Induction Of Alkaline Phosphatase Research Articles

Intestinal epithelial differentiation and barrier function is promoted in vitro by a Cynara cardunculus L. leaf extract through AMPK pathway activation

Gut epithelial barrier perturbation leads to leaky gut syndrome and permeation of substances activating immune response. Polyphenols can improve intestinal barrier function and represent candidates for preventing development of leaky gut. Herein, we evaluated in vitro the molecular mechanisms involved in the protective effects of a polyphenol-rich extract from leaves of Cynara cardunculus L. (CCLE) on intestinal barrier function and integrity on Caco-2 human epithelial cells. Treatment with CCLE from seeding until complete differentiation improved intestinal function by increasing trans-epithelial electrical resistance (TEER), reducing paracellular permeability to fluorescein, and promoting faster recovery of tight junctions (TJ) assembly in the Ca2+ switch assay. CCLE stimulated epithelial cell differentiation inducing alkaline phosphatase activity and TJ proteins. These CCLE-induced effects were attributed to activation of AMP-activated protein kinase (AMPK) pathway. Our data support the use of Cynara cardunculus L. leaves, an agricultural co-product rich in bioactive polyphenols, for the health of intestinal epithelium.

Odontogenic and anti-inflammatory effects of magnesium-doped bioactive glass in vital pulp therapy

This study aimed to investigate the effects of magnesium-doped bioactive glass (Mg-BG) on the mineralization, odontogenesis, and anti-inflammatory abilities of human dental pulp stem cells (hDPSCs). Mg-BG powders with different Mg concentrations were successfully synthesized via the sol-gel method and evaluated using x-ray diffraction, Fourier-transform infrared spectroscopy, scanning electron microscopy, and transmission electron microscopy. Apatite formation was observed on the surfaces of the materials after soaking in simulated body fluid. hDPSCs were cultured with Mg-BG powder extracts in vitro, and no evident cytotoxicity was observed. Mg-BG induced alkaline phosphatase (ALP) expression and mineralization of hDPSCs and upregulated the expression of odontogenic genes, including those encoding dentin sialophosphoprotein, dentin matrix protein 1, ALP, osteocalcin, and runt-related transcription factor 2. Moreover, Mg-BG substantially suppressed the secretion of inflammatory cytokines (interleukin [IL]-4, IL-6, IL-8, and tumor necrosis factor-alpha). Collectively, the results of this study suggest that Mg-BG has excellent in vitro bioactivity and is a potential material for vital pulp therapy of inflamed pulps.

Repair of Rat Calvarial Critical-Sized Defects Using Heparin-Conjugated Fibrin Hydrogel Containing BMP-2 and Adipose-Derived Pericytes.

The repair of critical-sized calvarial defects is a challenging problem for orthopedic surgery. One of the promising strategies of bone bioengineering to enhance the efficacy of large bone defect regeneration is the combined delivery of stem cells with osteoinductive factors within polymer carriers. The purpose of the research was to study the regenerative effects of heparin-conjugated fibrin (HCF) hydrogel containing bone morphogenetic protein 2 (BMP-2) and adipose-derived pericytes (ADPs) in a rat critical-sized calvarial defect model. In vitro analysis revealed that the HCF hydrogel was able to control the BMP-2 release and induce alkaline phosphatase (ALP) activity in neonatal rat osteoblasts. In addition, it was found that eluted BMP-2 significantly induced the osteogenic differentiation of ADPs. It was characterized by the increased ALP activity, osteocalcin expression and calcium deposits in ADPs. In vivo studies have shown that both HCF hydrogel with BMP-2 and HCF hydrogel with pericytes are able to significantly increase the regeneration of critical-sized calvarial defects in comparison with the control group. Nevertheless, the greatest regenerative effect was found after the co-delivery of ADPs and BMP-2 into a critical-sized calvarial defect. Thus, our findings suggest that the combined delivery of ADPs and BMP-2 in HCF hydrogel holds promise to be applied as an alternative biopolymer for the critical-sized bone defect restoration.

DP2, a Carbohydrate Derivative, Enhances In Vitro Osteoblast Mineralisation.

Bone fracture healing is a complex biological process involving four phases coordinated over time: hematoma formation, granulation tissue formation, bony callus formation, and bone remodelling. Bone fractures represent a significant health problem, particularly among the elderly population and patients with comorbidities. Therapeutic strategies proposed to treat such fractures include the use of autografts, allografts, and tissue engineering strategies. It has been shown that bone morphogenetic protein 2 (BMP-2) has a therapeutic potential to enhance fracture healing. Despite the clinical efficacy of BMP-2 in osteoinduction and bone repair, adverse side effects and complications have been reported. Therefore, in this in vitro study, we propose the use of a disaccharide compound (DP2) to improve the mineralisation process. We first evaluated the effect of DP2 on primary human osteoblasts (HOb), and then investigated the mechanisms involved. Our findings showed that (i) DP2 improved osteoblast differentiation by inducing alkaline phosphatase activity, osteopontin, and osteocalcin expression; (ii) DP2 induced earlier in vitro mineralisation in HOb cells compared to BMP-2 mainly by earlier activation of Runx2; and (iii) DP2 is internalized in HOb cells and activates the protein kinase C signalling pathway. Consequently, DP2 is a potential therapeutical candidate molecule for bone fracture repair.

Enzyme-induced morphological transformation of self-assembled peptide nanovehicles potentiates intratumoral aggregation and inhibits tumour immunosuppression

Conventional intratumoural drug delivery systems often struggle to achieve both high permeability and long-term retention. Smaller nanoparticles (NPs) readily diffuse into deep tumour regions, but their rapid clearance results in lower accumulation at tumour sites. Furthermore, although immunostimulatory molecules have been introduced for the immunotherapy of tumours, it is crucial to develop drug nanovehicles with immunomodulatory functions. Here, a novel self-assembled amphiphilic peptide, IpYR, self-assembled into spherical NPs after drug loading, which were transformed into nanofibres (NFs) with a high aspect ratio under the induction of alkaline phosphatase (ALP). This drug-loading system effectively prolonged the residence time of the chemotherapeutic drug in tumour tissues and displayed enhanced aggregation at tumour sites in vivo. Furthermore, the transformable peptide NPs not only inhibited tumour growth but also suppressed tumour metastasis. Importantly, the peptide nanovehicles inhibited the proliferation of Treg cells, downregulated the expression of immunosuppressive factors and PD-L1, and simultaneously stimulated the expression of immunogenic factors, effectively alleviating the immunosuppressive microenvironment. In conclusion, this transformable peptide-based nanodelivery system is a concise and promising nanoplatform for maintaining potent drug enrichment and inhibiting immunosuppression in the tumour microenvironment.

Zearalenone-Induced Mechanical Damage of Intestinal Barrier via the RhoA/ROCK Signaling Pathway in IPEC-J2 Cells.

Zearalenone (ZEN) is a widespread contaminant of cereals and agricultural products which causes food safety issues. Ingesting food or feed contaminated with ZEN can disrupt the intestinal epithelial barrier function. The RhoA/ROCK signaling pathway plays a key role in regulating the epithelial barrier function, but studies on such roles have rarely focused on the intestine. The aim of this experiment was to investigate the exact mechanism of ZEN-induced intestinal barrier damage and whether the RhoA/ROCK signaling pathway is involved. The results showed that ZEN significantly induced alkaline phosphatase (AP) activity and FITC–dextran (4 kDa) passage across the epithelial barrier, which significantly reduced the transepithelial resistance (TEER). Meanwhile, ZEN could induce the significantly down-regulated mRNA expression of tight junction proteins (occludin, claudin-1, ZO-1, and claudin-3) and redistribution of ZO-1 immunofluorescence. Further studies demonstrated that ZEN exposure activated the RhoA/ROCK signaling pathway, significantly up-regulated the mRNA expression of ROCK1, the main effector of the signaling pathway, the protein expression of phosphorylated myosin light chain (MLC) and myosin light chain kinase (MLCK), and relatively increased the activity of ATP in cells, simultaneously remodeling the cytoskeleton (F-actin). Overall, our study indicated that ZEN induced intestinal barrier dysfunction by activating the RhoA/ROCK signaling pathway.

8. Controlling the rate of presentation of BMP-2 from a highly structured composite for interbody fusion

<h3>BACKGROUND CONTEXT</h3> Clinical outcomes for patients with intervertebral disc injury or disease could be enhanced by a new generation of regenerative matrices that create tissue environments to control angiogenesis, chondrogenesis and bone formation. Regenerative matrices are a new class of controlled release biomaterials that use surface chemistry, materials architecture, and drug delivery kinetics to create optimised conditions for cell interactions that result in high quality tissue formation. A novel biodegradable polymer matrix that releases bone morphogenetic protein 2 (BMP-2) over a period of one month with a low Cmax and sustained presentation of active protein is investigated in this study. <h3>PURPOSE</h3> BMP-2 release kinetics and protein activity from a new regenerative matrix was quantified to develop an implant with 1-month release of the drug within a macroporous matrix. A rabbit posterolateral (PLF) vertebral fusion model was performed to assess bone formation and fusion outcomes as a function of dose and source of BMP-2 (E coli and CHO cells). <h3>STUDY DESIGN/SETTING</h3> The study was designed to investigate the effect of various biomaterial design factors and manufacturing methods on the rate of release of BMP-2, the activity of BMP-2 and the extent of in vivo bone formation. <h3>PATIENT SAMPLE</h3> This study did not involve patients. <h3>OUTCOME MEASURES</h3> Outcome measures were the mechanical properties of the regenerative matrix, the architecture and porosity of the matrix, BMP-2 release kinetics, BMP-2 activity as a function of release time and bone formation. <h3>METHODS</h3> BMP-2 activity was tested using two in vitro cell-based assays. BMP-2 solutions and regenerative matrix extracts were tested for their ability to induce alkaline phosphatase (ALP) activity in W-20-17 stromal cells using a modified version of the ASTM standard protocol for in vitro biological activity of recombinant human BMP-2 (F2131-02). Cells were exposed to serial dilutions of BMP-2 for 24 h, assayed for ALP activity and EC50s calculated allowing the relative potency to NIBSC WHO BMP-2 standard to be calculated. BMP-2 release was assessed with an in vitro assay of ALP activity in the mouse muscle myoblast cell line C2C12. Formulations were suspended above a C2C12 monolayer in an insert with a porous (8 µm) PET membrane. Following 3-4 days of exposure, the inserts containing LDGraft were transferred to fresh cells for a further 3-4 days. Cells exposed to LDGraft in this manner were assayed for ALP activity (normalised to protein content) once the inserts had been transferred to fresh cells. Fusion rate and new bone formation were compared to INFUSE in a single-level lumbar (L4-L5) posterolateral (PLF) vertebral fusion model in the New Zealand White rabbit based on standard protocols (Boden et al, 1995, ASTM F3207). Pre-formed implants (3 cc) of INFUSE (rhBMP-2 and absorbable collagen sponge) were implanted on decorticated dorsal surfaces of the transverse processes of L4 and L5 adjacent to the laminae. Fusion was assessed by radiograph at 6 weeks and by manual palpation, radiography and microCT at 12 weeks. <h3>RESULTS</h3> BMP-2 activity was retained over one month of release with the addition of protectants in the formulation and precise control of manufacturing processes. The matrix had a porosity of >70% with a high proportion of macropores (>200 micron diameter). Low doses of E. coli-produced BMP-2 induced fusion in the PLF model. <h3>CONCLUSIONS</h3> A one-month releasing BMP-2 formulation is described. This new regenerative matrix had internal architecture designed to host angiogenesis. Manufacturing and design challenges can be overcome to retain high biologic activity throughout the release period. <h3>FDA DEVICE/DRUG STATUS</h3> BMP-2, not approved for this indication.

Collagen-Based Osteogenic Nanocoating of Microrough Titanium Surfaces.

The aim of the present study was to develop a collagen/heparin-based multilayer coating on titanium surfaces for retarded release of recombinant human bone morphogenic protein 2 (rhBMP2) to enhance the osteogenic activity of implant surfaces. Polyelectrolyte multilayer (PEM) coatings were constructed on sandblasted/acid-etched surfaces of titanium discs using heparin and collagen. PEM films of ten double layers were produced and overlayed with 200 µL of a rhBMP2 solution containing 15 µg rhBMP2. Subsequently, cross-linking of heparin molecules was performed using EDC/NHS chemistry to immobilize the incorporated rhBMP2. Release characteristics for 3 weeks, induction of Alkaline Phosphatase (ALP) in C2C12 cells and proliferation of human mesenchymal stem cells (hMSCs) were evaluated to analyze the osteogenic capacity of the surface. The coating incorporated 10.5 µg rhBMP2 on average per disc and did not change the surface morphology. The release profile showed a delivery of 14.5% of the incorporated growth factor during the first 24 h with a decline towards the end of the observation period with a total release of 31.3%. Cross-linking reduced the release with an almost complete suppression at 100% cross-linking. Alkaline Phosphatase was significantly increased on day 1 and day 21, indicating that the growth factor bound in the coating remains active and available after 3 weeks. Proliferation of hMSCs was significantly enhanced by the non-cross-linked PEM coating. Nanocoating using collagen/heparin-based PEMs can incorporate clinically relevant amounts of rhBMP2 on titanium surfaces with a retarded release and a sustained enhancement of osteogenic activity without changing the surface morphology.

Phosphate-inducible poly-hydroxy butyrate production dynamics in CO2 supplemented upscaled cultivation of engineered Phaeodactylum tricornutum

Diatoms such as Phaeodactylum tricornutum are emerging as sustainable alternatives to traditional eukaryotic microbial cell factories. In order to facilitate a viable process for production of heterologous metabolites, a rational genetic design specifically tailored to metabolic requirements as well as optimised culture conditions are required. In this study we investigated the effect of constitutive and inducible expression of the heterologous poly-3-hydroxybutyrate (PHB) pathway in P. tricornutum using non-integrative episomes in 3 different configurations. Constitutive expression led to downregulation of at least one individual gene out of three (phaA, phaB and phaC) and was outperformed by inducible expression. To further asses and optimise the dynamics of PHB accumulation driven by the inducible alkaline phosphatase 1 promoter, we upscaled the production to lab-scale bioreactors and tested the effect of supplemented CO2 on biomass and PHB accumulation. While ambient CO2 cultivation resulted in a maximum PHB yield of 2.3% cell dry weight (CDW) on day 11, under elevated CO2 concentrations PHB yield peaked at 1.7% CDW on day 8, coincident with PHB titres at 27.9 mg L−1 that were approximately threefold higher than ambient CO2. With other more valuable bio-products in mind, these results highlight the importance of the genetic design as well as substrate availability to supply additional reduction equivalents to boost biomass accumulation and relieve potential enzymatic bottlenecks for improved product accumulation.

Discovery of prescopranone, a key intermediate in scopranone biosynthesis.

A key intermediate in scopranone biosynthesis, prescopranone, accumulated in the mycelium of Streptomyces avermitilis SUKA carrying the biosynthetic gene cluster for scopranone lacking the sprT encoding the monooxygenase. The structure of prescopranone was elucidated by NMR and other spectral data. Prescopranone consists of a 2-pyranone ring with two atypical scoop-like moieties (1-ethyl-1-propenyl and 2-ethylbutyl groups), which was deduced as a product of the modular polyketide syntheses encoded by sprA, sprB, and sprC. Prescopranone inhibited bone morphogenetic protein (BMP)-induced alkaline phosphatase activity in a BMP receptor mutant cell line.

Thermodynamic basis for comparative photobiomodulation dosing with multiple wavelengths to direct odontoblast differentiation.

Multiple wavelength devices are now available for photobiomodulation (PBM) treatments, but their dosimetry for individual or combinatorial use remains unclear. The present work investigated the effects of 447, 532, 658, 810, 980 and 1064 nm wavelengths on odontoblast differentiation at 10mW/cm2 using either equal treatment time for conventional fluence (300 seconds for 3J/cm2 ) or varying times to adjust for individual wavelength photon fluence (4.6p.J/cm2 ). Both 447 and 810 nm significantly increased alkaline phosphatase (ALP) activity, while 1064 nm showed reduced ALP activity at 3J/cm2 . However, ALP induction was significantly improved when equivalent photon fluence dosing was used. Other wavelengths did not show significant changes compared to untreated controls. The data suggest that accounting for wavelength-specific photon energy transfer during PBM dosing could improve clinical safety and efficacy.

Calcium Phosphate-Coated and Strontium-Incorporated Mesoporous Silica Nanoparticles Can Effectively Induce Osteogenic Stem Cell Differentiation.

Ceramic (nano)materials are promising materials for bone regeneration applications. The addition of bioinorganics such as strontium (Sr) and zinc (Zn) is a popular approach to further improve their biological performance. However, control over ion delivery is important to prevent off-target effects. Mesoporous silica nanoparticles (MSNs) are popular nanomaterials that can be designed to incorporate and controllably deliver multiple ions to steer specific regenerative processes. In this work, MSNs loaded with Sr (MSNSr ) and surface coated with a pH-sensitive calcium phosphate (MSNSr -CaP) or calcium phosphate zinc layer (MSNSr -CaZnP) are developed. The ability of the MSNs to promote osteogenesis in human mesenchymal stromal cells (hMSCs) under basic cell culture conditions is explored and compared to ion administration directly to the cell culture media. Here, it is shown that MSN-CaPs can effectively induce alkaline phosphatase (ALP) levels and osteogenic gene expression in the absence of other osteogenic stimulants, where an improved effect is observed for MSNs surface coated with multiple ions. Moreover, comparatively lower ion doses are needed when using MSNs as delivery vehicles compared to direct ion administration in the medium. In summary, the MSNs developed here represent promising vehicles to deliver (multiple) bioinorganics and promote hMSC osteogenesis in basic conditions.

Enzyme-Induced Transformable Peptide Nanocarriers with Enhanced Drug Permeability and Retention to Improve Tumor Nanotherapy Efficacy.

Temporal persistence is as important for nanocarriers as spatial accuracy. However, because of the insufficient aggreagtion and short retention time of chemotherapy drugs in tumors, their clinical application is greatly limited. A drug delivery approach dependent on the sensitivity to an enzyme present in the microenvironment of the tumor is designed to exhibit different sizes in different sites, achieving enhanced drug permeability and retention to improve tumor nanotherapy efficacy. In this work, we report a small-molecule peptide drug delivery system containing both tumor-targeting groups and enzyme response sites. This system enables the targeted delivery of peptide nanocarriers to tumor cells and a unique response to alkaline phosphatase (ALP) in the tumor microenvironment to activate morphological transformation and drug release. The amphiphilic peptide AYR self-aggregated into a spherical nanoparticle structure after encapsulating the lipid-soluble model drug doxorubicin (DOX) and rapidly converted to nanofibers via the induction of ALP. This morphological transformation toward a high aspect ratio allowed rapid, as well as effective drug release to tumor location while enhancing specific toxicity to tumor cells. Interestingly, this "transformer"-like drug delivery strategy can enhance local drug accumulation and effectively inhibit drug efflux. In vitro along with in vivo experiments further proved that the permeability and retention of antitumor drugs in tumor cells and tissues were significantly enhanced to reduce toxic side effects, and the therapeutic effect was remarkably improved compared with that of nondeformable drug-loaded peptide nanocarriers. The developed AYR nanoparticles with the ability to undergo morphological transformation in situ can improve local drug aggregation and retention time at the tumor site. Our findings provide a new and simple method for nanocarrier morphology transformation in novel cancer treatments.

Naringin Improves Osteoblast Mineralization and Bone Healing and Strength through Regulating Estrogen Receptor Alpha-Dependent Alkaline Phosphatase Gene Expression.

Phytoestrogens are strongly recommended for treating osteoporosis. Our previous study showed that naringin, a citrus flavonoid, can enhance the bone mass in ovariectomized rats. In this study, we further elucidated the mechanisms of naringin-induced osteoblast maturation and bone healing. Treatment of human osteoblasts with naringin increased cell viability and proliferation. In parallel, exposure to naringin enhanced translocation of estrogen receptor alpha (ERα) to nuclei and its transactivation activity. Sequentially, naringin induced alkaline phosphatase (ALP) mRNA and protein expression and its enzyme activity. Pretreatment with methylpiperidinopyrazole (MPP), a specific inhibitor of ERα, attenuated naringin-induced augmentations in ERα transactivation activity, ALP gene expression, and cell mineralization. The beneficial effects of naringin were also confirmed in mouse MC3T3-E1 cells. Moreover, administration of mice with a bone defect with naringin increased levels of ERα and ALP in damaged sites and simultaneously enhanced the healing rate and bone strength. Nevertheless, treatment with MPP weakened naringin-triggered expression of ERα and ALP and improved bone healing and mass. Therefore, naringin could improve osteoblast mineralization and bone healing via regulating ERα-dependent ALP gene expression. Naringin can be clinically applied for treatment of osteoporosis-related bone diseases.

Ecological Importance of Alkaline Phosphatase Activity in Changing Marine Environmental Conditions

The effects of changing hydrological and climatic conditions on the dynamics of phosphorus (P) were studied in the Northern Adriatic Sea (NA), a coastal system currently affected by these changes. P limitation is one of the key stresses in the NA and it is a globally important phenomenon in aquatic ecosystems. Therefore, the response to P stress by inducing alkaline phosphatase activity (APA) was studied in characteristic water types in the NA, formed by the changing freshwater input in different thermic conditions. APA was important in providing P for microbial growth in upper waters dominated by assimilation during the warmer part of the year in stratified conditions. Contrarily, APA was not important during mixing in the colder part of the year, as well as in waters dominated by regeneration. In waters influenced by freshwater, temperature had no effect on APA, while in high-salinity waters, temperature was an important factor for APA increase. The highest APA occurred during riverine nutrients supply, indicating that the alteration of anthropogenic nutrient loads might strongly change P status in this coastal system. Furthermore, predicted increases in winter sea temperature and summer Po River discharge could delay the water column mixing, prolonging periods of P limitation.

Effects of rice fermented extracts, "Sake Lees", on the functional activity of odontoblast-like cells (KN-3 cells).

This study was designed to investigate the effects of Sake Lees extracts (SLE, Sake Kasu) on the functional activity of odontoblastic cells and tooth pulp of the rats. For in vitro studies, a rat clonal odontoblast-like cell line, KN-3 cells were cultured. SLE significantly decreased KN-3 cell proliferation, but showed no significant cytotoxicity. SLE effects on several protein productions of KN-3 cells were compared with PBS. SLE and PBS increased alkaline phosphatase (ALP), dentin sialoprotein (DSP), and osterix in a day-course dependent manner, while SLE increased the induction of ALP on day 9-21 and DSP on day 15-21. SLE also increased Runx2 expression on day 3 and 9 compared to PBS. Alizarin Red stainings revealed that SLE showed a subtle increase in mineralization of KN-3 cells on day 15 and 21. A histological investigation was conducted to assess if SLE induced reparative dentin formation after direct capping at the exposed tooth pulp in rats, suggesting that SLE could increase the reparative dentin formation more than PBS. These findings suggest that Sake Lees could have functional roles in the alterations of odontoblastic activity, which might influence the physiology of the tooth pulp.

Enzyme-induced morphological transformation of drug carriers: Implications for cytotoxicity and the retention time of antitumor agents

Nanocarriers have been widely employed to deliver chemotherapeutic drugs for cancer treatment. However, the insufficient accumulation of nanoparticles in tumors is an important reason for the poor efficacy of nanodrugs. In this study, a novel drug delivery system with a self-assembled amphiphilic peptide was designed to respond specifically to alkaline phosphatase (ALP), a protease overexpressed in cancer cells. The amphiphilic peptide self-assembled into spherical and fibrous nanostructures, and it easily assembled into spherical drug-loaded peptide nanoparticles after loading of a hydrophobic chemotherapeutic drug. The cytotoxicity of the drug carriers was enhanced against tumor cells over time. These spherical nanoparticles transformed into nanofibers under the induction of ALP, leading to efficient release of the encapsulated drug. This drug delivery strategy relying on responsiveness to an enzyme present in the tumor microenvironment can enhance local drug accumulation at the tumor site. The results of live animal imaging showed that the residence time of the morphologically transformable drug-loaded peptide nanoparticles at the tumor site was prolonged in vivo, confirming their potential use in antitumor therapy. These findings can contribute to a better understanding of the influence of drug carrier morphology on intracellular retention.

Abstract 1159: Hexamethylene bisacetamide (HMBA) inhibits the induction of alkaline phosphatase by butyrate in some bladder and colon cancer cell lines

Abstract Induction of alkaline phosphatase in colon cancer cells by inhibitors of histone deacetylase activity has been widely used to monitor the induction of differentiation. We have previously identified three human bladder cancer cell lines in which alkaline phosphatase activity can be induced by inhibitors of histone deacetylase (5637, HT1197 and HT1376 cells). In earlier work it was observed that some bromodomain inhibitors inhibit growth and induction of alkaline phosphatase in these cells. After a recent report that HMBA acts as a bromodomain inhibitor, the effects of HMBA on growth and induction of alkaline phosphatase were investigated in two colon cancer cell lines (Caco-2 and HT29) and in 5637, HT1197 and HT1376 bladder cancer cells. Growth was monitored by staining with sulforhodamine B. Incubation with 2 mM butyrate for 72 hours caused induction of alkaline phosphatase in the five cell lines. In some cell lines there was inhibition of growth and of the induction of alkaline phosphatase activity by 2 mM HMBA. Increasing the HMBA concentration to 5 mM caused significant growth inhibition in all five cell lines and significant inhibition of induction of alkaline phosphatase activity by 2 mM butyrate in the 5637, Caco-2 and HT1376 cells, whether expressed as total activity or activity per unit protein. Although the effect of HMBA on induction of alkaline phosphatase in HT29 cells was not statistically significant there is evidence in the literature for inhibition with longer incubation times. The most marked effects on enzyme induction were seen with the HT1376 bladder cancer cells and this distinction was in line with previous studies on the combined effects of butyrate and the well-established bromodomain inhibitor, JQ1. HMBA has been found by several investigators to be an effective inducer of differentiation in erythroleukemia cells as judged by hemoglobin production. The lack of induction of alkaline phosphatase by HMBA as a single agent in any of the cell lines investigated in the present work suggests that different mechanisms of induction of differentiation operate in erythroleukemia cells than in bladder and colon cancer cells. Citation Format: Michael A. Lea. Hexamethylene bisacetamide (HMBA) inhibits the induction of alkaline phosphatase by butyrate in some bladder and colon cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1159.

Contributions of the Endothelium to Vascular Calcification.

Vascular calcification (VC) increases morbidity and mortality and constitutes a significant obstacle during percutaneous interventions and surgeries. On a cellular and molecular level, VC is a highly regulated process that involves abnormal cell transitions and osteogenic differentiation, re-purposing of signaling pathways normally used in bone, and even formation of osteoclast-like cells. Endothelial cells have been shown to contribute to VC through a variety of means. This includes direct contributions of osteoprogenitor cells generated through endothelial-mesenchymal transitions in activated endothelium, with subsequent migration into the vessel wall. The endothelium also secretes pro-osteogenic growth factors, such as bone morphogenetic proteins, inflammatory mediators and cytokines in conditions like hyperlipidemia, diabetes, and renal failure. High phosphate levels caused by renal disease have deleterious effects on the endothelium, and induction of tissue non-specific alkaline phosphatase adds to the calcific process. Furthermore, endothelial activation promotes proteolytic destruction of the internal elastic lamina that serves, among other things, as a stabilizer of the endothelium. Appropriate bone mineralization is highly dependent on active angiogenesis, but it is unclear whether the same relationship exists in VC. Through its location facing the vascular lumen, the endothelium is the first to encounter circulating factor and bone marrow-derived cells that might contribute to osteoclast-like versus osteoblast-like cells in the vascular wall. In the same way, the endothelium may be the easiest target to reach with treatments aimed at limiting calcification. This review provides a brief summary of the contributions of the endothelium to VC as we currently know them.

Rifampicin induces the bone form of alkaline phosphatase in humans.

Pregnane X receptor (PXR) is a xenobiotic-sensing nuclear receptor that regulates drug metabolism in the liver and intestine. In our clinical trials on healthy volunteers to discover novel metabolic functions of PXR activation, we observed that rifampicin, a well-established ligand for human PXR, 600mg daily for a week, increased the plasma alkaline phosphatase (ALP) significantly compared with the placebo. Further analysis with lectin affinity electrophoresis revealed that especially the bone form of ALP was elevated. To investigate the mechanism(s) of bone ALP induction, we employed osteoblast lineage differentiated from human primary bone marrow-derived mesenchymal stromal cells. Rifampicin treatment increased ALP activity and mRNA level of bone biomarker genes (ALP, MGP, OPN and OPG). PXR expression was detected in the cells, but the expression was very low compared with the human liver. To further investigate the potential role of PXR in the ALP induction, we treated mice and rats with a rodent PXR ligand pregnenolone 16α-carbonitrile (PCN). However, PCN treatment did not increase plasma ALP activity or bone ALP mRNA expression. In conclusion, rifampicin treatment induces the bone form of ALP in the serum of healthy human volunteers. Further studies are required to establish the mechanism of this novel finding.