Effects of ß-caryophyllene and oxygen availability on cholesterol and fatty acids in breast cancer cells.

Hypoxia is a common feature of most solid tumors, one that favors tumor progression and limits treatment effectiveness. Targeting hypoxia has long been a goal in cancer therapy, by identifying factors that reverse or ameliorate the effects of hypoxia on cancer cells. We, and others, have shown that ß-caryophyllene (BCP) exhibits anti-proliferative properties in cancer cells. We have further shown that non-cytotoxic concentrations of BCP affect cholesterol and lipid biosynthesis in hypoxic hBrC cells at both transcriptional and translational levels. This led us to hypothesize that BCP may reverse the hypoxic phenotype in hBrC cells. To test this, we determined the effect of BCP on hypoxic sensitive pathways, including oxygen consumption, glycolysis, oxidative stress, cholesterol and fatty acid biosynthesis, and ERK activation. While each of these studies revealed new information on the regulation by hypoxia and BCP, only the lipidomic studies showed reversal of hypoxic-dependent effects by BCP. These later studies showed that hypoxia-treated samples lowered monounsaturated fatty acid levels, shifting the saturation ratios of the fatty acid pools. This signature was ameliorated by sub-lethal concentrations of BCP, possibly through an effect on the C:16 fatty acid saturation ratios. This is consistent with BCP-induced upregulation of the stearoyl-CoA desaturase (SCD) gene, observed previously. This suggests that BCP may interfere with the lipid signature modulated by hypoxia which could have consequences for membrane biosynthesis or composition, both of which are important for cell replication.

Do you know when the inhaler is empty?

BACKGROUND: Medication review is recommended at asthma appointments. The presence of propellant in the metered dose inhalers (MDIs) makes it challenging to identify when the inhaler is empty. The COVID-19 pandemic has resulted in move towards more virtual monitoring of care. We aimed to evaluate if patients identify when the inhaler is empty and the method of inhaler disposal. METHODS: Prospective, multicentre quality improvement project. Data collected from children with asthma and other respiratory conditions. OUTCOME MEASURES: Children/carers attending hospital were asked how they identify an empty salbutamol inhaler; dose counters in the preventer inhalers and disposal practices were reviewed. RESULTS: 157 patients recruited. 125 (73.5%) patients deemed an empty inhaler as either full/partially full. 12 of 66 (18.2%) preventer inhalers with a dose counter were empty. 83% disposed their inhalers in a dustbin. CONCLUSIONS: Patients cannot reliably identify when their MDI is empty. There is an urgent need for improving inhaler technology and providing appropriate guidance on how to identify when an MDI is empty.

Inhibition of Carbonic Anhydrase Using SLC-149: Support for a Noncatalytic Function of CAIX in Breast Cancer.

Carbonic anhydrase IX (CAIX) is considered a target for therapeutic intervention in solid tumors. In this study, the efficacy of the inhibitor, 4-(3-(2,4-difluorophenyl)-oxoimidazolidin-1-yl)benzenesulfonamide (SLC-149), is evaluated on CAIX and a CAIX-mimic. We show that SLC-149 is a better inhibitor than acetazolamide against CAIX. Binding of SLC-149 thermally stabilizes CAIX-mimic at lower concentrations compared to that of CAII. Structural examinations of SLC-149 bound to CAIX-mimic and CAII explain binding preferences. In cell culture, SLC-149 is a more effective inhibitor of CAIX activity in a triple-negative breast cancer cell line than previously studied sulfonamide inhibitors. SLC-149 is also a better inhibitor of activity in cells expressing CAIX versus CAXII. However, SLC-149 has little effect on cytotoxicity, and high concentrations are required to inhibit cell growth, migration, and invasion. These data support the hypothesis that CAIX activity, shown to be important in regulating extracellular pH, does not underlie its ability to control cell growth.

A non-catalytic function of carbonic anhydrase IX contributes to the glycolytic phenotype and pH regulation in human breast cancer cells.

The most aggressive and invasive tumor cells often reside in hypoxic microenvironments and rely heavily on rapid anaerobic glycolysis for energy production. This switch from oxidative phosphorylation to glycolysis, along with up-regulation of the glucose transport system, significantly increases the release of lactic acid from cells into the tumor microenvironment. Excess lactate and proton excretion exacerbate extracellular acidification to which cancer cells, but not normal cells, adapt. We have hypothesized that carbonic anhydrases (CAs) play a role in stabilizing both intracellular and extracellular pH to favor cancer progression and metastasis. Here, we show that proton efflux (acidification) using the glycolytic rate assay is dependent on both extracellular pH (pHe) and CA IX expression. Yet, isoform-selective sulfonamide-based inhibitors of CA IX did not alter proton flux, which suggests that the catalytic activity of CA IX is not necessary for this regulation. Other investigators have suggested the CA IX co-operates with the MCT transport family to excrete protons. To test this possibility, we examined the expression patterns of selected ion transporters and show that members of this family are differentially expressed within the molecular subtypes of breast cancer. The most aggressive form of breast cancer, triple-negative breast cancer, appears to co-ordinately express the monocarboxylate transporter 4 (MCT4) and carbonic anhydrase IX (CA IX). This supports a possible mechanism that utilizes the intramolecular H+ shuttle system in CA IX to facilitate proton efflux through MCT4.

ß-caryophyllene enhances the transcriptional upregulation of cholesterol biosynthesis in breast cancer cells.

ß-caryophyllene (BCP) exhibits anti-proliferative properties in cancer cells. Here, we examine the hypothesis that BCP induces membrane remodeling. Our data show that high concentrations of BCP increase membrane permeability of human breast cells (hBrC) causing detachment and cell death. At a sub-lethal concentration of BCP, we show that BCP induces a striking upregulation of genes involved in cholesterol biosynthesis, including the gene that encodes for HMGCoA reductase (HMGCR), the rate-determining step in cholesterol biosynthesis. In addition, stearoyl-CoA desaturase (SCD) is also upregulated which would lead to the enhanced formation of monounsaturated fatty acids, specifically oleate and palmitoleate from stearoyl CoA and palmitoyl CoA, respectively. These fatty acids are major components of membrane phospholipids and cholesterol esters. Together, these data suggest that cells respond to BCP by increasing the synthesis of components found in membranes. These responses could be viewed as a repair mechanism and/or as a mechanism to mount resistance to the cytotoxic effect of BCP. Blocking HMGCR activity enhances the cytotoxicity of BCP, suggesting that BCP may provide an additional therapeutic tool in controlling breast cancer cell growth.

Selective inhibition of carbonic anhydrase IX over carbonic anhydrase XII in breast cancer cells using benzene sulfonamides: Disconnect between activity and growth inhibition.

Carbonic anhydrases (CAs) have been linked to tumor progression, particularly membrane-bound CA isoform IX (CA IX). The role of CA IX in the context of breast cancer is to regulate the pH of the tumor microenvironment. In contrast to CA IX, expression of CA XII, specifically in breast cancer, is associated with better outcome despite performing the same catalytic function. In this study, we have structurally modeled the orientation of bound ureido-substituted benzene sulfonamides (USBs) within the active site of CA XII, in comparison to CA IX and cytosolic off-target CA II, to understand isoform specific inhibition. This has identified specific residues within the CA active site, which differ between isoforms that are important for inhibitor binding and isoform specificity. The ability of these sulfonamides to block CA IX activity in breast cancer cells is less effective than their ability to block activity of the recombinant protein (by one to two orders of magnitude depending on the inhibitor). The same is true for CA XII activity but now they are two to three orders of magnitude less effective. Thus, there is significantly greater specificity for CA IX activity over CA XII. While the inhibitors block cell growth, without inducing cell death, this again occurs at two orders of magnitude above the Ki values for inhibition of CA IX and CA XII activity in their respective cell types. Surprisingly, the USBs inhibited cell growth even in cells where CA IX and CA XII expression was ablated. Despite the potential for these sulfonamides as chemotherapeutic agents, these data suggest that we reconsider the role of CA activity on growth potentiation.

Biophysical, Biochemical, and Cell Based Approaches Used to Decipher the Role of Carbonic Anhydrases in Cancer and to Evaluate the Potency of Targeted Inhibitors.

Carbonic anhydrases (CAs) are thought to be important for regulating pH in the tumor microenvironment. A few of the CA isoforms are upregulated in cancer cells, with only limited expression in normal cells. For these reasons, there is interest in developing inhibitors that target these tumor-associated CA isoforms, with increased efficacy but limited nonspecific cytotoxicity. Here we present some of the biophysical, biochemical, and cell based techniques and approaches that can be used to evaluate the potency of CA targeted inhibitors and decipher the role of CAs in tumorigenesis, cancer progression, and metastatic processes. These techniques include esterase activity assays, stop flow kinetics, and mass inlet mass spectroscopy (MIMS), all of which measure enzymatic activity of purified protein, in the presence or absence of inhibitors. Also discussed is the application of X-ray crystallography and Cryo-EM as well as other structure-based techniques and thermal shift assays to the studies of CA structure and function. Further, large-scale genomic and proteomic analytical methods, as well as cell based techniques like those that measure cell growth, apoptosis, clonogenicity, and cell migration and invasion, are discussed. We conclude by reviewing approaches that test the metastatic potential of CAs and how the aforementioned techniques have contributed to the field of CA cancer research.

Differential expression and function of CAIX and CAXII in breast cancer: A comparison between tumorgraft models and cells.

Carbonic anhydrase IX (CAIX) and XII (CAXII) are transmembrane proteins that are associated with cancer progression. We have previously described the catalytic properties of CAIX in MDA-MB-231 breast cancer cells, a line of cells that were derived from a patient with triple negative breast cancer. We chose this line because CAIX expression in breast cancer is a marker of hypoxia and a prognosticator for reduced survival. However, CAXII expression is associated with better survival statistics than those patients with low CAXII expression. Yet CAIX and CAXII have similar catalytic activities. Here we compare the potential roles of CAIX and CAXII in the context of TNBC and estrogen receptor (ER)-positive breast cancer. In tumor graft models, we show that CAIX and CAXII exhibit distinct expression patterns and non-overlapping. We find the same pattern across a panel of TNBC and luminal breast cancer cell lines. This affords an opportunity to compare directly CAIX and CAXII function. Our data suggest that CAIX expression is associated with growth potentiation in the tumor graft model and in a TNBC line using knockdown strategies and blocking activity with an impermeant sulfonamide inhibitor, N-3500. CAXII was not associated with growth potentiation. The catalytic activities of both CAIX and CAXII were sensitive to inhibition by N-3500 and activated at low pH. However, pH titration of activity in membrane ghosts revealed significant differences in the catalytic efficiency and pKa values. These features provide evidence that CAIX is a more efficient enzyme than CAXII at low pH and that CAIX shifts the equilibrium between CO2 and bicarbonate in favor of CO2 production by consuming protons. This suggests that in the acidic microenvironment of tumors, CAIX plays a role in stabilizing pH at a value that favors cancer cell survival.

Cancer Drug Development of Carbonic Anhydrase Inhibitors beyond the Active Site.

Carbonic anhydrases (CAs) catalyze the reversible hydration of carbon dioxide to produce bicarbonate and a proton. Multiple CA isoforms are implicated in a range of diseases, including cancer. In solid tumors, continuously dividing cells create hypoxic conditions that eventually lead to an acidic microenvironment. Hypoxic tumor cells have different mechanisms in place to regulate and adjust the surrounding microenvironment for survival. These mechanisms include expression of CA isoform IX (CA IX) and XII (CA XII). These enzymes help maintain a physiological intracellular pH while simultaneously contributing to an acidic extracellular pH, leading to tumor cell survival. Expression of CA IX and CA XII has also been shown to promote tumor cell invasion and metastasis. This review discusses the characteristics of CA IX and CA XII, their mechanism of action, and validates their prospective use as anticancer targets. We discuss the current status of small inhibitors that target these isoforms, both classical and non-classical, and their future design in order to obtain isoform-specificity for CA IX and CA XII. Biologics, such as monoclonal antibodies, monoclonal-radionuclide conjugated chimeric antibodies, and antibody-small molecule conjugates are also discussed.

Carbonic Anhydrases: Role in pH Control and Cancer.

The pH of the tumor microenvironment drives the metastatic phenotype and chemotherapeutic resistance of tumors. Understanding the mechanisms underlying this pH-dependent phenomenon will lead to improved drug delivery and allow the identification of new therapeutic targets. This includes an understanding of the role pH plays in primary tumor cells, and the regulatory factors that permit cancer cells to thrive. Over the last decade, carbonic anhydrases (CAs) have been shown to be important mediators of tumor cell pH by modulating the bicarbonate and proton concentrations for cell survival and proliferation. This has prompted an effort to inhibit specific CA isoforms, as an anti-cancer therapeutic strategy. Of the 12 active CA isoforms, two, CA IX and XII, have been considered anti-cancer targets. However, other CA isoforms also show similar activity and tissue distribution in cancers and have not been considered as therapeutic targets for cancer treatment. In this review, we consider all the CA isoforms and their possible role in tumors and their potential as targets for cancer therapy.

UFH-001 cells: A novel triple negative, CAIX-positive, human breast cancer model system.

Human cell lines are an important resource for research, and are often used as in vitro models of human diseases. In response to the mandate that all cells should be authenticated, we discovered that the MDA-MB-231 cells that were in use in our lab, did not validate based on the alleles of 9 different markers (STR Profile). We had been using this line as a model of triple negative breast cancer (TNBC) that has the ability to form tumors in immuno-compromised mice. Based on marker analysis, these cells most closely resembled the MCF10A line, which are a near diploid and normal mammary epithelial line. Yet, the original cells express carbonic anhydrase IX (CAIX) both constitutively and in response to hypoxia and are features that likely drive the aggressive nature of these cells. Thus, we sought to sub-purify CAIX-expressing cells using Fluorescence Activated Cell Sorting (FACS). These studies have revealed a new line of cells that we have name UFH-001, which have the TNBC phenotype, are positive for CAIX expression, both constitutively and in response to hypoxia, and behave aggressively in vivo. These cells may be useful for exploring mechanisms that underlie progression, migration, and metastasis of this phenotype. In addition, constitutive expression of CAIX allows its evaluation as a therapeutic target, both in vivo and in vitro.

Bioinspired Optical Sensor for Remote Measurement of Small Displacements at a Distance.

Identifying appropriate sites for landing a spacecraft or building permanent structures is critical for extraterrestrial exploration. By tracking the movement of land masses and structures on a planetary surface, scientists can better predict issues that could affect the integrity of the site or structures. A lightweight, low-cost, low-power bioinspired optical sensor is being developed at the National Aeronautics and Space Administration (NASA) Ames Research Center to remotely measure small displacements of land masses on either side of a fault. This paper describes the sensor, which is inspired by the compound eye vision system found in many insects, and the algorithms developed to estimate displacement. The results are presented for indoor and outdoor tests using the sensor to measure the displacement of a specially designed target that is located 0.35, 6, and 30 m from the sensor and is moved 10 mm to the left and right of a centered position, simulating the displacement of land masses on either side of a fault. Measurement uncertainties estimates were a few tenths of a millimeter when the target was located 0.35 and 6 m from the sensor. At the 30 m distance, corrections were required to obtain accuracies in the order of 1 mm.

Structure activity study of carbonic anhydrase IX: Selective inhibition with ureido-substituted benzenesulfonamides.

Ureido-substituted benzenesulfonamides (USBs) show great promise as selective and potent inhibitors for human carbonic anhydrase hCA IX and XII, with one such compound (SLC-0111/U-F) currently in clinical trials (clinical trials.gov, NCT02215850). In this study, the crystal structures of both hCA II (off-target) and an hCA IX-mimic (target) in complex with selected USBs (U-CH3, U-F, and U-NO2), at resolutions of 1.9 Å or better, are presented, and demonstrate differences in the binding modes within the two isoforms. The presence of residue Phe 131 in hCA II causes steric hindrance (U-CH3, 1765 nM; U-F, 960 nM; U-NO2, 15 nM) whereas in hCA IX (U-CH3, 7 nM; U-F, 45 nM; U-NO2, 1 nM) and hCA XII (U-CH3, 6 nM; U-F, 4 nM; U-NO2, 6 nM), 131 is a Val and Ala, respectively, allows for more favorable binding. Our results provide insight into the mechanism of USB selective inhibition and useful information for structural design and drug development, including synthesis of hybrid USB compounds with improved physiochemical properties.

The Structure of Carbonic Anhydrase IX Is Adapted for Low-pH Catalysis.

Human carbonic anhydrase IX (hCA IX) expression in many cancers is associated with hypoxic tumors and poor patient outcome. Inhibitors of hCA IX have been used as anticancer agents with some entering Phase I clinical trials. hCA IX is transmembrane protein whose catalytic domain faces the extracellular tumor milieu, which is typically associated with an acidic microenvironment. Here, we show that the catalytic domain of hCA IX (hCA IX-c) exhibits the necessary biochemical and biophysical properties that allow for low pH stability and activity. Furthermore, the unfolding process of hCA IX-c appears to be reversible, and its catalytic efficiency is thought to be correlated directly with its stability between pH 3.0 and 8.0 but not above pH 8.0. To rationalize this, we determined the X-ray crystal structure of hCA IX-c to 1.6 Å resolution. Insights from this study suggest an understanding of hCA IX-c stability and activity in low-pH tumor microenvironments and may be applicable to determining pH-related effects on enzymes.

Effects of acid-base variables and the role of carbonic anhydrase on oxalate secretion by the mouse intestine in vitro.

Hyperoxaluria is a major risk factor for calcium oxalate kidney stones and the intestine is recognized as an important extra-renal pathway for eliminating oxalate. The membrane-bound chloride/bicarbonate (Cl(-)/) exchangers are involved in the transcellular movement of oxalate, but little is understood about how they might be regulated. , CO2, and pH are established modulators of intestinal NaCl cotransport, involving Na(+)/H(+) and Cl(-)/ exchange, but their influence on oxalate transport is unknown. Measuring (14)C-oxalate and (36)Cl fluxes across isolated, short-circuited segments of the mouse distal ileum and distal colon we examined the role of these acid-base variables and carbonic anhydrase (CA) in oxalate and Cl(-) transport. In standard buffer both segments performed net oxalate secretion (and Cl(-) absorption), but only the colon, and the secretory pathway were responsive to and CO2. Ethoxzolamide abolished net oxalate secretion by the distal colon, and when used in tandem with an impermeant CA inhibitor, signaled an intracellular CA isozyme was required for secretion. There was a clear dependence on as their removal eliminated secretion, while at 42 mmol/L was also decreased and eradicated. Independent of pH, raising Pco2 from 28 to 64 mmHg acutely stimulated net oxalate secretion 41%. In summary, oxalate secretion by the distal colon was dependent on , CA and specifically modulated by CO2, whereas the ileum was remarkably unresponsive. These findings highlight the distinct segmental heterogeneity along the intestine, providing new insights into the oxalate transport mechanism and how it might be regulated.

Advances in Anti-Cancer Drug Development Targeting Carbonic Anhydrase IX and XII.

The microenvironment within a solid tumor is heterogeneous with regions being both acidic and hypoxic. As a result of this, cancer cells upregulate genes that allow survival in such environments. Some of these genes are pH regulatory factors, including carbonic anhydrase IX (CA IX) and in some cases XII (CA XII). CA IX helps to maintain normal cytoplasmic pH (pHi) while simultaneously contributing to the extracellular pH (pHe). CA XII is also thought to be responsible for stabilizing pHe at physiological conditions. Extracellular acidification of the tumor microenvironment promotes local invasion and metastasis while decreasing the effectiveness of adjuvant therapies, thus contributing to poor cancer clinical outcomes. In this review, we describe the properties of CA IX and CA XII that substantiate their potential use as anticancer targets. We also discuss the current status of CA isoform-selective inhibitor development and patents of CA IX/XII targeted inhibitors that show potential for treating aggressive tumors. Some of the recently published patents discussed include sulfonamide-based small molecule inhibitors including derivatives of boron cluster compounds; metal complexes of poly(carboxyl)amine-containing ligands; nitroi-midazole-, ureidosulfonamide-, and coumarin-based compounds; as well as G250 and A610 monoclonal antibodies for cancer treatment.

Localization of a moving target using a fly eye sensor.

An optical sensor based on the visual system of the common housefly (Musca domestica) has been developed by the researchers at the Wyoming Image and Signal Processing Research (WISPR) Laboratory. This optical sensor shows promising peformance for detection of an edge in real-time, with minimal processing overhead. An application of this sensor might be the deflection measurement of a moving target, such as the wing of an aircraft under test. In this paper, we have investiaged the possibility of localizing an edge, and hence the target, using the fly eye sensor, which is an important part of the deflection measurement process. A simulation program has been developed for this purpose, which simulates the sensor output, for a moving object on the target plane at a specified distance from the sensor. After successfully simulating the sensor output, it has been found that the location of an edge changes with the change in sensor to target distance, and their relative orientation. It also changes for the limited movement of the target, withing the field of view (FOV) of the sensor. On the contrary, the target-background contrast scenario, and shifting of the target normal to the target movement direction, do not affect the edge localization process. This paper shows and edge can be precisely detected for a target a aspecified distance from the sensor with proper sensor-target orientation, when the target moves all the way across the FOV of the sensor.

Effect of sensor-target-background distance on target tracking using a fly eye sensor.

A multi-aperture optical sensor, known as a fly eye sensor, has been developed at the Wyoming Image and Signal Processing Research (WISPR) Laboratory based on the visual system of the common housefly Musca domestical. This biomimetic sensor shows promising edge detection capability, in varying contrast scenarios, with minimal processing overhead. Use of this sensor for fast motion detection, and object tracking is appealing, but optimizing the use of such a sensor requires detailed study. This paper analyzes the effect of placing the background at various distances greater than the target, and provides visualization of these example scenarios. A computer simulationof the sensor using MATLAB demonstrates that the placdementof a target closer to the sensor, and further from the background, affects the sensor response. If not properly considered, this may introduce ambiguities and degrade the performance of a tracking system based ont he flye eye sensor that requires precise location of the target in front of the sensor. This paper shows how a peroperly designed low-pass filter can greatly mitigate this effect with only a small degradation of the relative response magnitude at different distances from the sensor.