Hypoxia-induced Ca Research Articles

Exploration of newly synthesized deferasirox derivatives as potential anti-cancer agents via in-vitro and in-silico approaches

Carbonic anhydrase IX (CA IX), upregulated by hypoxia-inducible factor (HIF), plays a crucial role in regulation of intracellular and extracellular pH, which is essential for the growth and spread of tumors. The overexpression of CA IX in breast cancer is linked to a low post-radiation patient survival rate. Under normoxic conditions, CA IX expression is relatively low, but hypoxia-inducible factors (HIFs) upregulate its expression when oxygen levels drop. This adaptation supports the tumor's acidic microenvironment, aiding processes like metastasis, immune evasion, and resistance to therapies. Due to these functions, CA IX is considered a promising target for cancer therapy, with inhibitors in development aimed at disrupting its activity and thus hindering tumor growth and survival. Thus, various derivatives of already reported anticancer drug i.e., deferasirox were synthesized and their effect on CA IX enzyme were assessed. Additionally, the binding affinities of deferasirox derivatives with three distinct receptor proteins i.e., Tumor Protein P53 (TP53), Nuclear factor kappa B (NF-κB) and caspase 3 (pdb: 3DCY, 1NFI, 3DEI) were also observed. Their anticancer effect was evaluated by using non-invasive human breast cancer cells i.e., MCF-7 and glioblastoma cells (U87). Among all derivatives, the four thioureas derivatives showed more anticancer potential. The 4-(3,5-bis(2-hydroxyphenyl)-1H-1,2,4-triazol-1-yl)-N-((3,4-dimethoxyphenyl)carbamothioyl) benzamide (6) derivative exhibited maximum anticancer potential (0.33 ± 0.02 μM) with greater binding affinity at different protein receptors. The MTT results further confirmed the enzyme inhibition results of deferasirox derivatives. In conclusion, targeting hypoxia-induced CA IX expression in breast cancer through the use of deferasirox-derived thiourea derivatives presents a promising therapeutic approach.

Combination of Betulinic Acid Fragments and Carbonic Anhydrase Inhibitors-A New Drug Targeting Approach.

Human carbonic anhydrase IX (hCA IX) is a zinc(II)-dependent metalloenzyme that plays a critical role in the conversion of carbon dioxide and water to protons and bicarbonate. It is a membrane-bound protein with an extracellular catalytic center that is predominantly overexpressed in solid hypoxic tumors. Sulfamates and sulfonamides, for example acetazolamide (AZA), have been used to inhibit hCA IX in order to improve the response to solid hypoxic tumors. In the present study, we propose a new drug targeting approach by attaching the natural cytotoxic substances betulin and betulinic acid (BA) via a linker to sulfonamides. The conjugate was designed with different spacer lengths to accumulate at the target site of hCA IX. Computational and cell biological studies suggest that the length of the linker may influence hCA IX inhibition. Cytotoxicity tests of the newly synthesized bifunctional conjugates 3, 5, and 9 show effective cytotoxicity in the range of 6.4 and 30.1 µM in 2D and 3D tumor models. The hCA IX inhibition constants of this conjugates, measured using an in vitro enzyme assay with p-nitrophenyl acetate, were determined in a low µM-range, and all compounds reveal a significant inhibition of hypoxia-induced CA activity in a cell-based assay using the Wilbur-Anderson method. In addition, the cells respond with G1 increase and apoptosis induction. Overall, the dual strategy to produce cytotoxic tumor therapeutics that inhibit tumor-associated hCA IX was successfully implemented.

Indoline-5-Sulfonamides: A Role of the Core in Inhibition of Cancer-Related Carbonic Anhydrases, Antiproliferative Activity and Circumventing of Multidrug Resistance.

The overexpression and activity of carbonic anhydrase (CA, EC 4.2.1.1) isoforms CA IX and CA XII promote the accumulation of exceeding protons and acidosis in the extracellular tumor environment. Sulfonamides are effective inhibitors of most families of CAs. In this study, using scaffold-hopping, indoline-5-sulfonamide analogs 4a-u of the CA IX-selective inhibitor 3 were designed and synthesized to evaluate their biological properties. 1-Acylated indoline-5-sulfonamides demonstrated inhibitory activity against tumor-associated CA IX and XII with KI values up to 132.8 nM and 41.3 nM. Compound 4f, as one of the most potent inhibitors of CA IX and XII, exhibits hypoxic selectivity, suppressing the growth of MCF7 cells at 12.9 µM, and causes partial inhibition of hypoxia-induced CA IX expression in A431 skin cancer cells. 4e and 4f reverse chemoresistance to doxorubicin of K562/4 with overexpression of P-gp.

Impairment of Hypoxia-Induced CA IX by Beta-Blocker Propranolol-Impact on Progression and Metastatic Potential of Colorectal Cancer Cells.

The coexistence of cancer and other concomitant diseases is very frequent and has substantial implications for treatment decisions and outcomes. Beta-blockers, agents that block the beta-adrenergic receptors, have been related also to cancers. In the model of multicellular spheroids formed by colorectal cancer cells we described a crosstalk between beta-blockade by propranolol and tumour microenvironment. Non-selective beta-blocker propranolol decreased ability of tumour cells to adapt to hypoxia by reducing levels of HIF1α and carbonic anhydrase IX in 3D spheroids. We indicated a double action of propranolol in the tumour microenvironment by inhibiting the stability of HIF1α, thus mediating decrease of CA IX expression and, at the same time, by its possible effect on CA IX activity by decreasing the activity of protein kinase A (PKA). Moreover, the inhibition of β-adrenoreceptors by propranolol enhanced apoptosis, decreased number of mitochondria and lowered the amount of proteins involved in oxidative phosphorylation (V-ATP5A, IV-COX2, III-UQCRC2, II-SDHB, I-NDUFB8). Propranolol reduced metastatic potential, viability and proliferation of colorectal cancer cells cultivated in multicellular spheroids. To choose the right treatment strategy, it is extremely important to know how the treatment of concomitant diseases affects the superior microenvironment that is directly related to the efficiency of anti-cancer therapy

CA IX Stabilizes Intracellular pH to Maintain Metabolic Reprogramming and Proliferation in Hypoxia.

Tumor hypoxia represents a severe microenvironmental stress that is frequently associated with acidosis. Cancer cells respond to these stresses with changes in gene expression that promote survival at least in part through pH regulation and metabolic reprogramming. Hypoxia-induced carbonic anhydrase IX (CA IX) plays a critical adaptive role in response to hypoxic and acidic environments by catalytically hydrating extracellular CO2 to produce bicarbonate for buffering intracellular pH (pHi). We used proteome-wide profiling to study the cellular response to transient CA IX knockdown in hypoxia and found a decrease in the levels of key glycolytic enzymes and lactate dehydrogenase A (LDHA). Interestingly, the activity of LDH was also decreased as demonstrated by native in-gel activity assay. These changes led to a significant reduction in glycolytic flux and extracellular lactate levels in cancer cells in vitro, contributing to a decrease in proliferation. Interestingly, addition of the alternative LDH substrate alpha-ketobutyrate restored LDHA activity, extracellular acidification, pHi, and cellular proliferation. These results indicate that in the absence of CA IX, reduction of pHi disrupts LDHA activity and hinders the cellular capacity to regenerate NAD+ and secrete protons to the extracellular space. Hypoxia-induced CA IX therefore mediates adaptation to microenvironmental hypoxia and acidosis directly, by enzymatically converting extracellular CO2 to bicarbonate, and indirectly, by maintaining glycolysis-permissive intracellular milieu.

Synthesis of novel isoindoline-1,3-dione-based oximes and benzenesulfonamide hydrazones as selective inhibitors of the tumor-associated carbonic anhydrase IX

The synthesis, characterization and biological evaluation of a library of isoindoline-1,3-dione-based oximes and benzenesulfonamide hydrazones is disclosed. The set of hydroxyiminoethyl aromatic derivatives 10–18 was designed to assess the potentiality as zinc-binder for a feebly studied functional group in the field of carbonic anhydrase (CA, EC 4.2.1.1) inhibition. Analogue phenylphthalimmides were linked to benzenesulfonamide scaffold by hydrazone spacers in the second subset of derivatives 20–28 to further investigate the application of the “tail approach” as tool to afford CA selective inhibition profiles. The compounds were assayed for the inhibition of physiologically relevant isoforms of human carbonic anhydrases (hCA, EC 4.2.1.1), the cytosolic CA I and II, and the membrane-bound CA IV and tumor-associated CA IX. The new zinc-binders, both of the oxime and sulfonamide types, showed a striking selective activity against the target hCA IX over ubiquitous hCA I and II, with diverse inhibitory ranges and ratio differing the two subsets. With CA IX being a strongly current antitumor/antimetastatic drug target, these series of compounds may be of interest for the development of new, both conventional and unconventional anticancer drugs targeting hypoxia-induced CA isoforms such as CA IX with minimum ubiquitous CAs-related side effects.

Novel sulfonamide incorporating piperazine, aminoalcohol and 1,3,5-triazine structural motifs with carbonic anhydrase I, II and IX inhibitory action

A new series of s-triazine derivatives incorporating sulfanilamide, homosulfanilamide, 4-aminoethyl-benzenesulfonamide and piperazine or aminoalcohol structural motifs is reported. Molecular docking was exploited to select compounds from virtual combinatorial library for synthesis and subsequent biological evaluation. The compounds were prepared by using step by step nucleophilic substitution of chlorine atoms from cyanuric chloride (2,4,6-trichloro-1,3,5-triazine). The compounds were tested as inhibitors of physiologically relevant carbonic anhydrase (CA, EC 4.2.1.1) isoforms. Specifically, against the cytosolic hCA I, II and tumor-associated hCA IX. These compounds show appreciable inhibition. hCA I was inhibited with KIs in the range of 8.5–2679.1 nM, hCA II with KIs in the range of 4.8–380.5 nM and hCA IX with KIs in the range of 0.4–307.7 nM. As other similar derivatives, some of the compounds showed good or excellent selectivity ratios for inhibiting hCA IX over hCA II, of 3.5–18.5. 4-[({4-Chloro-6-[(4-hydroxyphenyl)amino]-1,3,5-triazin-2-yl}amino)methyl] benzene sulfonamide demonstrated subnanomolar affinity for hCA IX (0.4 nM) and selectivity (18.50) over the cytosolic isoforms. This series of compounds may be of interest for the development of new, unconventional anticancer drugs targeting hypoxia-induced CA isoforms such as CA IX.

Hypoxia-Induced Cytosolic Calcium Decrease Is Mediated Primarily by the Forward Mode of Na+/Ca2+ Exchanger in Smooth Muscle Cells of Fetal Ductus Arteriosus

Closure of the ductus arteriosus (DA) after birth, essential for postnatal adaptation, is initiated by the transition from hypoxia to normoxia. The current study investigated how hypoxia affects the level of cytosolic calcium ([Ca(2+)](i)) in fetal lamb DA smooth muscle cells (DASMCs) and the role of calcium pumps in this process. The [Ca(2+)](i) variation in response to acute hypoxia was determined spectrofluorometrically with fura-3-AM in cultured fetal DASMCs. Interventions using chemicals or solutions including thapsigargin, vanadate, KB-R7943, alkaline PH9.0 solution, or Na(+)-free medium were administered when samples were exposed to acute hypoxia. The results show that [Ca(2+)](i) decreased dramatically under acute hypoxia. This decrease was not attenuated completely by an inhibitor of sarcoplasmic/endoplasmic reticulum Ca(2+) adenosine triphosphatase (ATPase) (SERCA), a blocker of plasma membrane Ca(2+) ATPase (PMCA), or an inhibitor and activator of the reserve mode of the Na(+)/Ca(2+) exchanger (NCX). In contrast, KT-R9743, an inhibitor of the forward mode of NCX at a high concentration (30 microm), greatly diminished the hypoxia-induced [Ca(2+)](i) decrease in fetal DASMCs. These results suggest that a hypoxia-induced Ca(2+) decrease in fetal DASMCs results from cytosolic Ca(2+) efflux mediated primarily by the forward mode of NCX.

ATP inhibits the hypoxia response in type I cells of rat carotid bodies

Summary During hypoxia, ATP was released from type I (glomus) cells in the carotid bodies. We studied the action of ATP on the intracellular Ca(2+) concentration ([Ca(2+)](i)) of type I cells dissociated from rat carotid bodies using a Ca(2+) imaging technique. ATP did not affect the resting [Ca(2+)](i) but strongly suppressed the hypoxia-induced [Ca(2+)](i) elevations in type I cells. The order of purinoreceptor agonist potency in inhibiting the hypoxia response was 2-methylthioATP > ATP > ADP >> alpha, beta-methylene ATP > UTP, implicating the involvement of P2Y(1) receptors. Simultaneous measurements of membrane potential and [Ca(2+)](i) show that ATP inhibited the hypoxia-induced Ca(2+) signal by reversing the hypoxia-triggered depolarization. However, ATP did not oppose the hypoxia-mediated inhibition of the oxygen-sensitive TASK-like K(+) background current. Neither the inhibition of the large-conductance Ca(2+)-activated K(+) (maxi-K) channels nor the removal of extracellular Na(+) could affect the inhibitory action of ATP. Under normoxic condition, ATP caused hyperpolarization and increase in cell input resistance. These results suggest that the inhibitory action of ATP is mediated via the closure of background conductance(s) other than the TASK-like K(+), maxi-K or Na(+) channels. In summary, ATP exerts strong negative feedback regulation on hypoxia signaling in rat carotid type I cells.

Hypoxia-induced Ca 2+-influx in cerebral cortical neuronal nuclei of newborn piglets

The present study was designed to investigate the effect of hypoxia on nuclear calcium-influx in the cerebral cortex of newborn piglets. Anesthetized and ventilated newborn piglets divided into normoxic ( n=4) and hypoxic groups with varying degrees of tissue hypoxia ( n=10) were studied. Nuclear Ca 2+-influx was determined using 45Ca 2+ and plotted against ATP and phosphocreatine levels. The plots were analyzed by non-linear regression (exponential) analysis that showed a curvilinear relationship ( r=0.92 for ATP and r=0.88 for phosphocreatine). These data suggest a threshold at which there is a sudden increase in the nuclear calcium-influx that then continues to increase with further decrease in the ATP and phosphocreatine levels. The results demonstrate an increase in the nuclear Ca 2+-influx during hypoxia in newborn piglets and that this increase correlates in a curvilinear fashion with the increase in the degree of cerebral tissue hypoxia. We propose that the hypoxia-induced increase in intranuclear Ca 2+ is due to altered nuclear membrane Ca 2+-influx mechanisms and will lead to Ca 2+-mediated alteration of apoptotic gene expression as well as Ca 2+-dependent activation of endonucleases that result in DNA fragmentation and subsequent programmed neuronal cell death.

Intracellular mechanisms of hypoxia-induced calcium increase in rat sensory neurons

Elevation of cytosolic level of Ca 2+ was measured by spatial screening of freshly isolated dorsal root ganglion neurons loaded with Fura-2AM after subjecting them to a moderate hypoxic solution ( pO 2=10–40 mm Hg ). Short exposure of neurons to hypoxia resulted in a reversible elevation of intracellular Ca 2+ to about 120% in the cell center and to 80% in the cell periphery. Such elevation could be almost completely eliminated by removal of Ca 2+ or Na + from external medium or application of nifedipine, an L-type calcium channel blocker. Remarkable antihypoxic efficiency (58%) was achieved by preapplication of mitochondrial protonophore CCCP. A conclusion is made that in sensory neurons the hypoxia-induced elevation of cytosolic Ca 2+ is induced by combined changes of function in three cell substructures: voltage-operated L-type Ca 2+ and Na + channels and Ca 2+ accumulation by mitochondria. Mitochondria are important for spatial difference in the hypoxia-induced Ca 2+ elevation due to their specific location in these neurons.