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1.
Function (Oxf) ; 3(4): zqac033, 2022.
Article in English | MEDLINE | ID: mdl-35910331

ABSTRACT

Cannabis sativa has long been known to affect numerous biological activities. Although plant extracts, purified cannabinoids, or synthetic cannabinoid analogs have shown therapeutic potential in pain, inflammation, seizure disorders, appetite stimulation, muscle spasticity, and treatment of nausea/vomiting, the underlying mechanisms of action remain ill-defined. In this study we provide the first comprehensive overview of the effects of whole-plant Cannabis extracts and various pure cannabinoids on store-operated calcium (Ca2+) entry (SOCE) in several different immune cell lines. Store-operated Ca2+ entry is one of the most significant Ca2+ influx mechanisms in immune cells, and it is critical for the activation of T lymphocytes, leading to the release of proinflammatory cytokines and mediating inflammation and T cell proliferation, key mechanisms for maintaining chronic pain. While the two major cannabinoids cannabidiol and trans-Δ9-tetrahydrocannabinol were largely ineffective in inhibiting SOCE, we report for the first time that several minor cannabinoids, mainly the carboxylic acid derivatives and particularly cannabigerolic acid, demonstrated high potency against SOCE by blocking calcium release-activated calcium currents. Moreover, we show that this inhibition of SOCE resulted in a decrease of nuclear factor of activated T-cells activation and Interleukin 2 production in human T lymphocytes. Taken together, these results indicate that cannabinoid-mediated inhibition of a proinflammatory target such as SOCE may at least partially explain the anti-inflammatory and analgesic effects of Cannabis.


Subject(s)
Cannabinoids , Cytokines , Humans , Cytokines/metabolism , Calcium/metabolism , Cannabinoids/pharmacology , Calcium Signaling , Inflammation/drug therapy
2.
Eur J Pharmacol ; 853: 299-307, 2019 Jun 15.
Article in English | MEDLINE | ID: mdl-30965058

ABSTRACT

Transient receptor potential melastatin type 2 (TRPM2) is a cation channel activated by free intracellular ADP-ribose and reactive oxygen species. TRPM2 signaling has been linked to the pathophysiology of CNS disorders such as neuropathic pain, bipolar disorder and Alzheimer's disease. In this manuscript, we describe the discovery of JNJ-28583113, a potent brain penetrant TRPM2 antagonist. Ca2+ flux assays in cells overexpressing TRPM2 and electrophysiological recordings were used to test the pharmacology of JNJ-28583113. JNJ-28583113 was assayed in vitro on GSK-3 phosphorylation levels, cell death, cytokine release in microglia and unbiased morphological phenotypic analysis. Finally, we dosed animals to evaluate its pharmacokinetic properties. Our results showed that JNJ-28583113 is a potent (126 ±â€¯0.5 nM) TRPM2 antagonist. Blocking TRPM2 caused phosphorylation of GSK3α and ß subunits. JNJ-28583113 also protected cells from oxidative stress induced cell death as well as morphological changes induced by non-cytotoxic concentrations of H2O2. In addition, inhibiting TRPM2 blunted cytokine release in response to pro-inflammatory stimuli in microglia. Lastly, we showed that JNJ-28583113 was brain penetrant but not suitable for systemic dosing as it was rapidly metabolized in vivo. While the in-vitro pharmacology of JNJ-28583113 is the best in class, its in-vivo properties would need optimization to assist in further probing key roles of TRPM2 in CNS pathophysiology.


Subject(s)
Drug Discovery , Pyrazoles/pharmacology , TRPM Cation Channels/antagonists & inhibitors , Animals , HEK293 Cells , HeLa Cells , Humans , Male , Mice , Rats
3.
Sci Rep ; 8(1): 1075, 2018 01 18.
Article in English | MEDLINE | ID: mdl-29348572

ABSTRACT

Betel nut consumption has significant implications for the public health globally, as the wide-spread habit of Areca chewing throughout Asia and the Pacific is associated with a high prevalence of oral carcinoma and other diseases. Despite a clear causal association of betel nut chewing and oral mucosal diseases, the biological mechanisms that link Areca nut-contained molecules, inflammation and cancer remain underexplored. In this study we show that the whole Areca nut extract (ANE) is capable of mobilizing Ca2+ in various immune cell lines. Interestingly, none of the four major alkaloids or a range of other known constituents of Areca nut were able to induce such Ca2+ signals, suggesting that the active components might represent novel or so far unappreciated chemical structures. The separation of ANE into aqueous and organic fractions has further revealed that the calcium-mobilizing molecules are exclusively present in the aqueous extract. In addition, we found that these calcium signals are associated with the activation of several immune cell lines as shown by the release of pro-inflammatory cytokines and increased cell proliferation. These results indicate that calcium-mobilizing molecules present in the aqueous fraction of the Areca nut may critically contribute to the inflammatory disorders affecting betel nut chewers.


Subject(s)
Areca/chemistry , Calcium/metabolism , Cytokines/metabolism , Inflammation Mediators/metabolism , Nuts/chemistry , Plant Extracts/pharmacology , Calcium Signaling/drug effects , Cell Line , Cell Proliferation , Cells, Cultured , Humans , Leukocytes, Mononuclear/drug effects , Leukocytes, Mononuclear/metabolism
5.
Cell Commun Signal ; 15(1): 30, 2017 08 15.
Article in English | MEDLINE | ID: mdl-28810912

ABSTRACT

BACKGROUND: Magnesium (Mg2+) is an essential cation implicated in carcinogenesis, solid tumor progression and metastatic potential. The Transient Receptor Potential Melastatin Member 7 (TRPM7) is a divalent ion channel involved in cellular and systemic Mg2+ homeostasis. Abnormal expression of TRPM7 is found in numerous cancers, including colon, implicating TRPM7 in this process. METHODS: To establish a possible link between systemic magnesium (Mg2+) status, the Mg2+ conducting channel TRPM7 in colon epithelial cells, and colon carcinogenesis, in vitro whole-cell patch clamp electrophysiology, qPCR, and pharmacological tools were used probing human colorectal adenocarcinoma HT-29 as well as normal primary mouse colon epithelial cells. This was extended to and combined with aberrant crypt foci development in an azoxymethane-induced colorectal cancer mouse model under hypomagnesemia induced by diet or pharmacologic intervention. RESULTS: We find that TRPM7 drives colon cancer cell proliferation in human HT-29 and expresses in normal primary mouse colon epithelia. This is linked to TRPM7's dominant role as Mg2+ transporter, since high extracellular Mg2+ supplementation cannot rescue inhibition of cell proliferation caused by suppressing TRPM7 either genetically or pharmacologically. In vivo experiments in mice provide evidence that the specific TRPM7 inhibitor waixenicin A, given as a single bolus injection, induces transient hypomagnesemia and increases intestinal absorption of calcium. Repeated injections of waixenicin A over 3 weeks cause hypomagnesemia via insufficient Mg2+ absorption by the colon. However, neither waixenicin A, nor a diet low in Mg2+, affect aberrant crypt foci development in an azoxymethane-induced colorectal cancer mouse model. CONCLUSION: Early stage colon cancer proceeds independent of systemic Mg2+ status and TRPM7, and waixenicin A is a useful pharmacological tool to study of TRPM7 in vitro and in vivo.


Subject(s)
Adenocarcinoma/metabolism , Cell Proliferation/drug effects , Colonic Neoplasms/metabolism , Magnesium Deficiency/metabolism , TRPM Cation Channels/antagonists & inhibitors , Acetates/pharmacology , Adenocarcinoma/etiology , Animals , Azoxymethane/toxicity , Calcium/metabolism , Cells, Cultured , Colonic Neoplasms/etiology , Diterpenes/pharmacology , HT29 Cells , Humans , Intestinal Absorption , Magnesium Deficiency/blood , Magnesium Deficiency/etiology , Male , Mice , Mice, Inbred C57BL
6.
J Physiol ; 595(10): 3165-3180, 2017 05 15.
Article in English | MEDLINE | ID: mdl-28130783

ABSTRACT

KEY POINTS: Pharmacological and molecular inhibition of transient receptor potential melastatin 7 (TRPM7) reduces store-operated calcium entry (SOCE). Overexpression of TRPM7 in TRPM7-/- cells restores SOCE. TRPM7 is not a store-operated calcium channel. TRPM7 kinase rather than channel modulates SOCE. TRPM7 channel activity contributes to the maintenance of store Ca2+ levels at rest. ABSTRACT: The transient receptor potential melastatin 7 (TRPM7) is a protein that combines an ion channel with an intrinsic kinase domain, enabling it to modulate cellular functions either by conducting ions through the pore or by phosphorylating downstream proteins via its kinase domain. In the present study, we report store-operated calcium entry (SOCE) as a novel target of TRPM7 kinase activity. TRPM7-deficient chicken DT40 B lymphocytes exhibit a strongly impaired SOCE compared to wild-type cells as a result of reduced calcium release activated calcium currents, and independently of potassium channel regulation, membrane potential changes or changes in cell-cycle distribution. Pharmacological blockade of TRPM7 with NS8593 or waixenicin A in wild-type B lymphocytes results in a significant decrease in SOCE, confirming that TRPM7 activity is acutely linked to SOCE, without TRPM7 representing a store-operated channel itself. Using kinase-deficient mutants, we find that TRPM7 regulates SOCE through its kinase domain. Furthermore, Ca2+ influx through TRPM7 is essential for the maintenance of endoplasmic reticulum Ca2+ concentration in resting cells, and for the refilling of Ca2+ stores after a Ca2+ signalling event. We conclude that the channel kinase TRPM7 and SOCE are synergistic mechanisms regulating intracellular Ca2+ homeostasis.


Subject(s)
Calcium Channels/physiology , Calcium/physiology , Protein Serine-Threonine Kinases/physiology , TRPM Cation Channels/physiology , Animals , B-Lymphocytes/physiology , Cell Line, Tumor , Chickens , HEK293 Cells , Humans , Protein Serine-Threonine Kinases/genetics , Rats , Stromal Interaction Molecule 1/physiology , Stromal Interaction Molecule 2/physiology , TRPM Cation Channels/genetics
7.
Oncotarget ; 7(24): 36419-36435, 2016 Jun 14.
Article in English | MEDLINE | ID: mdl-27183905

ABSTRACT

Intracellular Ca2+ levels are important regulators of cell cycle and proliferation. We, and others, have previously reported the role of KCa3.1 (KCNN4) channels in regulating the membrane potential and the Ca2+ entry in association with cell proliferation. However, the relevance of KC3.1 channels in cancer prognosis as well as the molecular mechanism of Ca2+ entry triggered by their activation remain undetermined. Here, we show that RNAi-mediated knockdown of KCa3.1 and/or TRPC1 leads to a significant decrease in cell proliferation due to cell cycle arrest in the G1 phase. These results are consistent with the observed upregulation of both channels in synchronized cells at the end of G1 phase. Additionally, knockdown of TRPC1 suppressed the Ca2+ entry induced by 1-EBIO-mediated KCa3.1 activation, suggesting a functional cooperation between TRPC1 and KCa3.1 in the regulation of Ca2+ entry, possibly within lipid raft microdomains where these two channels seem to co-localize. We also show significant correlations between KCa3.1 mRNA expression and poor patient prognosis and unfavorable clinical breast cancer parameters by mining large datasets in the public domain. Together, these results highlight the importance of KCa3.1 in regulating the proliferative mechanisms in breast cancer cells as well as in providing a promising novel target in prognosis and therapy.


Subject(s)
Breast Neoplasms/genetics , Cell Proliferation/genetics , Intermediate-Conductance Calcium-Activated Potassium Channels/genetics , TRPC Cation Channels/genetics , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Calcium/metabolism , Female , G1 Phase Cell Cycle Checkpoints/genetics , Gene Expression Profiling/methods , Gene Expression Regulation, Neoplastic , Humans , Intermediate-Conductance Calcium-Activated Potassium Channels/metabolism , Kaplan-Meier Estimate , MCF-7 Cells , Prognosis , RNA Interference , TRPC Cation Channels/metabolism
8.
Br J Pharmacol ; 172(21): 5161-73, 2015 Nov.
Article in English | MEDLINE | ID: mdl-26276903

ABSTRACT

BACKGROUND AND PURPOSE: Kv 1.3 potassium channels are promising pharmaceutical targets for treating immune diseases as they modulate Ca(2+) signalling in T cells by regulating the membrane potential and with it the driving force for Ca(2+) influx. The antimycobacterial drug clofazimine has been demonstrated to attenuate antigen-induced Ca(2+) oscillations, suppress cytokine release and prevent skin graft rejection by inhibiting Kv 1.3 channels with high potency and selectivity. EXPERIMENTAL APPROACH: We used patch-clamp methodology to investigate clofazimine's mechanism of action in Kv 1.3 channels expressed in HEK293 cells. KEY RESULTS: Clofazimine blocked Kv 1.3 channels by involving two discrete mechanisms, both of which contribute to effective suppression of channels: (i) a use-dependent open-channel block during long depolarizations, resulting in accelerated K(+) current inactivation and (ii) a block of closed deactivated channels after channels were opened by brief depolarizations. Both modes of block were use-dependent and state-dependent in that they clearly required prior channel opening. The clofazimine-sensitive closed-deactivated state of the channel was distinct from the resting closed state because channels at hyperpolarized voltages were not inhibited by clofazimine. Neither were channels in the C-type inactivated state significantly affected. Kv 1.3 channels carrying the H399T mutation and lacking C-type inactivation were insensitive to clofazimine block of the closed-deactivated state, but retained their susceptibility to open-channel block. CONCLUSIONS AND IMPLICATIONS: Given the prominent role of Kv 1.3 in shaping Ca(2+) oscillations, the use-dependent and state-dependent block of Kv 1.3 channels by clofazimine offers therapeutic potential for selective immunosuppression in the context of autoimmune diseases in which Kv 1.3-expressing T cells play a significant role.


Subject(s)
Calcium Channel Blockers/pharmacology , Clofazimine/pharmacology , Kv1.3 Potassium Channel/antagonists & inhibitors , Leprostatic Agents/pharmacology , HEK293 Cells , Humans , Ion Channel Gating/drug effects , Kinetics , Kv1.3 Potassium Channel/genetics , Mutation , Patch-Clamp Techniques
9.
Oncotarget ; 5(17): 7625-34, 2014 Sep 15.
Article in English | MEDLINE | ID: mdl-25277194

ABSTRACT

Intracellular levels of the divalent cations Ca2+ and Mg2+ are important regulators of cell cycle and proliferation. However, the precise mechanisms by which they are regulated in cancer remain incompletely understood. The channel kinases TRPM6 and TRPM7 are gatekeepers of human Ca2+/Mg2+ metabolism. Here, we investigated the human neuroblastoma cell line SHEP-21N in which the MYCN oncogene (encoding N-Myc) can be reversibly expressed under control of an inducible repressor. We report that N-Myc expression increases cell growth and up-regulates both TRPM6 and TRPM7 expression. Membrane current analyses reveal that endogenous TRPM6/TRPM7 currents exhibit reduced Mg·ATP suppression, increased Mg2+ sensitivity, and diminished sensitivity to 2-APB inhibition. These properties are consistent with N-Myc-induced increase of heteromeric TRPM7/TRPM6 channels promoting Ca2+ and Mg2+ uptake. Genetic suppression of TRPM6/TRPM7 through siRNA inhibits cell proliferation, suggesting that N-Myc can promote neuroblastoma cell proliferation through up-regulation of divalent cation-transporting channels.


Subject(s)
Gene Expression Regulation, Neoplastic/genetics , Neuroblastoma/genetics , Protein Serine-Threonine Kinases/biosynthesis , Proto-Oncogene Proteins c-myc/metabolism , TRPM Cation Channels/biosynthesis , Cations, Divalent/metabolism , Cell Line, Tumor , Cell Proliferation , Humans , Neuroblastoma/metabolism , Patch-Clamp Techniques , RNA, Small Interfering , Real-Time Polymerase Chain Reaction , Transfection , Up-Regulation
10.
Handb Exp Pharmacol ; 222: 403-26, 2014.
Article in English | MEDLINE | ID: mdl-24756715

ABSTRACT

TRPM2 is the second member of the transient receptor potential melastatin-related (TRPM) family of cation channels. The protein is widely expressed including in the brain, immune system, endocrine cells, and endothelia. It embodies both ion channel functionality and enzymatic ADP-ribose (ADPr) hydrolase activity. TRPM2 is a Ca(2+)-permeable nonselective cation channel embedded in the plasma membrane and/or lysosomal compartments that is primarily activated in a synergistic fashion by intracellular ADP-ribose (ADPr) and Ca(2+). It is also activated by reactive oxygen and nitrogen species (ROS/NOS) and enhanced by additional factors, such as cyclic ADPr and NAADP, while inhibited by permeating protons (acidic pH) and adenosine monophosphate (AMP). Activation of TRPM2 leads to increases in intracellular Ca(2+) levels, which can serve signaling roles in inflammatory and secretory cells through release of vesicular mediators (e.g., cytokines, neurotransmitters, insulin) and in extreme cases can induce apoptotic and necrotic cell death under oxidative stress.


Subject(s)
TRPM Cation Channels/metabolism , Animals , Cell Membrane Permeability , Gene Expression Regulation , Genetic Predisposition to Disease , Humans , Ion Channel Gating , Membrane Potentials , Mice , Mice, Knockout , Phenotype , Protein Conformation , Signal Transduction , Structure-Activity Relationship , TRPM Cation Channels/chemistry , TRPM Cation Channels/genetics
11.
J Biol Chem ; 289(8): 5217-27, 2014 Feb 21.
Article in English | MEDLINE | ID: mdl-24385424

ABSTRACT

The transient receptor potential melastatin member 7 (TRPM7) and member 6 (TRPM6) are divalent cation channel kinases essential for magnesium (Mg(2+)) homeostasis in vertebrates. It remains unclear how TRPM6 affects divalent cation transport and whether this involves functional homomeric TRPM6 plasma membrane channels or heteromeric channel assemblies with TRPM7. We show that homomeric TRPM6 is highly sensitive to intracellular free Mg(2+) and therefore unlikely to be active at physiological levels of [Mg(2+)]i. Co-expression of TRPM7 and TRPM6 produces heteromeric TRPM7/M6 channels with altered pharmacology and sensitivity to intracellular Mg·ATP compared with homomeric TRPM7. Strikingly, the activity of heteromeric TRPM7/M6 channels is independent of intracellular Mg·ATP concentrations, essentially uncoupling channel activity from cellular energy status. Disruption of TRPM6 kinase phosphorylation activity re-introduces Mg·ATP sensitivity to the heteromeric channel similar to that of TRPM7. Thus, TRPM6 modulates the functionality of TRPM7, and the TRPM6 kinase plays a critical role in tuning the phenotype of the TRPM7·M6 channel complex.


Subject(s)
Adenosine Triphosphate/pharmacology , TRPM Cation Channels/chemistry , TRPM Cation Channels/metabolism , Boron Compounds/pharmacology , HEK293 Cells , Humans , Intracellular Space/drug effects , Intracellular Space/metabolism , Ion Channel Gating/drug effects , Osmolar Concentration , Phosphotransferases/metabolism , Point Mutation/genetics , Protein Serine-Threonine Kinases , Protein Structure, Tertiary , Solutions , Structure-Activity Relationship
12.
Biochim Biophys Acta ; 1833(3): 752-60, 2013 Mar.
Article in English | MEDLINE | ID: mdl-23266555

ABSTRACT

Members of the Orai family are highly selective calcium ion channels that play an important role in store-operated calcium entry. Among the three known Orai isoforms, Orai3 has gained increased attention, notably for its emerging role in cancer. We recently demonstrated that Orai3 channels are over-expressed in breast cancer (BC) biopsies, and involved specifically in proliferation, cell cycle progression and survival of MCF-7 BC cells. Here, we investigate the downstream signaling mechanisms affected by Orai3 silencing, leading to the subsequent functional impact specifically seen in MCF-7 cancer cells. We report a correlation between Orai3 and c-myc expression in tumor tissues and in the MCF-7 cancer cell line by demonstrating that Orai3 down-regulation reduces both expression and activity of the proto-oncogene c-myc. This is likely mediated through the MAP Kinase pathway, as we observed decreased pERK1/2 levels and cell-cycle arrest in G1 phase after Orai3 silencing. Our results provide strong evidence that the c-myc proto-oncogene is influenced by the store-operated calcium entry channel Orai3 through the MAP kinase pathway. This connection provides new clues in the downstream mechanism linking Orai3 channels and proliferation, cell cycle progression and survival of MCF-7 BC cells.


Subject(s)
Breast Neoplasms/pathology , Calcium Channels/metabolism , Cell Proliferation , G1 Phase/physiology , Proto-Oncogene Proteins c-myc/metabolism , Adenocarcinoma , Apoptosis , Blotting, Western , Breast/cytology , Breast/metabolism , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Calcium/metabolism , Calcium Channels/chemistry , Calcium Channels/genetics , Cells, Cultured , Female , Fluorescent Antibody Technique , Gene Expression Regulation, Neoplastic , Gene Silencing , Humans , Immunoenzyme Techniques , Mitogen-Activated Protein Kinases/genetics , Mitogen-Activated Protein Kinases/metabolism , Proto-Oncogene Mas , Proto-Oncogene Proteins c-myc/genetics , RNA, Messenger/genetics , RNA, Small Interfering/genetics , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction , Tissue Array Analysis
13.
Cell Physiol Biochem ; 28(5): 813-22, 2011.
Article in English | MEDLINE | ID: mdl-22178934

ABSTRACT

BACKGROUND: Transient Receptor Potential (TRP) channels are expressed in many solid tumors. However, their expression in breast cancer remains largely unknown. Here, we investigated the profile expression of 13 TRP channels in human breast ductal adenocarcinoma (hBDA) and performed a correlation between their overexpression and pathological parameters. METHODS: The TRP channels expression was determined by RT-PCR in hBDA tissue, in human breast cancer epithelial (hBCE) primary culture and in MCF-7 cell line. The TRP protein level was evaluated by immunohistochemistry in hBDA tissue samples of 59 patients. RESULTS: TRPC1, TRPC6, TRPM7, TRPM8, and TRPV6 channels were overexpressed in hBDA compared to the adjacent non-tumoral tissue. Most interestingly, TRPC1, TRPM7 and TRPM8 expression strongly correlated with proliferative parameters (SBR grade, Ki67 proliferation index, and tumor size), and TRPV6 was mainly overexpressed in the invasive breast cancer cells. Using laser capture microdissection, we found that TRPV6 expression was higher in invasive areas, compared to the corresponding non-invasive ones. Moreover, TRPV6 silencing inhibited MDA-MB-231 migration and invasion, and MCF-7 migration. CONCLUSION: TRP channels are aberrantly expressed in hBDA, hBCE primary cultures, and cell lines, and associated with pathological parameters. The high expression of TRP channels in tumors suggests the potential of these channels for diagnostic, prognosis and/or therapeutic approaches in human breast ductal adenocarcinoma.


Subject(s)
Breast Neoplasms/pathology , Gene Expression Regulation, Neoplastic , Transient Receptor Potential Channels/metabolism , Breast Neoplasms/metabolism , Cells, Cultured , Female , Humans , Immunohistochemistry , Neoplasm Staging , Protein Serine-Threonine Kinases , RNA Interference , RNA, Small Interfering/metabolism , TRPC Cation Channels/metabolism , TRPC6 Cation Channel , TRPM Cation Channels/metabolism , TRPV Cation Channels/antagonists & inhibitors , TRPV Cation Channels/genetics , TRPV Cation Channels/metabolism , Transient Receptor Potential Channels/antagonists & inhibitors , Transient Receptor Potential Channels/genetics
14.
J Cell Physiol ; 226(2): 542-51, 2011 Feb.
Article in English | MEDLINE | ID: mdl-20683915

ABSTRACT

Breast cancer (BC) is the leading cancer in the world in terms of incidence and mortality in women. However, the mechanism by which BC develops remains largely unknown. The increase in cytosolic free Ca(2+) can result in different physiological changes including cell growth and death. Orai isoforms are highly Ca(2+) selective channels. In the present study, we analyzed Orai3 expression in normal and cancerous breast tissue samples, and its role in MCF-7 BC and normal MCF-10A mammary epithelial cell lines. We found that the expression of Orai3 mRNAs was higher in BC tissues and MCF-7 cells than in normal tissues and MCF-10A cells. Down-regulation of Orai3 by siRNA inhibited MCF-7 cell proliferation and arrested cell cycle at G1 phase. This phenomenon is associated with a reduction in CDKs 4/2 (cyclin-dependent kinases) and cyclins E and D1 expression and an accumulation of p21(Waf1/Cip1) (a cyclin-dependent kinase inhibitor) and p53 (a tumor-suppressing protein). Orai3 was also involved in MCF-7 cell survival. Furthermore, Orai3 mediated Ca(2+) entry and contributed to intracellular calcium concentration ([Ca(2+)](i)). In MCF-10A cells, silencing Orai3 failed to modify [Ca(2+)](i), cell proliferation, cell-cycle progression, cyclins (D1, E), CDKs (4, 2), and p21(Waf1/Cip1) expression. Our results provide strong evidence for a significant effect of Orai3 on BC cell growth in vitro and show that this effect is associated with the induction of cell cycle and apoptosis resistance. Our study highlights a possible role of Orai3 as therapeutic target in BC therapy.


Subject(s)
Apoptosis/physiology , Breast Neoplasms , Breast/cytology , Calcium Channels/metabolism , Cell Cycle/physiology , Epithelial Cells/physiology , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Calcium Channels/genetics , Cell Proliferation , Cell Survival , Cells, Cultured , Cyclin-Dependent Kinases/genetics , Cyclin-Dependent Kinases/metabolism , Down-Regulation , Epithelial Cells/cytology , Female , Humans , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism
15.
Histol Histopathol ; 25(10): 1247-55, 2010 10.
Article in English | MEDLINE | ID: mdl-20712009

ABSTRACT

K+ channels are key molecules in the progression of several cancer types and are considered to be potential targets for cancer therapy. In this study, we investigated the intermediate- conductance Ca2+-activated K+ channels (hKCa3.1) expression in both breast carcinoma (BC) specimens and human breast cancer epithelial primary cell cultures (hBCE) using immuno-histochemistry (60 samples), quantitative Real-Time RT-PCR (30 samples) and Western blot assay (30 samples). We also looked at whether or not the expression of these channels is correlated with breast carcinomas grade tumours and metastasis status. Furthermore, we characterized the hKCa3.1 channel activity in hBCE cells by using the Whole Cell Patch Clamp Technique. We found that hKCa3.1 transcripts and proteins were expressed in both BC samples and hBCE cells. Clinicopathologic evaluation indicated a significant correlation between hKCa3.1-expression and tumour grade. hKCa3.1 mRNA and protein were more highly expressed in grade III tumours than in both grades I and II. However, the hKCa3.1 expression-increase according to grade was only observed in tumours with negative metastasis status. Moreover, the hKCa3.1 channels expressed in hBCE cells are functional. This was attested by patch-clamp recordings showing typical hKCa3.1-mediated currents in these cells. In conclusion, these data suggest that hKCa3.1 might contribute to breast tumour-progression and can serve as a useful prognostic marker for breast cancer.


Subject(s)
Adenocarcinoma/metabolism , Biomarkers, Tumor/metabolism , Breast Neoplasms/metabolism , Intermediate-Conductance Calcium-Activated Potassium Channels/metabolism , Adenocarcinoma/genetics , Adenocarcinoma/secondary , Biomarkers, Tumor/genetics , Blotting, Western , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Chi-Square Distribution , Female , Gene Expression Regulation, Neoplastic , Humans , Immunohistochemistry , Intermediate-Conductance Calcium-Activated Potassium Channels/genetics , Membrane Potentials , Neoplasm Staging , Patch-Clamp Techniques , Potassium/metabolism , Prognosis , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Tumor Cells, Cultured
16.
J Membr Biol ; 234(1): 47-56, 2010 Mar.
Article in English | MEDLINE | ID: mdl-20177667

ABSTRACT

Prolactin (PRL) is a polypeptidic hormone which acts both systemically and locally to cause lactation by interacting with the PRL receptor, a Janus kinase (JAK2)-coupled cytokine receptor family member. Several studies have reported that serum PRL level elevation is associated with an increased risk for breast cancer, and evidence has suggested that PRL is one actor in the pathogenesis and progression of this cancer. We previously reported the involvement of hIKCa1 in breast cell cycle progression and cell proliferation. However, mechanisms by which PRL cooperates with these channels to modulate breast epithelial cell proliferation remain unknown. Our results showed that, in the MCF-7 breast cancer cell line, PRL increased hIKCa1 current density. These channels were functional and regulated the resting membrane potential. The PRL effects were inhibited by TRAM-34 and clotrimazole, the most used hIKCa1 blockers. Moreover, PRL increased proliferation in a dose-dependent manner without overexpressing hIKCa1. To determine whether PRL-induced proliferation and hIKCa1 activity involved the JAK2 pathway, we used pharmacological JAK2 inhibitors (AG490 and JAK inhibitor I). Indeed, PRL-induced JAK2 phosphorylation was required for both cell proliferation and hIKCa1 activity. In the presence of either hIKCa1 blockers or siRNA-hIKCa1, PRL failed to increase cell proliferation and hIKCa1 activity. Taken together, our results demonstrate that PRL plays a role in breast cancer cell proliferation by increasing hIKCa1 activity through the JAK2 signaling pathway.


Subject(s)
Intermediate-Conductance Calcium-Activated Potassium Channels/physiology , Janus Kinase 2/physiology , Receptors, Prolactin/physiology , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Calcium/pharmacology , Cell Line, Tumor , Cell Proliferation/drug effects , Clotrimazole/pharmacology , Female , Humans , Intermediate-Conductance Calcium-Activated Potassium Channels/drug effects , Prolactin/pharmacology , Pyrazoles , Tyrphostins/pharmacology
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