, LCA treatment decreased the proportions of cancer stem cells. Finally, LCA induced total and ATP-linked mitochondrial oxidation and fatty acid oxidation. LCA exerted effects through the farnesoid X receptor (FXR), vitamin D receptor (VDR), and constitutive androstane receptor (CAR). LCA did not interfere with cytostatic agents used in the chemotherapy of pancreatic adenocarcinoma. Taken together, LCA is a non-toxic compound and has antineoplastic effects in pancreatic adenocarcinoma.

Hypoxic Conditions Modulate Chondrogenesis through the Circadian Clock: The Role of Hypoxia-Inducible Factor-1α.

Hypoxia-inducible factor-1 (HIF-1) is a heterodimer transcription factor composed of an alpha and a beta subunit. HIF-1α is a master regulator of cellular response to hypoxia by activating the transcription of genes that facilitate metabolic adaptation to hypoxia. Since chondrocytes in mature articular cartilage reside in a hypoxic environment, HIF-1α plays an important role in chondrogenesis and in the physiological lifecycle of articular cartilage. Accumulating evidence suggests interactions between the HIF pathways and the circadian clock. The circadian clock is an emerging regulator in both developing and mature chondrocytes. However, how circadian rhythm is established during the early steps of cartilage formation and through what signaling pathways it promotes the healthy chondrocyte phenotype is still not entirely known. This narrative review aims to deliver a concise analysis of the existing understanding of the dynamic interplay between HIF-1α and the molecular clock in chondrocytes, in states of both health and disease, while also incorporating creative interpretations. We explore diverse hypotheses regarding the intricate interactions among these pathways and propose relevant therapeutic strategies for cartilage disorders such as osteoarthritis.

Combining biomechanical stimulation and chronobiology: a novel approach for augmented chondrogenesis?

The unique structure and composition of articular cartilage is critical for its physiological function. However, this architecture may get disrupted by degeneration or trauma. Due to the low intrinsic regeneration properties of the tissue, the healing response is generally poor. Low-grade inflammation in patients with osteoarthritis advances cartilage degradation, resulting in pain, immobility, and reduced quality of life. Generating neocartilage using advanced tissue engineering approaches may address these limitations. The biocompatible microenvironment that is suitable for cartilage regeneration may not only rely on cells and scaffolds, but also on the spatial and temporal features of biomechanics. Cell-autonomous biological clocks that generate circadian rhythms in chondrocytes are generally accepted to be indispensable for normal cartilage homeostasis. While the molecular details of the circadian clockwork are increasingly well understood at the cellular level, the mechanisms that enable clock entrainment by biomechanical signals, which are highly relevant in cartilage, are still largely unknown. This narrative review outlines the role of the biomechanical microenvironment to advance cartilage tissue engineering via entraining the molecular circadian clockwork, and highlights how application of this concept may enhance the development and successful translation of biomechanically relevant tissue engineering interventions.

The pro- and antineoplastic effects of deoxycholic acid in pancreatic adenocarcinoma cell models.

BACKGROUND: Commensal bacteria secrete metabolites that reach distant cancer cells through the circulation and influence cancer behavior. Deoxycholic acid (DCA), a hormone-like metabolite, is a secondary bile acid specifically synthesized by intestinal microbes. DCA may have both pro- and antineoplastic effects in cancers. METHODS AND RESULTS: The pancreatic adenocarcinoma cell lines, Capan-2 and BxPC-3, were treated with 0.7 µM DCA, which corresponds to the reference concentration of DCA in human serum. DCA influenced the expression of epithelial to mesenchymal transition (EMT)-related genes, significantly decreased the expression level of the mesenchymal markers, transcription factor 7- like 2 (TCF7L2), snail family transcriptional repressor 2 (SLUG), CLAUDIN-1, and increased the expression of the epithelial genes, zona occludens 1 (ZO-1) and E-CADHERIN, as shown by real-time PCR and Western blotting. Consequently, DCA reduced the invasion capacity of pancreatic adenocarcinoma cells in Boyden chamber experiments. DCA induced the protein expression of oxidative/nitrosative stress markers. Moreover, DCA reduced aldehyde dehydrogenase 1 (ALDH1) activity in an Aldefluor assay and ALDH1 protein level, suggesting that DCA reduced stemness in pancreatic adenocarcinoma. In Seahorse experiments, DCA induced all fractions of mitochondrial respiration and glycolytic flux. The ratio of mitochondrial oxidation and glycolysis did not change after DCA treatment, suggesting that cells became hypermetabolic. CONCLUSION: DCA induced antineoplastic effects in pancreatic adenocarcinoma cells by inhibiting EMT, reducing cancer stemness, and inducing oxidative/nitrosative stress and procarcinogenic effects such as hypermetabolic bioenergetics.

The clusterin connectome: Emerging players in chondrocyte biology and putative exploratory biomarkers of osteoarthritis.

Introduction: Clusterin is a moonlighting protein that has many functions. It is a multifunctional holdase chaperone glycoprotein that is present intracellularly and extracellularly in almost all bodily fluids. Clusterin is involved in lipid transport, cell differentiation, regulation of apoptosis, and clearance of cellular debris, and plays a protective role in ensuring cellular survival. However, the possible involvement of clusterin in arthritic disease remains unclear. Given the significant potential of clusterin as a biomarker of osteoarthritis (OA), a more detailed analysis of its complex network in an inflammatory environment, specifically in the context of OA, is required. Based on the molecular network of clusterin, this study aimed to identify interacting partners that could be developed into biomarker panels for OA. Methods: The STRING database and Cytoscape were used to map and visualize the clusterin connectome. The Qiagen Ingenuity Pathway Analysis (IPA) software was used to analyze and study clusterin associated signaling networks in OA. We also analyzed transcription factors known to modulate clusterin expression, which may be altered in OA. Results: The top hits in the clusterin network were intracellular chaperones, aggregate-forming proteins, apoptosis regulators and complement proteins. Using a text-mining approach in Cytoscape, we identified additional interacting partners, including serum proteins, apolipoproteins, and heat shock proteins. Discussion: Based on known interactions with proteins, we predicted potential novel components of the clusterin connectome in OA, including selenoprotein R, semaphorins, and meprins, which may be important for designing new prognostic or diagnostic biomarker panels.

Ca2+-Activated K+ Channels in Progenitor Cells of Musculoskeletal Tissues: A Narrative Review.

Musculoskeletal disorders represent one of the main causes of disability worldwide, and their prevalence is predicted to increase in the coming decades. Stem cell therapy may be a promising option for the treatment of some of the musculoskeletal diseases. Although significant progress has been made in musculoskeletal stem cell research, osteoarthritis, the most-common musculoskeletal disorder, still lacks curative treatment. To fine-tune stem-cell-based therapy, it is necessary to focus on the underlying biological mechanisms. Ion channels and the bioelectric signals they generate control the proliferation, differentiation, and migration of musculoskeletal progenitor cells. Calcium- and voltage-activated potassium (KCa) channels are key players in cell physiology in cells of the musculoskeletal system. This review article focused on the big conductance (BK) KCa channels. The regulatory function of BK channels requires interactions with diverse sets of proteins that have different functions in tissue-resident stem cells. In this narrative review article, we discuss the main ion channels of musculoskeletal stem cells, with a focus on calcium-dependent potassium channels, especially on the large conductance BK channel. We review their expression and function in progenitor cell proliferation, differentiation, and migration and highlight gaps in current knowledge on their involvement in musculoskeletal diseases.

The temporal transcriptomic signature of cartilage formation.

Chondrogenesis is a multistep process, in which cartilage progenitor cells generate a tissue with distinct structural and functional properties. Although several approaches to cartilage regeneration rely on the differentiation of implanted progenitor cells, the temporal transcriptomic landscape of in vitro chondrogenesis in different models has not been reported. Using RNA sequencing, we examined differences in gene expression patterns during cartilage formation in micromass cultures of embryonic limb bud-derived progenitors. Principal component and trajectory analyses revealed a progressively different and distinct transcriptome during chondrogenesis. Differentially expressed genes (DEGs), based on pairwise comparisons of samples from consecutive days were classified into clusters and analysed. We confirmed the involvement of the top DEGs in chondrogenic differentiation using pathway analysis and identified several chondrogenesis-associated transcription factors and collagen subtypes that were not previously linked to cartilage formation. Transient gene silencing of ATOH8 or EBF1 on day 0 attenuated chondrogenesis by deregulating the expression of key osteochondrogenic marker genes in micromass cultures. These results provide detailed insight into the molecular mechanism of chondrogenesis in primary micromass cultures and present a comprehensive dataset of the temporal transcriptomic landscape of chondrogenesis, which may serve as a platform for new molecular approaches in cartilage tissue engineering.

Cyclic uniaxial mechanical load enhances chondrogenesis through entraining the molecular circadian clock.

The biomechanical environment plays a key role in regulating cartilage formation, but the current understanding of mechanotransduction pathways in chondrogenic cells is incomplete. Among the combination of external factors that control chondrogenesis are temporal cues that are governed by the cell-autonomous circadian clock. However, mechanical stimulation has not yet directly been proven to modulate chondrogenesis via entraining the circadian clock in chondroprogenitor cells. The purpose of this study was to establish whether mechanical stimuli entrain the core clock in chondrogenic cells, and whether augmented chondrogenesis caused by mechanical loading was at least partially mediated by the synchronised, rhythmic expression of the core circadian clock genes, chondrogenic transcription factors, and cartilage matrix constituents at both transcript and protein levels. We report here, for the first time, that cyclic uniaxial mechanical load applied for 1 h for a period of 6 days entrains the molecular clockwork in chondroprogenitor cells during chondrogenesis in limb bud-derived micromass cultures. In addition to the several core clock genes and proteins, the chondrogenic markers SOX9 and ACAN also followed a robust sinusoidal rhythmic expression pattern. These rhythmic conditions significantly enhanced cartilage matrix production and upregulated marker gene expression. The observed chondrogenesis-promoting effect of the mechanical environment was at least partially attributable to its entraining effect on the molecular clockwork, as co-application of the small molecule clock modulator longdaysin attenuated the stimulatory effects of mechanical load. This study suggests that an optimal biomechanical environment enhances tissue homoeostasis and histogenesis during chondrogenesis at least partially through entraining the molecular clockwork.

The role of bile acids in carcinogenesis.

Bile acids are soluble derivatives of cholesterol produced in the liver that subsequently undergo bacterial transformation yielding a diverse array of metabolites. The bulk of bile acid synthesis takes place in the liver yielding primary bile acids; however, other tissues have also the capacity to generate bile acids (e.g. ovaries). Hepatic bile acids are then transported to bile and are subsequently released into the intestines. In the large intestine, a fraction of primary bile acids is converted to secondary bile acids by gut bacteria. The majority of the intestinal bile acids undergo reuptake and return to the liver. A small fraction of secondary and primary bile acids remains in the circulation and exert receptor-mediated and pure chemical effects (e.g. acidic bile in oesophageal cancer) on cancer cells. In this review, we assess how changes to bile acid biosynthesis, bile acid flux and local bile acid concentration modulate the behavior of different cancers. Here, we present in-depth the involvement of bile acids in oesophageal, gastric, hepatocellular, pancreatic, colorectal, breast, prostate, ovarian cancer. Previous studies often used bile acids in supraphysiological concentration, sometimes in concentrations 1000 times higher than the highest reported tissue or serum concentrations likely eliciting unspecific effects, a practice that we advocate against in this review. Furthermore, we show that, although bile acids were classically considered as pro-carcinogenic agents (e.g. oesophageal cancer), the dogma that switch, as lower concentrations of bile acids that correspond to their serum or tissue reference concentration possess anticancer activity in a subset of cancers. Differences in the response of cancers to bile acids lie in the differential expression of bile acid receptors between cancers (e.g. FXR vs. TGR5). UDCA, a bile acid that is sold as a generic medication against cholestasis or biliary surge, and its conjugates were identified with almost purely anticancer features suggesting a possibility for drug repurposing. Taken together, bile acids were considered as tumor inducers or tumor promoter molecules; nevertheless, in certain cancers, like breast cancer, bile acids in their reference concentrations may act as tumor suppressors suggesting a Janus-faced nature of bile acids in carcinogenesis.

Pituitary Adenylate Cyclase Activating Polypeptide Has Inhibitory Effects on Melanoma Cell Proliferation and Migration In Vitro.

Pituitary adenylate cyclase activating polypeptide (PACAP) is an endogenous neuropeptide which is distributed throughout the body. PACAP influences development of various tissues and exerts protective function during cellular stress and in some tumour formation. No evidence is available on its role in neural crest derived melanocytes and its malignant transformation into melanoma. Expression of PACAP receptors was examined in human skin samples, melanoma lesions and in a primary melanocyte cell culture. A2058 and WM35 melanoma cell lines, representing two different stages of melanoma progression, were used to investigate the effects of PACAP. PAC1 receptor was identified in melanocytes in vivo and in vitro and in melanoma cell lines as well as in melanoma lesions. PACAP administration did not alter viability but decreased proliferation of melanoma cells. With live imaging random motility, average speed, vectorial distance and maximum distance of migration of cells were reduced upon PACAP treatment. PACAP administration did not alter viability but decreased proliferation capacity of melanoma cells. On the other hand, PACAP administration decreased the migration of melanoma cell lines towards fibronectin chemoattractant in the Boyden chamber. Furthermore, the presence of the neuropeptide inhibited the invasion capability of melanoma cell lines in Matrigel chambers. In summary, we provide evidence that PACAP receptors are expressed in melanocytes and in melanoma cells. Our results also prove that various aspects of the cellular motility were inhibited by this neuropeptide. On the basis of these results, we propose PACAP signalling as a possible target in melanoma progression.

Lithocholic Acid, a Metabolite of the Microbiome, Increases Oxidative Stress in Breast Cancer.

In breast cancer patients, the diversity of the microbiome decreases, coinciding with decreased production of cytostatic bacterial metabolites like lithocholic acid (LCA). We hypothesized that LCA can modulate oxidative stress to exert cytostatic effects in breast cancer cells. Treatment of breast cancer cells with LCA decreased nuclear factor-2 (NRF2) expression and increased Kelch-like ECH associating protein 1 (KEAP1) expression via activation of Takeda G-protein coupled receptor (TGR5) and constitutive androstane receptor (CAR). Altered NRF2 and KEAP1 expression subsequently led to decreased expression of glutathione peroxidase 3 (GPX3), an antioxidant enzyme, and increased expression of inducible nitric oxide synthase (iNOS). The imbalance between the pro- and antioxidant enzymes increased cytostatic effects via increased levels of lipid and protein oxidation. These effects were reversed by the pharmacological induction of NRF2 with RA839, tBHQ, or by thiol antioxidants. The expression of key components of the LCA-elicited cytostatic pathway (iNOS and 4HNE) gradually decreased as the breast cancer stage advanced. The level of lipid peroxidation in tumors negatively correlated with the mitotic index. The overexpression of iNOS, nNOS, CAR, KEAP1, NOX4, and TGR5 or the downregulation of NRF2 correlated with better survival in breast cancer patients, except for triple negative cases. Taken together, LCA, a metabolite of the gut microbiome, elicits oxidative stress that slows down the proliferation of breast cancer cells. The LCA-oxidative stress protective pathway is lost as breast cancer progresses, and the loss correlates with poor prognosis.

Cadaverine, a metabolite of the microbiome, reduces breast cancer aggressiveness through trace amino acid receptors.

Recent studies showed that changes to the gut microbiome alters the microbiome-derived metabolome, potentially promoting carcinogenesis in organs that are distal to the gut. In this study, we assessed the relationship between breast cancer and cadaverine biosynthesis. Cadaverine treatment of Balb/c female mice (500 nmol/kg p.o. q.d.) grafted with 4T1 breast cancer cells ameliorated the disease (lower mass and infiltration of the primary tumor, fewer metastases, and lower grade tumors). Cadaverine treatment of breast cancer cell lines corresponding to its serum reference range (100-800 nM) reverted endothelial-to-mesenchymal transition, inhibited cellular movement and invasion, moreover, rendered cells less stem cell-like through reducing mitochondrial oxidation. Trace amino acid receptors (TAARs), namely, TAAR1, TAAR8 and TAAR9 were instrumental in provoking the cadaverine-evoked effects. Early stage breast cancer patients, versus control women, had reduced abundance of the CadA and LdcC genes in fecal DNA, both responsible for bacterial cadaverine production. Moreover, we found low protein expression of E. coli LdcC in the feces of stage 1 breast cancer patients. In addition, higher expression of lysine decarboxylase resulted in a prolonged survival among early-stage breast cancer patients. Taken together, cadaverine production seems to be a regulator of early breast cancer.

Lithocholic acid, a bacterial metabolite reduces breast cancer cell proliferation and aggressiveness.

Our study aimed at finding a mechanistic relationship between the gut microbiome and breast cancer. Breast cancer cells are not in direct contact with these microbes, but disease could be influenced by bacterial metabolites including secondary bile acids that are exclusively synthesized by the microbiome and known to enter the human circulation. In murine and bench experiments, a secondary bile acid, lithocholic acid (LCA) in concentrations corresponding to its tissue reference concentrations (< 1 µM), reduced cancer cell proliferation (by 10-20%) and VEGF production (by 37%), aggressiveness and metastatic potential of primary tumors through inducing mesenchymal-to-epithelial transition, increased antitumor immune response, OXPHOS and the TCA cycle. Part of these effects was due to activation of TGR5 by LCA. Early stage breast cancer patients, versus control women, had reduced serum LCA levels, reduced chenodeoxycholic acid to LCA ratio, and reduced abundance of the baiH (7α/ß-hydroxysteroid dehydroxylase, the key enzyme in LCA generation) gene in fecal DNA, all suggesting reduced microbial generation of LCA in early breast cancer.