Estrogen regulation of TRPM8 expression in breast cancer cells

<p>Abstract</p> <p>Background</p> <p>The calcium-permeable cation channel TRPM8 (melastatin-related transient receptor potential member 8) is over-expressed in several cancers. The present study aimed at investigating the expression, function and potential regulation of TRPM8 channels by ER alpha (estrogen receptor alpha) in breast cancer.</p> <p>Methods</p> <p>RT-PCR, Western blot, immuno-histochemical, and siRNA techniques were used to investigate TRPM8 expression, its regulation by estrogen receptors, and its expression in breast tissue. To investigate the channel activity in MCF-7 cells, we used the whole cell patch clamp and the calcium imaging techniques.</p> <p>Results</p> <p>TRPM8 channels are expressed at both mRNA and protein levels in the breast cancer cell line MCF-7. Bath application of the potent TRPM8 agonist Icilin (20 μM) induced a strong outwardly rectifying current at depolarizing potentials, which is associated with an elevation of cytosolic calcium concentration, consistent with established TRPM8 channel properties. RT-PCR experiments revealed a decrease in TRPM8 mRNA expression following steroid deprivation for 48 and 72 hours. In steroid deprived medium, addition of 17-beta-estradiol (E₂, 10 nM) increased both TRPM8 mRNA expression and the number of cells which respond to Icilin, but failed to affect the Ca²⁺entry amplitude. Moreover, silencing ERα mRNA expression with small interfering RNA reduced the expression of TRPM8. Immuno-histochemical examination of the expression of TRPM8 channels in human breast tissues revealed an over-expression of TRPM8 in breast adenocarcinomas, which is correlated with estrogen receptor positive (ER⁺) status of the tumours.</p> <p>Conclusion</p> <p>Taken together, these results show that TRPM8 channels are expressed and functional in breast cancer and that their expression is regulated by ER alpha.</p

Expression of TRPC6 channels in human epithelial breast cancer cells

<p>Abstract</p> <p>Background</p> <p>TRP channels have been shown to be involved in tumour generation and malignant growth. However, the expression of these channels in breast cancer remains unclear. Here we studied the expression and function of endogenous TRPC6 channels in a breast cancer cell line (MCF-7), a human breast cancer epithelial primary culture (hBCE) and in normal and tumour breast tissues.</p> <p>Methods</p> <p>Molecular (Western blot and RT-PCR), and immunohistochemical techniques were used to investigate TRPC6 expression. To investigate the channel activity in both MCF-7 cells and hBCE we used electrophysiological technique (whole cell patch clamp configuration).</p> <p>Results</p> <p>A non selective cationic current was activated by the oleoyl-2-acetyl-sn-glycerol (OAG) in both hBCE and MCF-7 cells. OAG-inward current was inhibited by 2-APB, SK&F 96365 and La³⁺. TRPC6, but not TRPC7, was expressed both in hBCE and in MCF-7 cells. TRPC3 was only expressed in hBCE. Clinically, TRPC6 mRNA and protein were elevated in breast carcinoma specimens in comparison to normal breast tissue. Furthermore, we found that the overexpression of TRPC6 protein levels were not correlated with tumour grades, estrogen receptor expression or lymph node positive tumours.</p> <p>Conclusion</p> <p>Our results indicate that TRPC6 channels are strongly expressed and functional in breast cancer epithelial cells. Moreover, the overexpression of these channels appears without any correlation with tumour grade, ER expression and lymph node metastasis. Our findings support the idea that TRPC6 may have a role in breast carcinogenesis.</p

Rôle de la lactoferrine dans la maturation des cellules T (induction d'un signal de transduction aboutissant à l'expression du CD4 dans les cellules lymphoblastiques T Jurkat)

La lactoferrine est une glycoproteine secretee dans les liquides de secretion, notamment dans le lait, et liberee dans le plasma par degranulation des neutrophiles. Les principales proprietes biologiques de la lactoferrine concernent les processus inflammatoire et immunitaire. La lactoferrine est, en effet, capable d'accelerer la maturation des cellules t en augmentant l'expression de l'antigene de surface cd4. Afin d'approfondir cet effet de la lactoferrine humaine sur les cellules t, nous avons utilise la lignee humaine lymphoblastique t jurkat. Nous avons ainsi montre que la lactoferrine augmente la densite de surface du cd4 en modulant l'expression du gene de ce marqueur : la synthese des arnm ainsi que l'activite du promoteur du gene du cd4 sont stimulees par la lactoferrine. Le mecanisme d'action aboutissant a cette regulation a ete elucide en etudiant le signal de transduction induit par fixation de la lactoferrine a son recepteur. Nous avons observe que la lactoferrine stimule la phosphorylation de nombreuses proteines cytosoliques, et qu'elle active une seule isoforme de la map kinase ( mitogen-activated protein kinase ). L'utilisation des inhibiteurs genisteine et pd98059 a ensuite permis de correler ces deux evenements a l'expression du cd4.Enfin, en utilisant des cellules jurkat deficientes en proteine lck, les cellules j.cam1.6, nous avons demontre que la kinase p56 l c k est necessaire a la regulation du cd4 par la lactoferrine. La derniere partie de nos travaux a consiste a comparer les effets des lactoferrines d'origine humaine et bovine sur certaines cellules du systeme immunitaire. Les deux proteines presentent les memes activites sur la cytotoxicite des cellules nk ( natural killer ) envers des lignees tumorales, et sur la regulation de la densite du cd4 a la surface des cellules jurkat. Par ailleurs, nos resultats indiquent que la proteine bovine regule l'expression du cd4 en stimulant egalement l'activite de la map kinase.LILLE1-BU (590092102) / SudocSudocFranceF

TRP channels: diagnostic markers and therapeutic targets for breast cancer?

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Tumor necrosis factor-α increases lactoferrin transcytosis through the blood-brain barrier

International audienceLactoferrin (Lf) is an iron-binding protein involved in host defense against infection and severe inflammation, which accumulates in the brain during neurodegenerative disorders. Prior to determining Lf function in pathological brain tissues, we investigated its transport through the blood–brain barrier (BBB) in inflammatory conditions. For this purpose, we used a reconstituted BBB model consisting of the coculture of bovine brain capillary endothelial cells (BBCECs) and astrocytes in the presence of tumor necrosis factor-α (TNF-α). As TNF-α can be either synthesized by brain glial cells or present in circulating blood, BBCECs were exposed to this cytokine at their luminal or abluminal side. We have been able to demonstrate that in the presence of TNF-α, whatever the type of exposure, BBCECs were activated and Lf transport through the activated BBCECs was markedly increased. Lf was recovered intact at the abluminal side of the cells, suggesting that increased Lf accumulation may occur in immune-mediated pathophysiology. This process was transient as 20 h later, cells were in a resting state and Lf transendothelial traffic was back to normal. The enhancement of Lf transcytosis seems not to involve the up-regulation of the Lf receptor but rather an increase in the rate of transendothelial transport

Evidence that TRPM7 is required for breast cancer cell proliferation

International audienceBecause transient receptor potential (TRP) channels have been implicated in tumor progression, we have investigated the potential role of TRPM7 channel in breast cancer cell proliferation. Under whole cell patch clamp, a Mg 2+ -inhibited cationic (MIC) current was observed in MCF-7 cells. This current was characterized by an inward current and a strong outward rectifying current that were both inhibited in a concentration-dependent manner by the presence of intracellular Mg 2+ or Mg 2+ -ATP. The inward current was reduced by La 3+ , and the outward current was sensitive to 2-aminoethoxydiphenyl borate (2-APB), spermine, La 3+ , and flufenamic acid. Importantly, a similar MIC current was also recorded in the primary culture of human breast cancerous epithelial cells (hBCE). Moreover, TRPM7 transcripts were found in both hBCE and MCF-7 cells. In MCF-7 cells, the MIC current was inhibited by TRPM7 small interfering RNA. Interestingly, we found that cell proliferation and intracellular Ca 2+ concentration were also reduced by TRPM7 silencing in MCF-7 cells. TRPM7 channels were also found in both human breast cancer and healthy tissues. Importantly, TRPM7 channel was overexpressed in grade III breast cancer samples associated with important Ki67 or tumor size. Our findings strongly suggest that TRPM7 is involved in the proliferative potentiality of breast cancer cells, probably by regulating Ca 2+ influx

The Tumor Suppressor hTid1 Inhibits STAT5b Activity via Functional Interaction

International audienceSTAT5a and -5b (signal transducers and activators of transcription 5a and 5b) proteins play an essential role in hematopoietic cell proliferation and survival and are frequently constitutively active in hematologic neoplasms and solid tumors. Because STAT5a and STAT5b differ mainly in the carboxyl-terminal transactivation domain, we sought to identify new proteins that bind specifically to this domain by using a bacterial two-hybrid screening. We isolated hTid1, a human DnaJ protein that acts as a tumor suppressor in various solid tumors. hTid1 interacts specifically with STAT5b but not with STAT5a in hematopoietic cell lines. This interaction involves the cysteine-rich region of the hTid1 DnaJ domain. We also demonstrated that hTid1 negatively regulates the expression and transcriptional activity of STAT5b and suppresses the growth of hematopoietic cells transformed by an oncogenic form of STAT5b. Our findings define hTid1 as a novel partner and negative regulator of STAT5b