Type 1 Sodium Calcium Exchanger Forms a Complex with Carbonic Anhydrase IX and Via Reverse Mode Activity Contributes to pH Control in Hypoxic Tumors.

Hypoxia and acidosis are among the key microenvironmental factors that contribute to cancer progression. We have explored a possibility that the type 1Na+/Ca2+ exchanger (NCX1) is involved in pH control in hypoxic tumors. We focused on changes in intracellular pH, co-localization of NCX1, carbonic anhydrase IX (CA IX), and sodium proton exchanger type 1 (NHE1) by proximity ligation assay, immunoprecipitation, spheroid formation assay and migration of cells due to treatment with KB-R7943, a selective inhibitor of the reverse-mode NCX1. In cancer cells exposed to hypoxia, reverse-mode NCX1 forms a membrane complex primarily with CA IX and also with NHE1. NCX1/CA IX/NHE1 assembly operates as a metabolon with a potent ability to extrude protons to the extracellular space and thereby facilitate acidosis. KB-R7943 prevents formation of this metabolon and reduces cell migration. Thus, we have shown that in hypoxic cancer cells, NCX1 operates in a reverse mode and participates in pH regulation in hypoxic tumors via cooperation with CAIX and NHE1.

Sodium/calcium exchanger is involved in apoptosis induced by H2S in tumor cells through decreased levels of intracellular pH.

We explored possibility that sodium/calcium exchanger 1 (NCX1) is involved in pH modulation and apoptosis induction in GYY4137 treated cells. We have shown that although 10 days treatment with GYY4137 did not significantly decreased volume of tumors induced by colorectal cancer DLD1 cells in nude mice, it already induced apoptosis in these tumors. Treatment of DLD1 and ovarian cancer A2780 cells with GYY4137 resulted in intracellular acidification in a concentration-dependent manner. We observed increased mRNA and protein expression of both, NCX1 and sodium/hydrogen exchanger 1 (NHE1) in DLD1-induced tumors from GYY4137-treated mice. NCX1 was coupled with NHE1 in A2780 and DLD1 cells and this complex partially disintegrated after GYY4137 treatment. We proposed that intracellular acidification is due to uncoupling of NCX1/NHE1 complex rather than blocking of the reverse mode of NCX1, probably due to internalization of NHE1. Results might contribute to understanding molecular mechanism of H2S-induced apoptosis in tumor cells.

Type 3 inositol 1,4,5-trisphosphate receptor has antiapoptotic and proliferative role in cancer cells.

Although the involvement of type 1 (IP3R1) and type 2 (IP3R2) inositol 1,4,5-trisphosphate receptors in apoptosis induction has been well documented in different cancer cells and tissues, the function of type 3 IP3R (IP3R3) is still elusive. Therefore, in this work we focused on the role of IP3R3 in tumor cells in vitro and in vivo. We determined increased expression of this receptor in clear cell renal cell carcinoma compared to matched unaffected part of the kidney from the same patient. Thus, we hypothesized about different functions of IP3R3 compared to IP3R1 and IP3R2 in tumor cells. Silencing of IP3R1 prevented apoptosis induction in colorectal cancer DLD1 cells, ovarian cancer A2780 cells, and clear cell renal cell carcinoma RCC4 cells, compared to apoptosis in cells treated with scrambled siRNA. As expected, silencing of IP3R3 and subsequent apoptosis induction resulted in increased levels of apoptosis in all these cells. Further, we prepared a DLD1/IP3R3_del cell line using CRISPR/Cas9 gene editing method. These cells were injected into nude mice and tumor's volume was compared with tumors induced by DLD1 cells. Lower volume of tumors originated from DLD1/IP3R3_del cells was observed after 12 days, compared to wild type DLD1 cells. Also, the migration of these cells was lesser compared to wild type DLD1 cells. Apoptosis under hypoxic conditions was more pronounced in DLD1/IP3R3_del cells than in DLD1 cells. These results clearly show that IP3R3 has proliferative and anti-apoptotic effect in tumor cells, on contrary to the pro-apoptotic effect of IP3R1.

Endogenous H2S producing enzymes are involved in apoptosis induction in clear cell renal cell carcinoma.

BACKGROUND: Knowledge about the expression and thus a role of enzymes that produce endogenous H2S - cystathionine-ß-synthase, cystathionine γ-lyase and mercaptopyruvate sulfurtransferase - in renal tumors is still controversial. In this study we aimed to determine the expression of these enzymes relatively to the expression in unaffected part of kidney from the same patient and to found relation of these changes to apoptosis. To evaluate patient's samples, microarray and immunohistochemistry was used. METHODS: To determine the physiological importance, we used RCC4 stable cell line derived from clear cell renal cell carcinoma, where apoptosis induction by a mixture of five chemotherapeutics with/without silencing of H2S-producing enzymes was detected. Immunofluorescence was used to determine each enzyme in the cells. RESULTS: In clear cell renal cell carcinomas, expression of H2S-producing enzymes was mostly decreased compared to a part of kidney that was distal from the tumor. To evaluate a potential role of H2S-producing enzymes in the apoptosis induction, we used RCC4 stable cell line. We have found that silencing of cystathionine-ß-synthase and cystathionine γ-lyase prevented induction of apoptosis. Immunofluorescence staining clearly showed that these enzymes were upregulated during apoptosis in RCC4 cells. CONCLUSION: Based on these results we concluded that in clear cell renal cell carcinoma, reduced expression of the H2S-producing enzymes, mainly cystathionine γ-lyase, might contribute to a resistance to the induction of apoptosis. Increased production of the endogenous H2S, or donation from the external sources might be of a therapeutic importance in these tumors.

Haloperidol Affects Plasticity of Differentiated NG-108 Cells Through σ1R/IP3R1 Complex.

Haloperidol is an antipsychotic agent that primarily acts as an antagonist of D2 dopamine receptors. Besides other receptor systems, it targets sigma 1 receptors (σ1Rs) and inositol 1,4,5-trisphosphate receptors (IP3Rs). Aim of this work was to investigate possible changes in IP3Rs and σ1Rs resulting from haloperidol treatment and to propose physiological consequences in differentiated NG-108 cells, i.e., effect on cellular plasticity. Haloperidol treatment resulted in up-regulation of both type 1 IP3Rs (IP3R1s) and σ1Rs at mRNA and protein levels. Haloperidol treatment did not alter expression of other types of IP3Rs. Calcium release from endoplasmic reticulum (ER) mediated by increased amount of IP3R1s elevated cytosolic calcium and generated ER stress. IP3R1s were bound to σ1Rs, and translocation of this complex from ER to nucleus occurred in the group of cells treated with haloperidol, which was followed by increased nuclear calcium levels. Haloperidol-induced changes in cytosolic, reticular, and nuclear calcium levels were similar when specific σ1 blocker -BD 1047- was used. Changes in calcium levels in nucleus, ER, and cytoplasm might be responsible for alterations in cellular plasticity, because length of neurites increased and number of neurites decreased in haloperidol-treated differentiated NG-108 cells.

Melatonin-Induced Changes in Cytosolic Calcium Might be Responsible for Apoptosis Induction in Tumour Cells.

BACKGROUND/AIMS: Melatonin is a hormone transferring information about duration of darkness to the organism and is known to modulate several signaling pathways in the cells, e.g. generation of endoplasmic reticulum stress, oxidative status of the cells, etc. Melatonin has been shown to exert antiproliferative and cytotoxic effects on various human cancers. We proposed that this hormone can differently affect tumour cells and healthy cells. METHODS: We compared the effect of 24 h melatonin treatment on calcium transport (by fluorescent probes FLUO-3AM and Rhod-5N), ER stress (determined as changes in the expression of CHOP, XBP1 and fluorescently, using Thioflavin T), ROS formation (by CellROX® Green/Orange Reagent) and apoptosis induction (by Annexin-V-FLUOS/propidiumiodide) in two tumour cell lines - ovarian cancer cell line A2780 and stable cell line DLD1 derived from colorectal carcinoma, with non-tumour endothelial cell line EA.hy926. RESULTS: Melatonin increased apoptosis in both tumour cell lines more than twice, while in EA.hy926 cells the apoptosis was increased only by 30%. As determined by silencing with appropriate siRNAs, both, type 1 sodium/calcium exchanger and type 1 IP3 receptor are involved in the apoptosis induction. Antioxidant properties of melatonin were significantly increased in EA.hy926 cells, while in tumour cell lines this effect was much weaker. CONCLUSION: Taken together, melatonin has different antioxidative effects on tumour cells compared to non-tumour ones; it also differs in the ability to induce apoptosis through the type 1 sodium/calcium exchanger, and type 1 IP3 receptor. Different targeting of calcium transport systems in tumour and normal, non-tumour cells is suggested as a key mechanism how melatonin can exert its anticancer effects. Therefore, it might have a potential as a novel therapeutic implication in cancer treatment.

Hypoxic conditions increases H2S-induced ER stress in A2870 cells.

Hypoxia - a state of lower oxygen demand-is responsible for a higher aggressiveness of tumors and therefore a worse prognosis. During hypoxia, several metabolic pathways are re-organized, e.g., energetic metabolism, modulation of pH, and calcium transport. Calcium is an important second messenger that regulates variety of processes in the cell. Thus, aim of this work was to compare H2S modulation of the intracellular calcium transport systems in hypoxia and in cells grown in standard culture conditions. For all experiments, we used ovarian cancer cell line (A2780). H2S is a novel gasotransmitter, known to be involved in a modulation of several calcium transport systems, thus resulting in altered calcium signaling. Two models of hypoxia were used in our study-chemical (induced by dimethyloxallyl glycine) and 2 % O2 hypoxia, both combined with a treatment using a slow H2S donor GYY4137. In hypoxia, we observed rapid changes in cytosolic and reticular calcium levels compared to cells grown in standard culture conditions, and these changes were even more exagerrated when combined with the GYY4137. Changes in a calcium homeostasis result from IP3 receptor´s up-regulation and down-regulation of the SERCA 2, which leads to a development of the endoplasmic reticulum stress. Based on our results, we propose a higher vulnerability of calcium transport systems to H2S regulation under hypoxia.

Sodium/calcium exchanger is upregulated by sulfide signaling, forms complex with the ß1 and ß3 but not ß2 adrenergic receptors, and induces apoptosis.

Hydrogen sulfide (H2S) as a novel gasotransmitter regulates variety of processes, including calcium transport systems. Sodium calcium exchanger (NCX) is one of the key players in a regulation calcium homeostasis. Thus, the aims of our work were to determine effect of sulfide signaling on the NCX type 1 (NCX1) expression and function in HeLa cells, to investigate the relationship of ß-adrenergic receptors with the NCX1 in the presence and/or absence of H2S, and to determine physiological importance of this potential communication. As a H2S donor, we used morpholin-4-ium-4-methoxyphenyl(morpholino) phosphinodithioate-GYY4137. We observed increased levels of the NCX1 mRNA, protein, and activity after 24 h of GYY4137 treatment. This increase was accompanied by elevated cAMP due to the GYY4137 treatment, which was completely abolished, when NCX1 was silenced. Increased cAMP levels would point to upregulation of ß-adrenergic receptors. Indeed, GYY4137 increased expression of ß1 and ß3 (but not ß2) adrenergic receptors. These receptors co-precipitated, co-localized with the NCX1, and induced apoptosis in the presence of H2S. Our results suggest that sulfide signaling plays a role in regulation of the NCX1, ß1 and ß3 adrenergic receptors, their co-localization, and stimulation of apoptosis, which might be of a potential importance in cancer treatment.

Isoproterenol accelerates apoptosis through the over-expression of the sodium/calcium exchanger in HeLa cells.

Apoptosis induction causes over-expression of the Na+/Ca2+ exchanger of type 1 (NCX1) in the HeLa cell line. During induction of apoptosis and in the presence of isoproterenol hydrochloride (I; ß-adrenergic agonist), increase in the NCX1 is even more pronounced. Anti-apoptotic Bcl-2 mRNA and protein is markedly reduced during apoptosis and in the presence of I, which causes a rapid increase in the Bax/Bcl-2 ratio. During apoptosis induction by apoptosis inducing kit (A), both with and without I, the active form of caspase-3, which is the executive enzyme in apoptosis, becomes visible on Western blots. Silencing NCX1 resulted in the reversal of the Bax/Bcl-2 ratio, it prevented a decrease in mitochondrial membrane potential compared to the AI group and it decreased the level of AI-induced apoptosis in HeLa cells. Based on the experiments with single apoptotic inducers camptothecin, cycloheximide and dexamethasone, it might be proposed that potentiated apoptotic effect in I-treated cells is due to the inhibition of nuclear topoisomerase. As illustrated in immunofluorescence and Western blot analysis, calnexin increased significantly during induction of the apoptosis in the presence of I. In addition, further decrease in sarco/endoplasmic ATPase 2 (SERCA2), decrease in reticular calcium and mitochondrial membrane potential was observed, which suggests development of the endoplasmic reticulum (ER) stress. Based on these results, we propose that I further enhanced NCX1 expression in apoptotic cells through the development of ER stress.

IP(3) receptors, stress and apoptosis.

Inositol 1,4,5-trisphosphate (IP3) receptors are intracellular calcium channels that are able to release calcium from intracellular stores upon activation by IP3 and modulation by calcium. IP3 receptors are involved in variety of processes during physiological, but also in the pathophysiological states. Unraveling their regulation and function, especially under the pathological situations can result in a development of new therapeutic strategies based on the IP3 receptor´s activation and/or blocking. To the stimuli that can modulate IP3 receptors belong several stress factors (e.g. immobilization stress, oxidative stress and hypoxia) and also apoptosis. Depending on the length and strength of the stress stimulus, expression of IP3 receptors can be increased, or decreased. Therefore, in this minireview modulation of IP3 receptors by some stressors is discussed. Since it was already shown that strong hypoxia might lead to the apoptosis induction, special focus will be given to the hypoxic stress and induction of apoptosis.

NF-κB inhibition significantly upregulates the norepinephrine transporter system, causes apoptosis in pheochromocytoma cell lines and prevents metastasis in an animal model.

Pheochromocytomas (PHEOs) and paragangliomas (PGLs) are specific types of neuroendocrine tumors that originate in the adrenal medulla or sympathetic/parasympathetic paraganglia, respectively. Although these tumors are intensively studied, a very effective treatment for metastatic PHEO or PGL has not yet been established. Preclinical evaluations of novel therapies for these tumors are very much required. Therefore, in this study we tested the effect of triptolide (TTL), a potent nuclear factor-kappaB (NF-κB) inhibitor, on the cell membrane norepinephrine transporter (NET) system, considered to be the gatekeeper for the radiotherapeutic agent 131I-metaiodobenzylguanidine (131I-MIBG). We measured changes in the mRNA and protein levels of NET and correlated them with proapoptotic factors and metastasis inhibition. The study was performed on three different stable PHEO cell lines. We found that blocking NF-κB with TTL or capsaicin increased both NET mRNA and protein levels. Involvement of NF-κB in the upregulation of NET was verified by mRNA silencing of this site and also by using NF-κB antipeptide. Moreover, in vivo treatment with TTL significantly reduced metastatic burden in an animal model of metastatic PHEO. The present study for the first time shows how NF-κB inhibitors could be successfully used in the treatment of metastatic PHEO/PGL by a significant upregulation of NET to increase the efficacy of 131I-MIBG and by the induction of apoptosis.

Chemically mimicked hypoxia modulates gene expression and protein levels of the sodium calcium exchanger in HEK 293 cell line via HIF-1α.

Up to now a little is known about the effect of hypoxia on the sodium calcium exchanger type 1 (NCX1) expression and function. Therefore, we studied how dimethyloxallyl glycine (DMOG), an activator and stabilizer of the hypoxia-inducible factor (HIF)-1α, could affect expression of the NCX1 in HEK 293 cell line. We also tried to determine whether this activation can result in the induction of apoptosis in HEK 293 cells. We have found that DMOG treatment for 3 hours significantly increased gene expression and also protein levels of the NCX1. This increase was accompanied by a decrease in intracellular pH. Wash-out of DMOG did not result in reduction of the NCX1 mRNA and protein to original - control levels, although pH returned to physiological values. Using luciferase reporter assay we observed increase in the NCX1 promoter activity after DMOG treatment and using wild-type mouse embryonic fibroblast (MEF)-HIF-1(+/+) and HIF-1-deficient MEF-HIF-1(-/-) cells we have clearly shown that in the promoter region, HIF-1α is involved in DMOG induced upregulation of the NCX1. Moreover, we also showed that an increase in the NCX1 mRNA due to the apoptosis induction is not regulated by HIF-1α.

Apoptosis induced clustering of IP(3)R1 in nuclei of non-differentiated PC12 cells.

Inositol 1,4,5-trisphosphate (IP(3)) receptors are emerging as key sites for regulation by pro- and anti-apoptotic factors. Induction of apoptosis for 3 h increased mRNA and protein levels of type 1 IP(3) receptors in non-differentiated (ND), but not in differentiated (D) PC12 cells. Inhibitors of the IP(3) R's calcium release-2-aminoethoxydiphenyl borate (2-APB) and xestospongin-completely prevented Bax and caspase-3 mRNA increase after treatment with the apoptosis inducer set (AIK), and this reinforces the importance of IP(3) R1 in the apoptosis of ND PC12 cells. Apoptosis induction not only increases the IP(3) R1 protein, but it also causes formation of IP(3) R1 clusters in the nucleus which most likely result from fusion of the nucleoplasmic reticulum and/or IP(3) R1 translocation to the nucleus. This is quite similar to the observations noted after overexpression of IP(3) R1 in PC12 cells. The amount of IP(3) induced calcium release was higher in control than in AIK-treated cells. From our results we propose that after the apoptosis induction the amount of intranuclear calcium decreased dramatically due to the increase of calcium permeability of the nuclear calcium store vesicles. Therefore, increase of the calcium permeability may result from IP(3) receptors translocation to nuclei that can boost the calcium transport through IP(3) receptors.

Expressional changes of RyR1 and RyR2 in PC12 cells after induction of apoptosis.

Ca2+ released from endoplasmic reticulum through ryanodine receptors (RyRs) and inositol 1,4,5-trisphosphate receptors (IP3Rs) can trigger apoptotic or necrotic pathways in cooperation with proapoptotic and/or prosurvival proteins, as those of Bcl-2 family. In such regulatory pathways expressional modulation of these Ca2+ transporters could also be expected. Therefore, our aim was to determine the expressional changes of RyR1 and RyR2 after experimental induction of apoptosis in PC12 cells. Our results showed significant decrease of RyR1 and RyR2 expressions, while caspase-3 and Bax expression significantly increased. We conclude that induction of apoptosis in PC12 cells could result in RyR expression down regulation.

Changes and role of adrenoceptors in PC12 cells after phenylephrine administration and apoptosis induction.

The present study addresses the hypothesis that adrenergic regulation modulates the effect of apoptosis. Therefore we studied, whether α1-adrenergic receptor's agonist phenylephrine (PE) can affect or induce apoptosis in rat pheochromocytoma (PC12) cells. We have shown that PE treatment did not increase level of the apoptosis, or level of the caspase 3 mRNA. When apoptosis was induced in the presence of PE, caspase 3 mRNA was significantly increased, while the percentage of apoptotic cells remained unchanged compared to apoptotic group without PE. During this process, α1D-, ß2- and ß3-adrenergic receptors (ARs) were upregulated. Since all these three types of ARs are differently localized in the cell, we assume that mutual communication of all three ARs is crucial to participate in this signaling and during development of apoptosis, some of these systems might translocate. Another important system in handling noradrenaline during apoptosis might be noradrenaline transporter (NET), since it was downregulated in apoptotic cells treated with PE, compared to untreated apoptotic cells. However, precise mechanism of mutual communication among all these systems remains to be elucidated.

Plasma membrane-cytoskeleton-endoplasmic reticulum complexes in neurons and astrocytes.

The possibility that certain integral plasma membrane (PM) proteins involved in Ca(2+) homeostasis form junctional units with adjacent endoplasmic reticulum (ER) in neurons and glia was explored using immunoprecipitation and immunocytochemistry. Rat brain membranes were solubilized with the mild, non-ionic detergent, IGEPAL CA-630. Na(+)/Ca(2+) exchanger type 1 (NCX1), a key PM Ca(2+) transporter, was immunoprecipitated from the detergent-soluble fraction. Several abundant PM proteins co-immunoprecipitated with NCX1, including the alpha2 and alpha3 isoforms of the Na(+) pump catalytic (alpha) subunit, and the alpha2 subunit of the dihydropyridine receptor. The adaptor protein, ankyrin 2 (Ank 2), and the cytoskeletal proteins, alpha-fodrin and beta-spectrin, also selectively co-immunoprecipitated with NCX1, as did the ER proteins, Ca(2+) pump type 2 (SERCA 2), and inositol-trisphosphate receptor type 1 (IP(3)R-1). In contrast, a number of other abundant PMs, adaptors, and cytoskeletal proteins did not co-immunoprecipitate with NCX1, including the Na(+) pump alpha1 isoform, PM Ca(2+) pump type 1 (PMCA1), beta-fodrin, and Ank 3. In reciprocal experiments, immunoprecipitation with antibodies to the Na(+) pump alpha2 and alpha3 isoforms, but not alpha1, co-immunoprecipitated NCX1; the antibodies to alpha1 did, however, co-immunoprecipitate PMCA1. Antibodies to Ank 2, alpha-fodrin, beta-spectrin and IP(3)R-1 all co-immunoprecipitated NCX1. Immunocytochemistry revealed partial co-localization of beta-spectrin with NCX1, Na(+) pump alpha3, and IP(3)R-1 in neurons and of alpha-fodrin with NCX1 and SERCA2 in astrocytes. The data support the idea that in neurons and glia PM microdomains containing NCX1 and Na(+) pumps with alpha2 or alpha3 subunits form Ca(2+) signaling complexes with underlying ER containing SERCA2 and IP(3)R-1. These PM and ER components appear to be linked through the cytoskeletal spectrin network, to which they are probably tethered by Ank 2.

Immobilization stress elevates IP(3) receptor mRNA in adult rat hearts in a glucocorticoid-dependent manner.

Gene expression of the type 1 and 2 inositol 1,4,5-trisphosphate (IP(3)) receptors in the rat cardiac atria and ventricles and their possible modulation by single immobilization stress was studied. Single immobilization stress significantly elevated mRNA levels for both types of these receptors. To evaluate the involvement of glucocorticoids in the modulation of the gene expression of IP(3) receptors by immobilization stress, we used adrenalectomized and/or hypophysectomized rats. Since adrenalectomy and/or hypophysectomy completely abolished increase in IP(3) receptor's mRNA levels after the immobilization, we conclude that immobilization stress elevates mRNA of type 1 and 2 IP(3) receptors, mainly through the glucocorticoid responsive element.

Organization of Ca2+ stores in vascular smooth muscle: functional implications.

Much evidence suggests that caffeine/ryanodine (Caf/Ry)-releasable and inositol-1,4,5-trisphosphate (InsP3)-releasable Ca2+ stores in the sarcoplasmic reticulum (SR) of smooth muscles are at least partially distinct. We directly visualized SR stores in primary-cultured rat mesenteric artery myocytes with high-resolution digital imaging and the low-affinity Ca2, indicator, Furaptra (Kd = 75.6 microM). The SR appears to be a continuous tubular network. Nevertheless, SR Ca2+ stores are organized into small, separate, functionally independent compartments. Cyclopiazonic acid (CPA; inhibits SR (Ca2+ pump) and Caf (or Ry) release Ca2+ from different, spatially distinct compartments. Similar heterogeneity is seen with serotonin (acts via InsP3), which unloads only the CPA-sensitive compartments. Some of the SR ('junctional' SR; jSR) lies within 12-15 nm of the plasmalemma (PL). The jSR, the overlying PL microdomains, and the intervening, tiny volume of cytosol form junctional complexes ('PLasmERosomes'). Na+ pumps with high-ouabain-affinity alpha2 or alpha3 subunits, Na+/Ca2+ exchangers, and store-operated channels are confined to these PL microdomains, whereas Na+ pumps with low-ouabain-affinity alpha1 subunits and plasma membrane Ca2+ pumps are uniformly distributed. As a result of this organization, low-dose ouabain can selectively modulate Na+ and Ca2+ concentrations in the PLasmERosomes and jSR Ca2+ stores, and can thereby regulate Ca2+ signalling.