Extended Synaptotagmins 1 and 2 Are Required for Store-Operated Calcium Entry, Cell Migration and Viability in Breast Cancer Cells.

Extended synaptotagmins (E-Syts) are endoplasmic reticulum (ER)-associated proteins that facilitate the tethering of the ER to the plasma membrane (PM), participating in lipid transfer between the membranes and supporting the Orai1-STIM1 interaction at ER-PM junctions. Orai1 and STIM1 are the core proteins of store-operated Ca2+ entry (SOCE), a major mechanism for Ca2+ influx that regulates a variety of cellular functions. Aberrant modulation of SOCE in cells from different types of cancer has been reported to underlie the development of several tumoral features. Here we show that estrogen receptor-positive (ER+) breast cancer MCF7 and T47D cells and triple-negative breast cancer (TNBC) MDA-MB-231 cells overexpress E-Syt1 and E-Syt2 at the protein level; the latter is also overexpressed in the TNBC BT20 cell line. E-Syt1 and E-Syt2 knockdown was without effect on SOCE in non-tumoral MCF10A breast epithelial cells and ER+ T47D breast cancer cells; however, SOCE was significantly attenuated in ER+ MCF7 cells and TNBC MDA-MB-231 and BT20 cells upon transfection with siRNA E-Syt1 or E-Syt2. Consistent with this, E-Syt1 and E-Syt2 knockdown significantly reduced cell migration and viability in ER+ MCF7 cells and the TNBC cells investigated. To summarize, E-Syt1 and E-Syt2 play a relevant functional role in breast cancer cells.

Orai1α and Orai1ß support calcium entry and mammosphere formation in breast cancer stem cells.

Orai1 is the pore-forming subunit of the Ca2+-release activated Ca2+ channels that mediate store-operated Ca2+ entry (SOCE) in excitable and non-excitable cells. Two Orai1 forms have been identified in mammalian cells, the full-length variant Orai1α, and the short form Orai1ß, lacking the N-terminal 63 amino acids. Stem cells were isolated from non-tumoral breast epithelial cells of the MCF10A cell line, and the most representative ER+ , HER2 or triple negative breast cancer cell lines MCF7, SKBR3 and MDA-MB-231, respectively. Orai and TRPC family members expression was detected by RT-PCR and Western blotting. Changes in cytosolic Ca2+ concentration were analyzed by confocal microscopy using Fluo 4 and the spheroid-forming ability and self-renewal was estimated in culture plates coated with pHEMA using a cell imaging system. Here, we have characterized the expression of Orai family members and several TRPC channels at the transcript level in breast stem cells (BSC) derived from the non-tumoral breast epithelial cell line MCF10A and breast cancer stem cells (BCSC) derived from the well-known estrogen receptor positive (ER+), HER2 and triple negative cell lines MCF7, SKBR3 and MDA-MB-231, respectively. Furthermore, we have evaluated the mammosphere formation efficiency and self-renewal of the BSC and BCSC. Next, through a combination of Orai1 knockdown by iRNA and the use of MDA-MB-231 KO cells, missing the native Orai1, transfected with plasmids encoding for either Orai1α or Orai1ß, we show that Orai1 is essential for mammosphere formation and self-renewal efficiency in BCSC derived from triple negative and HER2 subtypes cell cultures, while this channel has a negligible effect in BCSC derived from ER+ cells as well as in non-tumoral BSC. Both, Orai1α, and Orai1ß support SOCE in MDA-MB-231-derived BCSC with similar efficiency, as well as COX activation and mammosphere formation. These findings provide evidence of the functional role of Orai1α and Orai1ß in spheroid forming efficiency and self-renewal in breast cancer stem cells.

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 [...].

Store-Operated Calcium Entry and Its Implications in Cancer Stem Cells.

Tumors are composed by a heterogeneous population of cells. Among them, a sub-population of cells, termed cancer stem cells, exhibit stemness features, such as self-renewal capabilities, disposition to differentiate to a more proliferative state, and chemotherapy resistance, processes that are all mediated by Ca2+. Ca2+ homeostasis is vital for several physiological processes, and alterations in the patterns of expressions of the proteins and molecules that modulate it have recently become a cancer hallmark. Store-operated Ca2+ entry is a major mechanism for Ca2+ entry from the extracellular medium in non-excitable cells that leads to increases in the cytosolic Ca2+ concentration required for several processes, including cancer stem cell properties. Here, we focus on the participation of STIM, Orai, and TRPC proteins, the store-operated Ca2+ entry key components, in cancer stem cell biology and tumorigenesis.

Store-Operated Calcium Entry in Breast Cancer Cells Is Insensitive to Orai1 and STIM1 N-Linked Glycosylation.

N-linked glycosylation is a post-translational modification that affects protein function, structure, and interaction with other proteins. The store-operated Ca2+ entry (SOCE) core proteins, Orai1 and STIM1, exhibit N-glycosylation consensus motifs. Abnormal SOCE has been associated to a number of disorders, including cancer, and alterations in Orai1 glycosylation have been related to cancer invasiveness and metastasis. Here we show that treatment of non-tumoral breast epithelial cells with tunicamycin attenuates SOCE. Meanwhile, tunicamycin was without effect on SOCE in luminal MCF7 and triple negative breast cancer (TNBC) MDA-MB-231 cells. Ca2+ imaging experiments revealed that expression of the glycosylation-deficient Orai1 mutant (Orai1N223A) did not alter SOCE in MCF10A, MCF7 and MDA-MB-231 cells. However, expression of the non-glycosylable STIM1 mutant (STIM1N131/171Q) significantly attenuated SOCE in MCF10A cells but was without effect in SOCE in MCF7 and MDA-MB-231 cells. In non-tumoral cells impairment of STIM1 N-linked glycosylation attenuated thapsigargin (TG)-induced caspase-3 activation while in breast cancer cells, which exhibit a smaller caspase-3 activity in response to TG, expression of the non-glycosylable STIM1 mutant (STIM1N131/171Q) was without effect on TG-evoked caspase-3 activation. Summarizing, STIM1 N-linked glycosylation is essential for full SOCE activation in non-tumoral breast epithelial cells; by contrast, SOCE in breast cancer MCF7 and MDA-MB-231 cells is insensitive to Orai1 and STIM1 N-linked glycosylation, and this event might participate in the development of apoptosis resistance.

Role of Orai3 in the Pathophysiology of Cancer.

The mammalian exclusive Orai3 channel participates in the generation and/or modulation of two independent Ca2+ currents, the store-operated current, Icrac, involving functional interactions between the stromal interaction molecules (STIM), STIM1/STIM2, and Orai1/Orai2/Orai3, as well as the store-independent arachidonic acid (AA) (or leukotriene C4)-regulated current Iarc, which involves Orai1, Orai3 and STIM1. Overexpression of functional Orai3 has been described in different neoplastic cells and cancer tissue samples as compared to non-tumor cells or normal adjacent tissue. In these cells, Orai3 exhibits a cell-specific relevance in Ca2+ influx. In estrogen receptor-positive breast cancer cells and non-small cell lung cancer (NSCLC) cells store-operated Ca2+ entry (SOCE) is strongly dependent on Orai3 expression while in colorectal cancer and pancreatic adenocarcinoma cells Orai3 predominantly modulates SOCE. On the other hand, in prostate cancer cells Orai3 expression has been associated with the formation of Orai1/Orai3 heteromeric channels regulated by AA and reduction in SOCE, thus leading to enhanced proliferation. Orai3 overexpression is associated with supporting several cancer hallmarks, including cell cycle progression, proliferation, migration, and apoptosis resistance. This review summarizes the current knowledge concerning the functional role of Orai3 in the pathogenesis of cancer.

SARAF and EFHB Modulate Store-Operated Ca2+ Entry and Are Required for Cell Proliferation, Migration and Viability in Breast Cancer Cells.

Breast cancer is among the most common malignancies in women. From the molecular point of view, breast cancer can be grouped into different categories, including the luminal (estrogen receptor positive (ER+)) and triple negative subtypes, which show distinctive features and, thus, are sensitive to different therapies. Breast cancer cells are strongly dependent on Ca2+ influx. Store-operated Ca2+ entry (SOCE) has been found to support a variety of cancer hallmarks including cell viability, proliferation, migration, and metastasis. The Ca2+ channels of the Orai family and the endoplasmic reticulum Ca2+ sensor STIM1 are the essential components of SOCE, but the extent of Ca2+ influx is fine-tuned by several regulatory proteins, such as the STIM1 modulators SARAF and EFHB. Here, we show that the expression and/or function of SARAF and EFHB is altered in breast cancer cells and both proteins are required for cell proliferation, migration, and viability. EFHB expression is upregulated in luminal and triple negative breast cancer (TNBC) cells and is essential for full SOCE in these cells. SARAF expression was found to be similar in breast cancer and pre-neoplastic breast epithelial cells, and SARAF knockdown was found to result in enhanced SOCE in pre-neoplastic and TNBC cells. Interestingly, silencing SARAF expression in ER+ MCF7 cells led to attenuation of SOCE, thus suggesting a distinctive role for SARAF in this cell type. Finally, we used a combination of approaches to show that molecular knockdown of SARAF and EFHB significantly attenuates the ability of breast cancer cells to proliferate and migrate, as well as cell viability. In aggregate, SARAF and EFHB are required for the fine modulation of SOCE in breast cancer cells and play an important role in the maintenance of proliferation, migration, and viability in these cells.

Melatonin Induces Apoptosis and Modulates Cyclin Expression and MAPK Phosphorylation in Pancreatic Stellate Cells Subjected to Hypoxia.

In certain diseases of the pancreas, pancreatic stellate cells form an important part of fibrosis and are critical for the development of cancer cells. A hypoxic condition develops within the tumor, to which pancreatic stellate cells adapt and are able to proliferate. The consequence is the growth of the tumor. Melatonin, the product of the pineal gland, is gaining attention as an agent with therapeutic potential against pancreatic cancers. Its actions on tumor cells lead, in general, to a reduction in cell viability and proliferation. However, its effects on pancreatic stellate cells subjected to hypoxia are less known. In this study, we evaluated the actions of pharmacological concentrations of melatonin (1 mM-1 µM) on pancreatic stellate cells subjected to hypoxia. The results show that melatonin induced a decrease in cell viability at the highest concentrations tested. Similarly, the incorporation of BrdU into DNA was diminished by melatonin. The expression of cyclins A and D also was decreased in the presence of melatonin. Upon treatment of cells with melatonin, increases in the expression of major markers of ER stress, namely BIP, phospho-eIF2α and ATF-4, were detected. Modulation of apoptosis was noticed as an increase in caspase-3 activation. In addition, changes in the phosphorylated state of p44/42, p38 and JNK MAPKs were detected in cells treated with melatonin. A slight decrease in the content of α-smooth muscle actin was detected in cells treated with melatonin. Finally, treatment of cells with melatonin decreased the expression of matrix metalloproteinases 2, 3, 9 and 13. Our observations suggest that melatonin, at pharmacological concentrations, diminishes the proliferation of pancreatic stellate cells subjected to hypoxia through modulation of cell cycle, apoptosis and the activation of crucial MAPKs. Cellular responses might involve certain ER stress regulator proteins. In view of the results, melatonin could be taken into consideration as a potential therapeutic agent for pancreatic fibrosis.

Melatonin Modulates the Antioxidant Defenses and the Expression of Proinflammatory Mediators in Pancreatic Stellate Cells Subjected to Hypoxia.

Pancreatic stellate cells (PSC) play a major role in the formation of fibrotic tissue in pancreatic tumors. On its side, melatonin is a putative therapeutic agent for pancreatic cancer and inflammation. In this work, the actions of melatonin on PSC subjected to hypoxia were evaluated. Reactive oxygen species (ROS) generation reduced (GSH) and oxidized (GSSG) levels of glutathione, and protein and lipid oxidation were analyzed. The phosphorylation of nuclear factor erythroid 2-related factor (Nrf2), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB), and the regulatory protein nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor-alpha (IκBα) was studied. The expression of Nrf2-regulated antioxidant enzymes, superoxide dismutase (SOD) enzymes, cyclooxygenase 2 (COX-2), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were also studied. Total antioxidant capacity (TAC) was assayed. Finally, cell viability was studied. Under hypoxia and in the presence of melatonin generation of ROS was observed. No increases in the oxidation of proteins or lipids were detected. The phosphorylation of Nrf2 and the expression of the antioxidant enzymes catalytic subunit of glutamate-cysteine ligase, catalase, NAD(P)H-quinone oxidoreductase 1, heme oxygenase-1, SOD1, and of SOD2 were augmented. The TAC was increased. Protein kinase C was involved in the effects of melatonin. Melatonin decreased the GSH/GSSG ratio at the highest concentration tested. Cell viability dropped in the presence of melatonin. Finally, melatonin diminished the phosphorylation of NF-kB and the expression of COX-2, IL-6, and TNF-α. Our results indicate that melatonin, at pharmacological concentrations, modulates the red-ox state, viability, and the expression of proinflammatory mediators in PSC subjected to hypoxia.

Cross-Talk Between the Adenylyl Cyclase/cAMP Pathway and Ca2+ Homeostasis.

Cyclic AMP and Ca2+ are the first second or intracellular messengers identified, unveiling the cellular mechanisms activated by a plethora of extracellular signals, including hormones. Cyclic AMP generation is catalyzed by adenylyl cyclases (ACs), which convert ATP into cAMP and pyrophosphate. By the way, Ca2+, as energy, can neither be created nor be destroyed; Ca2+ can only be transported, from one compartment to another, or chelated by a variety of Ca2+-binding molecules. The fine regulation of cytosolic concentrations of cAMP and free Ca2+ is crucial in cell function and there is an intimate cross-talk between both messengers to fine-tune the cellular responses. Cancer is a multifactorial disease resulting from a combination of genetic and environmental factors. Frequent cases of cAMP and/or Ca2+ homeostasis remodeling have been described in cancer cells. In those tumoral cells, cAMP and Ca2+ signaling plays a crucial role in the development of hallmarks of cancer, including enhanced proliferation and migration, invasion, apoptosis resistance, or angiogenesis. This review summarizes the cross-talk between the ACs/cAMP and Ca2+ intracellular pathways with special attention to the functional and reciprocal regulation between Orai1 and AC8 in normal and cancer 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.

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.

PGRMC1 Inhibits Progesterone-Evoked Proliferation and Ca2+ Entry Via STIM2 in MDA-MB-231 Cells.

Progesterone receptor membrane component 1 (PGRMC1) has been shown to regulate some cancer hallmarks. Progesterone (P4) evokes intracellular calcium (Ca2+) changes in the triple-negative breast cancer cell lines (MDA-MB-231, MDA-MB-468, and BT-20) and in other breast cancer cell lines like the luminal MCF7 cells. PGRMC1 expression is elevated in MDA-MB-231 and MCF7 cells as compared to non-tumoral MCF10A cell line, and PGRMC1 silencing enhances P4-evoked Ca2+ mobilization. Here, we found a new P4-dependent Ca2+ mobilization pathway in MDA-MB-231 cells and other triple-negative breast cancer cells, as well as in MCF7 cells that involved Stromal interaction molecule 2 (STIM2), Calcium release-activated calcium channel protein 1 (Orai1), and Transient Receptor Potential Channel 1 (TRPC1). Stromal interaction molecule 1 (STIM1) was not involved in this novel Ca2+ pathway, as evidenced by using siRNA STIM1. PGRMC1 silencing reduced the negative effect of P4 on cell proliferation and cell death in MDA-MB-231 cells. In line with the latter observation, Nuclear Factor of Activated T-Cells 1 (NFAT1) nuclear accumulation due to P4 incubation for 48 h was enhanced in cells transfected with the small hairpin siRNA against PGRMC1 (shPGRMC1). These results provide evidence for a novel P4-evoked Ca2+ entry pathway that is downregulated by PGRMC1.

TRPC6 channel and its implications in breast cancer: an overview.

TRPC6 channel is widely expressed in most human tissues and participates in a number of physiological processes. TRPC6 belongs to the DAG-activated subfamily of channels, but has also been postulated as a mediator in the store-operated calcium entry pathway. The recent characterization of TRPC6 crystal structure has granted a wonderful tool to finally dissect and understand TRPC6 physiological and biophysical properties. Growing evidences have demonstrated that the pattern of expression of TRPC6 proteins is upregulated in several pathophysiological conditions, including breast cancer. However, the real role of TRPC6 in breast cancer persists still unknown. Here we present the current state of the art concerning the function and significance of TRPC6 in this disease. Future investigations should be focus in the creation and identification of compounds that specifically target the channel to ameliorate TRPC6-related diseases.

Functional role of TRPC6 and STIM2 in cytosolic and endoplasmic reticulum Ca2+ content in resting estrogen receptor-positive breast cancer cells.

TRPC6 forms non-selective cation channels activated by a variety of stimuli that are involved in a wide number of cellular functions. In estrogen receptor-positive (ER+) breast cancer cells, the store-operated Ca2+ entry has been reported to be dependent on STIM1, STIM2 and Orai3, with TRPC6 playing a key role in the activation of store-operated Ca2+ entry as well as in proliferation, migration and viability of breast cancer cells. We have used a combination of biotinylation, Ca2+ imaging as well as protein knockdown and overexpression of a dominant-negative TRPC6 mutant (TRPC6dn) to show that TRPC6 and STIM2 are required for the maintenance of cytosolic and endoplasmic reticulum Ca2+ content under resting conditions in ER+ breast cancer MCF7 cells. These cells exhibit a greater plasma membrane expression of TRPC6 under resting conditions than non-tumoral breast epithelial cells. Attenuation of STIM2, TRPC6 and Orai3, alone or in combination, results in impairment of resting cytosolic and endoplasmic reticulum Ca2+ homeostasis. Similar results were observed when cells were transfected with expression plasmid for TRPC6dn. TRPC6 co-immunoprecipitates with STIM2 in resting MCF7 cells, a process that is impaired by rises in cytosolic Ca2+ concentration. Impairment of TRPC6 function leads to abnormal Ca2+ homeostasis and endoplasmic reticulum stress, thus, suggesting that TRPC6 might be a potential target for the development of anti-tumoral therapies.

Pancreatic stellate cells exhibit adaptation to oxidative stress evoked by hypoxia.

BACKGROUND INFORMATION: Pancreatic stellate cells play a key role in the fibrosis that develops in diseases such as pancreatic cancer. In the growing tumour, a hypoxia condition develops under which cancer cells are able to proliferate. The growth of fibrotic tissue contributes to hypoxia. In this study, the effect of hypoxia (1% O2 ) on pancreatic stellate cells physiology was investigated. Changes in intracellular free-Ca2+ concentration, mitochondrial free-Ca2+ concentration and mitochondrial membrane potential were studied by fluorescence techniques. The status of enzymes responsible for the cellular oxidative state was analyzed by quantitative reverse transcription-polymerase chain reaction, high-performance liquid chromatography, spectrophotometric and fluorimetric methods and by Western blotting analysis. Cell viability and proliferation were studied by crystal violet test, 5-bromo-2-deoxyuridine cell proliferation test and Western blotting analysis. Finally, cell migration was studied employing the wound healing assay. RESULTS: Hypoxia induced an increase in intracellular and mitochondrial free-Ca2+ concentration, whereas mitochondrial membrane potential was decreased. An increase in mitochondrial reactive oxygen species production was observed. Additionally, an increase in the oxidation of proteins and lipids was detected. Moreover, cellular total antioxidant capacity was decreased. Increases in the expression of superoxide dismutase 1 and 2 were observed and superoxide dismutase activity was augmented. Hypoxia evoked a decrease in the oxidized/reduced glutathione ratio. An increase in the phosphorylation of nuclear factor erythroid 2-related factor and in expression of the antioxidant enzymes catalytic subunit of glutamate-cysteine ligase, catalase, NAD(P)H-quinone oxidoreductase 1 and heme oxygenase-1 were detected. The expression of cyclin A was decreased, whereas expression of cyclin D and the content of 5-bromo-2-deoxyuridine were increased. This was accompanied by an increase in cell viability. The phosphorylation state of c-Jun NH2 -terminal kinase was increased, whereas that of p44/42 and p38 was decreased. Finally, cells subjected to hypoxia maintained migration ability. CONCLUSIONS AND SIGNIFICANCE: Hypoxia creates pro-oxidant conditions in pancreatic stellate cells to which cells adapt and leads to increased viability and proliferation.

Melatonin modulates proliferation of pancreatic stellate cells through caspase-3 activation and changes in cyclin A and D expression.

In this study, the effects of melatonin (1 µM-1 mM) on pancreatic stellate cells (PSC) have been examined. Cell viability and proliferation, caspase-3 activation, and the expression of cyclin A and cyclin D were analyzed. Our results show that melatonin decreased PSC viability in a time- and concentration-dependent manner. This effect was not inhibited by treatment of cells with MT1, MT2, calmodulin, or ROR-alpha inhibitors prior to melatonin addition. Activation of caspase-3 in response to melatonin was detected. The expression of cyclin A and cyclin D was decreased in cells treated with melatonin. Finally, changes in BrdU incorporation into the newly synthesized DNA of proliferating cells were also observed in the presence of melatonin. We conclude that melatonin, at pharmacological concentrations, modulates proliferation of PSC through activation of apoptosis and involving crucial regulators of the cell cycle. These actions might not require specific melatonin receptors. Our observations suggest that melatonin, at high doses, could potentially exert anti-fibrotic effects and, thus, could be taken into consideration as supportive treatment in the therapy of pancreatic diseases.

Arachidonic Acid Attenuates Cell Proliferation, Migration and Viability by a Mechanism Independent on Calcium Entry.

Arachidonic acid (AA) is a phospholipase A2 metabolite that has been reported to mediate a plethora of cellular mechanisms involved in healthy and pathological states such as platelet aggregation, lymphocyte activation, and tissue inflammation. AA has been described to activate Ca2+ entry through the arachidonate-regulated Ca2+-selective channels (ARC channels). Here, the analysis of the changes in the intracellular Ca2+ homeostasis revealed that, despite MDA-MB-231 cells expressing the ARC channel components Orai1, Orai3, and STIM1, AA does not evoke Ca2+ entry in these cells. We observed that AA evokes Ca2+ entry in MDA-MB-231 cells transiently expressing ARC channels. Nevertheless, MDA-MB-231 cell treatment with AA reduces cell proliferation and migration while inducing cell death through apoptosis. The latter mostly likely occurs via mitochondria membrane depolarization and the activation of caspases-3, -8, and -9. Altogether, our results indicate that AA exerts anti-tumoral effects on MDA-MB-231 cells, without having any effect on non-tumoral breast epithelial cells, by a mechanism that is independent on the activation of Ca2+ influx via ARC channels.

Melatonin modulates red-ox state and decreases viability of rat pancreatic stellate cells.

In this work we have studied the effects of pharmacological concentrations of melatonin (1 µM-1 mM) on pancreatic stellate cells (PSC). Cell viability was analyzed by AlamarBlue test. Production of reactive oxygen species (ROS) was monitored following CM-H2DCFDA and MitoSOX Red-derived fluorescence. Total protein carbonyls and lipid peroxidation were analyzed by HPLC and spectrophotometric methods respectively. Mitochondrial membrane potential (ψm) was monitored by TMRM-derived fluorescence. Reduced (GSH) and oxidized (GSSG) levels of glutathione were determined by fluorescence techniques. Quantitative reverse transcription-polymerase chain reaction was employed to detect the expression of Nrf2-regulated antioxidant enzymes. Determination of SOD activity and total antioxidant capacity (TAC) were carried out by colorimetric methods, whereas expression of SOD was analyzed by Western blotting and RT-qPCR. The results show that melatonin decreased PSC viability in a concentration-dependent manner. Melatonin evoked a concentration-dependent increase in ROS production in the mitochondria and in the cytosol. Oxidation of proteins was detected in the presence of melatonin, whereas lipids oxidation was not observed. Depolarization of ψm was noted with 1 mM melatonin. A decrease in the GSH/GSSG ratio was observed, that depended on the concentration of melatonin used. A concentration-dependent increase in the expression of the antioxidant enzymes catalytic subunit of glutamate-cysteine ligase, catalase, NAD(P)H-quinone oxidoreductase 1 and heme oxygenase-1 was detected in cells incubated with melatonin. Finally, decreases in the expression and in the activity of superoxide dismutase were observed. We conclude that pharmacological concentrations melatonin modify the redox state of PSC, which might decrease cellular viability.

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.