Correction: Sanchez-Collado et al. Orai2 Modulates Store-Operated Ca2+ Entry and Cell Cycle Progression in Breast Cancer Cells. Cancers 2021, 14, 114.

In the original publication [...].

Melatonin downregulates TRPC6, impairing store-operated calcium entry in triple-negative breast cancer cells.

Melatonin has been reported to induce effective reduction in growth and development in a variety of tumors, including breast cancer. In triple-negative breast cancer (TNBC) cells, melatonin attenuates a variety of cancer features, such as tumor growth and apoptosis resistance, through a number of still poorly characterized mechanisms. One biological process that is important for TNBC cells is store-operated Ca2+ entry (SOCE), which is modulated by TRPC6 expression and function. We wondered whether melatonin might intersect with this pathway as part of its anticancer activity. We show that melatonin, in the nanomolar range, significantly attenuates TNBC MDA-MB-231 cell viability, proliferation, and migration in a time- and concentration-dependent manner, without having any effect on nontumoral breast epithelial MCF10A cells. Pretreatment with different concentrations of melatonin significantly reduced SOCE in MDA-MB-231 cells without altering Ca2+ release from the intracellular stores. By contrast, SOCE in MCF10A cells was unaffected by melatonin. In the TNBC MDA-MB-468 cell line, melatonin not only attenuated viability, migration, and SOCE, but also reduced TRPC6 expression in a time- and concentration-dependent manner, without altering expression or function of the Ca2+ channel Orai1. The expression of exogenous TRPC6 overcame the effect of melatonin on SOCE and cell proliferation, and silencing or inhibition of TRPC6 impaired the inhibitory effect of melatonin on SOCE. These findings indicate that TRPC6 downregulation might be involved in melatonin's inhibitory effects on Ca2+ influx and the maintenance of cancer hallmarks and point toward a novel antitumoral mechanism of melatonin in TNBC cells.

Orai2 Modulates Store-Operated Ca2+ Entry and Cell Cycle Progression in Breast Cancer Cells.

Breast cancer is a heterogeneous disease from the histological and molecular expression point of view, and this heterogeneity determines cancer aggressiveness. Store-operated Ca2+ entry (SOCE), a major mechanism for Ca2+ entry in non-excitable cells, is significantly remodeled in cancer cells and plays an important role in the development and support of different cancer hallmarks. The store-operated CRAC (Ca2+ release-activated Ca2+) channels are predominantly comprised of Orai1 but the participation of Orai2 and Orai3 subunits has been reported to modulate the magnitude of Ca2+ responses. Here we provide evidence for a heterogeneous expression of Orai2 among different breast cancer cell lines. In the HER2 and triple negative breast cancer cell lines SKBR3 and BT20, respectively, where the expression of Orai2 was greater, Orai2 modulates the magnitude of SOCE and sustain Ca2+ oscillations in response to carbachol. Interestingly, in these cells Orai2 modulates the activation of NFAT1 and NFAT4 in response to high and low agonist concentrations. Finally, we have found that, in cells with high Orai2 expression, Orai2 knockdown leads to cell cycle arrest at the G0-G1 phase and decreases apoptosis resistance upon cisplatin treatment. Altogether, these findings indicate that, in breast cancer cells with a high Orai2 expression, Orai2 plays a relevant functional role in agonist-evoked Ca2+ signals, cell proliferation and apoptosis resistance.

Adenylyl Cyclase Type 8 Overexpression Impairs Phosphorylation-Dependent Orai1 Inactivation and Promotes Migration in MDA-MB-231 Breast Cancer Cells.

Orai1 plays a major role in store-operated Ca2+ entry (SOCE) in triple-negative breast cancer (TNBC) cells. This channel is inactivated via different mechanisms, including protein kinase C (PKC) and protein kinase A (PKA)-dependent phosphorylation at Ser-27 and Ser-30 or Ser-34, respectively, which shapes the Ca2+ responses to agonists. The Ca2+ calmodulin-activated adenylyl cyclase type 8 (AC8) was reported to interact directly with Orai1, thus mediating a dynamic interplay between the Ca2+- and cyclic adenosine monophosphate (cAMP)-dependent signaling pathways. Here, we show that the breast cancer cell lines MCF7 and MDA-MB-231 exhibit enhanced expression of Orai1 and AC8 as compared to the non-tumoral breast epithelial MCF10A cell line. In these cells, AC8 interacts with the Orai1α variant in a manner that is not regulated by Orai1 phosphorylation. AC8 knockdown in MDA-MB-231 cells, using two different small interfering RNAs (siRNAs), attenuates thapsigargin (TG)-induced Ca2+ entry and also Ca2+ influx mediated by co-expression of Orai1 and the Orai1-activating small fragment (OASF) of STIM1 (stromal interaction molecule-1). Conversely, AC8 overexpression enhances SOCE, as well as Ca2+ entry, in cells co-expressing Orai1 and OASF. In MDA-MB-231 cells, we found that AC8 overexpression reduces the Orai1 phosphoserine content, thus suggesting that AC8 interferes with Orai1 serine phosphorylation, which takes place at residues located in the AC8-binding site. Consistent with this, the subset of Orai1 associated with AC8 in naïve MDA-MB-231 cells is not phosphorylated in serine residues in contrast to the AC8-independent Orai1 subset. AC8 expression knockdown attenuates migration of MCF7 and MDA-MB-231 cells, while this maneuver has no effect in the MCF10A cell line, which is likely attributed to the low expression of AC8 in these cells. We found that AC8 is required for FAK (focal adhesion kinase) phosphorylation in MDA-MB-231 cells, which might explain its role in cell migration. Finally, we found that AC8 is required for TNBC cell proliferation. These findings indicate that overexpression of AC8 in breast cancer MDA-MB-231 cells impairs the phosphorylation-dependent Orai1 inactivation, a mechanism that might support the enhanced ability of these cells to migrate.

Flavonoids and Platelet-Derived Thrombotic Disorders.

Thrombotic disorders are characterized by an increase in the probability of the formation of unnecessary thrombi that might be due to the activation of the coagulation cascade or the circulating platelets. Platelets or thrombocytes play an essential role in hemostasis but abnormal platelet function leads to the development of a number of cardiovascular complications, including thrombotic disorders. Under pathological conditions, platelets are associated with the development of different thrombotic disorders, including atherosclerosis, arterial thrombosis and stroke, deep venous thrombosis and pulmonary embolism; therefore, platelets are the target of a number of anti-thrombotic strategies. Flavonoids, a large group of polyphenols ubiquitously expressed in fruits and vegetables that have attracted considerable attention because of their benefits in human health, including the reduction of the risk of cardiovascular disease. Flavonoids have been reported to reduce platelet activity by attenuating agonist-induced GPIIb/IIIa receptor activation, mobilization of intracellular free Ca2+, granule exocytosis, as well as activation of different signaling molecules such as mitogen- activated protein kinases or phospholipases. This review summarizes the current studies concerning the modulation of platelet activation by flavonoids, giving especial attention to those events associated to thrombotic disorders.

TRP Channels in Angiogenesis and Other Endothelial Functions.

Angiogenesis is the growth of blood vessels mediated by proliferation, migration, and spatial organization of endothelial cells. This mechanism is regulated by a balance between stimulatory and inhibitory factors. Proangiogenic factors include a variety of VEGF family members, while thrombospondin and endostatin, among others, have been reported as suppressors of angiogenesis. Transient receptor potential (TRP) channels belong to a superfamily of cation-permeable channels that play a relevant role in a number of cellular functions mostly derived from their influence in intracellular Ca2+ homeostasis. Endothelial cells express a variety of TRP channels, including members of the TRPC, TRPV, TRPP, TRPA, and TRPM families, which play a relevant role in a number of functions, including endothelium-induced vasodilation, vascular permeability as well as sensing hemodynamic and chemical changes. Furthermore, TRP channels have been reported to play an important role in angiogenesis. This review summarizes the current knowledge and limitations concerning the involvement of particular TRP channels in growth factor-induced angiogenesis.

Melatonin enhances responsiveness to Dichelobacter nodosus vaccine in sheep and increases peripheral blood CD4 T lymphocytes and IgG-expressing B lymphocytes.

The immunomodulatory functions mediated by melatonin support its use as vaccine adjuvant. Previously, we have demonstrated that melatonin enhances antibody responses in sheep vaccinated against Dichelobacter nodosus. Here, we analyze the effect of melatonin on T and B lymphocyte subsets in peripheral blood of sheep vaccinated against D. nodosus. We also compare the use of melatonin in implants and in injections. Melatonin administration either as implants or by injection produced higher antibody titers against A1 and C serotypes compared to those animals that received only the vaccine. These results support the use of melatonin as an adjuvant in vaccination against D. nodosus. Firstly, melatonin induces higher antibody titer than the vaccine alone, secondly, melatonin increase IgG+ B lymphocytes and CD4+ T lymphocytes in vaccinated sheep. These results suggest that melatonin enhances T CD4 cell activation and subsequently secondary humoral immune responses. Further studies are required to determine the mechanism underlining the immunomodulatory role of melatonin in the context of vaccination.

Role of STIM1 in the surface expression of SARAF.

The store-operated Ca2+ entry-associated regulatory factor (SARAF), a protein expressed both in the endoplasmic reticulum and the plasma membrane, has been presented as a STIM1-interacting protein with the ability to modulate intracellular Ca2+ homeostasis. SARAF negatively modulates store-operated Ca2+ entry (SOCE) by preventing STIM1 spontaneous activation and regulating STIM1-Orai1 complex formation. In addition, SARAF is a negative regulator of Ca2+ entry through the arachidonate-regulated Ca2+ (ARC) channels. Here we explored the possible role of the surface expression of SARAF on the location of STIM1 in the plasma membrane. In NG115-401L cells, lacking a detectable expression of native STIM1, transfection with pHluorin-STIM1, which is able to translocate to the cell surface, enhances the plasma membrane location of SARAF as compared to cells transfected with YFP-STIM1, lacking the ability to translocate to the cell surface. These findings suggest that the surface location of SARAF is dependent on the expression of STIM1 in the plasma membrane.

Vaccination prepartum enhances the beneficial effects of melatonin on the immune response and reduces platelet responsiveness in sheep.

BACKGROUND: Melatonin regulates several physiological processes and its powerful action as antioxidant has been widely reported. Melatonin acts modulating the immune system, showing a protective effect on the cardiovascular system and improving vaccine administration as an adjuvant-like agent. Here, we have investigated the role of melatonin as an adjuvant of the Clostridium perfringens vaccine in prepartum sheep and whether melatonin modulates platelet physiology during peripartum. RESULTS: The experiments were carried out in peripartum sheep from a farm located in an area of Mediterranean-type ecosystem. Plasma melatonin levels were determined by ELISA and sheep platelet aggregation was monitored using an aggregometer. Here we demonstrated for the first time that plasma melatonin concentration were higher in pregnant (125 pg/mL) than in non-pregnant sheep (15 pg/mL; P < 0.05). Administration of melatonin prepartum did not significantly modify platelet function but significantly improved the immune response to vaccination against C. perfringens. CONCLUSION: Administration of melatonin as an adjuvant provides a significant improvement in the immune response to vaccine administration prepartum against C. perfringens.

Evolution of oxidative/nitrosative stress biomarkers during an open-field vaccination procedure in sheep: effect of melatonin.

Melatonin has been shown to exert immunomodularory properties with broad application in veterinary medicine. In previous work we have described that subcutaneous coadministration of melatonin to seeps vaccinated against two stumps of A1 and C strains of Dichelobacter nodosus enhanced both the antibody titer and serum IgG levels to A1 and C strains of D. nodosus compared to vaccinated animals not treated with melatonin. Following a similar protocol here we have investigated the effect of a higher dose of melatonin (36mg/animal) in the improvement of the immune response and in the possible oxidative/nitrosative stress produced during the immunization protocol. Our results show that footrot vaccine application induced nitrosative but not oxidative stress at 42 days post-vaccination, which was neutralized by melatonin administration. On the other hand, melatonin improved the immune response with respect to our previous data increasing the time of permanence of antibodies in serum, opening new perspectives for melatonin as prophylactic drug.

Melatonin, as an adjuvant-like agent, enhances platelet responsiveness.

Melatonin exerts immunomodulatory actions that enhance the magnitude and quality of immune responses specific for certain antigens; this has raised the possibility of using melatonin to design novel vaccine adjuvant systems. The present study investigated the effect of subcutaneous slow-release melatonin implants and subcutaneous melatonin injections on the responsiveness of circulating platelets in sheep after vaccination against Dichelobacter nodosus (A1 and C serotypes), the bacterium that causes ovine footrot, a major cause of lameness in sheep. The experiments were carried out in sheep from a farm located in an area of Mediterranean-type ecosystem. Plasma melatonin levels were determined by radioimmunoassay, sheep platelet aggregation was monitored using an aggregometer and Ca2+ mobilization was determined by spectrofluorimetry using fura-2. Administration of melatonin either by implants or subcutaneous injections increased plasma melatonin concentrations, an effect that was found to be greater and more sustained when melatonin was administered via implants. Vaccination per se, as well as melatonin, increased the percentage and rate of platelet aggregation and reduced the lag-time in response to the physiological agonist thrombin, an effect that was found to be significantly greater when melatonin was administered to vaccinated animals. Melatonin enhanced thrombin-evoked Ca2+ release and entry and further increased Ca2+ mobilization observed in platelets from vaccinated sheep. These observations suggest that the use of melatonin, as a novel adjuvant, induces beneficial effects on platelet function and haemostasis, and opens new perspectives for therapeutic manipulation of immune responses to vaccination.

Melatonin enhances the immune response to vaccination against A1 and C strains of Dichelobacter nodosus.

Melatonin has been shown to exert immunomodulatory properties with broad application in veterinary medicine. Here we have investigated the effect of exogenous melatonin in the improvement of the immune response to administration of an immune-preparation of two stumps of A1 and C strains of Dichelobacter nodosus in sheep. Subcutaneous administration of melatonin enhanced plasma levels of melatonin from days 42 to 120. Administration of melatonin to vaccinated animals enhanced both the titer of antibodies and serum IgG levels to A1 and C strains of D. nodosus compared to vaccinated animals not treated with melatonin. Our results suggest that melatonin increased the immune response to vaccination and open new perspectives in the design of prophylactic strategies.

The use of melatonin as a vaccine agent.

Molecules with immunomodulatory properties determine the magnitude and quality of immune responses specific for the coadministered antigen. Melatonin is considered a biological-response modifier of the immune system with broad application in veterinary medicine. In seasonally-breeding animals, the indolamine is able to improve reproductive performance. With the purpose of expanding new advantageous roles for melatonin, we investigated the effect of subcutaneous slow-release melatonin implants in the humoral response after a vaccination. We reported here a new feature of melatonin as an adjuvant-like system towards Dichelobacter nodosus (A1 and C serotypes)--the bacterium which cause ovine footrot--the most important cause of lameness in sheep. Antibody titres determined by both agglutination and ELISA techniques were substantially higher and were sustained for a longer duration than non-implanted animals. Remarkably, the effect of melatonin was completely dependent on the presence of aluminium hydroxide. The finding that melatonin enhances a defined immune response in vivo opens new perspectives for the improvement of Th2-biased immune responses by alum adjuvants.