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Introduction: Upon activation at low pH, TMEM206 conducts Cl- ions across plasma and vesicular membranes. In a (patho)physiological context, TMEM206 was reported to contribute to acid-induced cell death in neurons, kidney and cervical epithelial cells. We investigated the role of TMEM206 in acid-induced cell death in colorectal cancer cells. In addition, we studied CBA as a new small molecule inhibitor for TMEM206. Methods: The role of TMEM206 in acid-induced cell death was studied with CRISPR/Cas9-mediated knockout and FACS analysis. The pharmacology of TMEM206 was determined with the patch clamp technique. Results: In colorectal cancer cells, TMEM206 is not a critical mediator of acid-induced cell death. CBA is a small molecule inhibitor of TMEM206 (IC50 = 9.55 µM) at low pH, at pH 6.0 inhibition is limited. Conclusion: CBA demonstrates effective and specific inhibition of TMEM206; however, its inhibitory efficacy is limited at pH 6.0. Despite this limitation, CBA is a potent inhibitor for functional studies at pH 4.5 and may be a promising scaffold for the development of future TMEM206 inhibitors.
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Inflammatory bowel disease (IBD) is a collective term for inflammatory diseases of the human gastrointestinal (GI) tract that are characterized by perturbations in the intestinal immune responses. In their study, Letizia et al (2022) found an enrichment of CD4+ effector T cells, interferon gamma (IFNγ) producing CD8+ T cells, regulatory T cells, and innate lymphoid cells (ILC) in the lamina propria (LP) of IBD patients. In these cells, pharmacological inhibition of store-operated calcium entry (SOCE) reduced cytokine production. In addition, in a murine IBD model, systemic SOCE inhibition reduced IBD severity and weight loss.
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Canales de Calcio Activados por la Liberación de Calcio , Enfermedades Inflamatorias del Intestino , Animales , Linfocitos T CD8-positivos , Humanos , Inmunidad Innata , Mucosa Intestinal , Linfocitos , RatonesRESUMEN
Under physiological conditions, the widely expressed calcium-activated TRPM4 channel conducts sodium into cells. This sodium influx depolarizes the plasma membrane and reduces the driving force for calcium entry. The aberrant expression or function of TRPM4 has been reported in various diseases, including different types of cancer. TRPM4 is mainly localized in the plasma membrane, but it is also found in intracellular vesicles, which can undergo exocytosis. In this study, we show that calcium-induced exocytosis in the colorectal cancer cell line HCT116 is dependent on TRPM4. In addition, the findings from some studies of prostate cancer cell lines suggest a more general role of TRPM4 in calcium-induced exocytosis in cancer cells. Furthermore, calcium-induced exocytosis depends on TRPM4 ion conductivity. Additionally, an increase in intracellular calcium results in the delivery of TRPM4 to the plasma membrane. This process also depends on TRPM4 ion conductivity. TRPM4-dependent exocytosis and the delivery of TRPM4 to the plasma membrane are mediated by SNARE proteins. Finally, we provide evidence that calcium-induced exocytosis depends on TRPM4 ion conductivity, not within the plasma membrane, but rather in TRPM4-containing vesicles.
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Exocitosis , Canales Catiónicos TRPM , Calcio/metabolismo , Línea Celular Tumoral , Humanos , Sodio/metabolismo , Canales Catiónicos TRPM/metabolismoRESUMEN
Altered expression of transient receptor potential channel melastatin 4 (TRPM4) contributes to several diseases, including cardiac conduction disorders, immune diseases, and cancer. Yet the underlying mechanisms of TRPM4 expression changes remain elusive. In this study, we report that loss of tumor suppressor protein p53 or p63γ function or mutation of a putative p53 response element in the TRPM4 promoter region increase TRPM4 promoter activity in the colorectal cancer cell line HCT 116. In cells that lack p53 expression, we observed increased TRPM4 mRNA and protein levels and TRPM4-mediated Na+ currents. This phenotype can be reversed by transient overexpression of p53. In the prostate cancer cell line LNCaP, which expresses p53 endogenously, p53 overexpression decreases TRPM4-mediated currents. As in other cancer cells, CRISPR-Cas9 mediated knockout of TRPM4 in p53 deficient HCT 116 cells results in increased store-operated Ca2+entry. The effect of the TRPM4 knockout is mimicked by p53 mediated suppression of TRPM4 in the parental cell line expressing TRPM4. In addition, a TRPM4 knockout-mediated shift in cell cycle is abolished upon loss of p53. Taken together, these findings indicate that p53 represses TRPM4 expression, thereby altering cellular Ca2+ signaling and that TRPM4 adds to cell cycle shift dependent on p53 signaling. One sentence summary: TRPM4 is repressed in the p53 pathway leading to reduced currents and increased calcium signaling.
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Neoplasias de la Próstata , Canales Catiónicos TRPM , Calcio/metabolismo , Señalización del Calcio , Ciclo Celular , Humanos , Masculino , Neoplasias de la Próstata/metabolismo , Canales Catiónicos TRPM/genética , Canales Catiónicos TRPM/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Proteína p53 Supresora de Tumor/farmacologíaRESUMEN
Transient receptor potential melastatin 4 (TRPM4) is a broadly expressed Ca2+ activated monovalent cation channel that contributes to the pathophysiology of several diseases. For this study, we generated stable CRISPR/Cas9 TRPM4 knockout (K.O.) cells from the human prostate cancer cell line DU145 and analyzed the cells for changes in cancer hallmark functions. Both TRPM4-K.O. clones demonstrated lower proliferation and viability compared to the parental cells. Migration was also impaired in the TRPM4-K.O. cells. Additionally, analysis of 210 prostate cancer patient tissues demonstrates a positive association between TRPM4 protein expression and local/metastatic progression. Moreover, a decreased adhesion rate was detected in the two K.O. clones compared to DU145 cells. Next, we tested three novel TRPM4 inhibitors with whole-cell patch clamp technique for their potential to block TRPM4 currents. CBA, NBA and LBA partially inhibited TRPM4 currents in DU145 cells. However, none of these inhibitors demonstrated any TRPM4-specific effect in the cellular assays. To evaluate if the observed effect of TRPM4 K.O. on migration, viability, and cell cycle is linked to TRPM4 ion conductivity, we transfected TRPM4-K.O. cells with either TRPM4 wild-type or a dominant-negative mutant, non-permeable to Na+. Our data showed a partial rescue of the viability of cells expressing functional TRPM4, while the pore mutant was not able to rescue this phenotype. For cell cycle distribution, TRPM4 ion conductivity was not essential since TRPM4 wild-type and the pore mutant rescued the phenotype. In conclusion, TRPM4 contributes to viability, migration, cell cycle shift, and adhesion; however, blocking TRPM4 ion conductivity is insufficient to prevent its role in cancer hallmark functions in prostate cancer cells.
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Neoplasias de la Próstata/tratamiento farmacológico , Bibliotecas de Moléculas Pequeñas/farmacología , Canales Catiónicos TRPM/antagonistas & inhibidores , Calcio/metabolismo , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Humanos , Masculino , Técnicas de Placa-Clamp/métodos , Neoplasias de la Próstata/metabolismoRESUMEN
Store-operated heteromeric Orai1/Orai3 channels have been discussed in the context of aging, cancer, and immune cell differentiation. In contrast to homomeric Orai1 channels, they exhibit a different pharmacology upon application of reactive oxygen species (ROS) or 2-aminoethoxydiphenyl borate (2-APB) in various cell types. In endogenous cells, subunit composition and arrangement may vary and cannot be defined precisely. In this study, we used patch-clamp electrophysiology to investigate the 2-APB profile of store-operated and store-independent homomeric Orai1 and heteromeric Orai1/Orai3 concatenated channels with defined subunit compositions. As has been shown previous, one or more Orai3 subunit(s) within the channel result(s) in decreased Ca2+ release activated Ca2+ current (ICRAC). Upon application of 50 µM 2-APB, channels with two or more Orai3 subunits exhibit large outward currents and can be activated by 2-APB independent from storedepletion and/or the presence of STIM1. The number and position of Orai3 subunits within the heteromeric store-operated channel change ion conductivity of 2-APB-activated outward current. Compared to homomeric Orai1 channels, one Orai3 subunit within the channel does not alter 2-APB pharmacology. None of the concatenated channel constructs were able to exactly simulate the complex 2-APB pharmacology observed in prostate cancer cells. However, 2-APB profiles of prostate cancer cells are similar to those of concatenated channels with Orai3 subunit(s). Considering the presented and previous results, this indicates that distinct subtypes of heteromeric SOCE channels may be selectively activated or blocked. In the future, targeting distinct heteromeric SOCE channel subtypes may be the key to tailored SOCE-based therapies.
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Canales de Calcio/metabolismo , Activación del Canal Iónico , Multimerización de Proteína , Canales de Calcio/química , Línea Celular Tumoral , Humanos , Activación del Canal Iónico/efectos de los fármacos , Masculino , Potenciales de la Membrana , Proteína ORAI1/química , Proteína ORAI1/metabolismo , Unión Proteica , Multimerización de Proteína/efectos de los fármacosRESUMEN
Cancers of the digestive tract are among the most prevalent types of cancer. These types of cancers are often diagnosed at a late stage, which results in a poor prognosis. Currently, many biomedical studies focus on the role of ion channels, in particular transient receptor potential (TRP) channels, in cancer pathophysiology. TRP channels show mostly non-selective permeability to monovalent and divalent cations. TRP channels are often dysregulated in digestive tract cancers, which can result in alterations of cancer hallmark functions, such as enhanced proliferation, migration, invasion and the inability to induce apoptosis. Therefore, TRP channels could serve as potential diagnostic biomarkers. Moreover, TRP channels are mostly expressed on the cell surface and ion channel targeting drugs do not need to enter the cell, making them attractive candidate drug targets. In this review, we summarize the current knowledge about TRP channels in connection to digestive tract cancers (oral cancer, esophageal cancer, liver cancer, pancreatic cancer, gastric cancer and colorectal cancer) and give an outlook on the potential of TRP channels as cancer biomarkers or therapeutic targets.
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Neoplasias Gastrointestinales/metabolismo , Tracto Gastrointestinal/metabolismo , Canales de Potencial de Receptor Transitorio/metabolismo , Animales , HumanosRESUMEN
Transient receptor potential melastatin-4 channel (TRPM4) dysregulation contributes to heart conditions, immune diseases, and cervical and prostate cancer. Up to now, the involvement of TRPM4 in colorectal cancer (CRC) pathophysiology remains unknown. Here, we investigated tumor tissue microarrays from 379 CRC patients and analyzed TRPM4 protein expression, tumor characteristics, and clinical outcome. High TRPM4 protein expression was associated with unfavorable tumor features characteristic for epithelial-mesenchymal transition and infiltrative growth patterns, that is, a high number of tumor buds and a low percentage in tumor border configuration. Compared to CRC cells representing early cancer stages, TRPM4 protein expression was the highest in cells representing late-stage metastatic cancer. Investigation of CRC cell line HCT116 and five CRISPR/cas9 TRPM4 knockout clones demonstrated that TRPM4 exhibited large Na+ current densities (~ 60 pA/pF). In addition, CRISPR/cas9 TRPM4 knockout clones showed a tendency toward decreased migration and invasion, cell viability, and proliferation and exhibited a shift in cell cycle when compared to HCT116. Stable overexpression of TRPM4 (TRPM4 wild-type) in two CRISPR/cas9 TRPM4 knockout clones rescued the decrease in cell viability and cell cycle shift. Stable overexpression of a nonconducting, dominant-negative TRPM4 mutant (TRPM4 D894A) did not rescue the decrease in viability or cell cycle shift. Taken together, these findings pointed to TRPM4 ion channel conductivity as the underlying mechanism for decreased viability and cell cycle shift in the TRPM4 knockout clones. Together with previous findings, our present data suggest that TRPM4 plays a versatile role in cancer cell proliferation, cell cycle, and invasion.
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Ciclo Celular , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Canales Catiónicos TRPM/metabolismo , Línea Celular Tumoral , Proliferación Celular , Humanos , Activación del Canal Iónico , Invasividad NeoplásicaRESUMEN
Precise intracellular calcium signaling is crucial to numerous cellular functions. In non-excitable cells, store-operated calcium entry (SOCE) is a key step in the generation of intracellular calcium signals. Tight regulation of SOCE is important, and dysregulation is involved in several pathophysiological cellular malfunctions. The current underlying SOCE, calcium release-activated calcium current (ICRAC), was first discovered almost three decades ago. Since its discovery, the molecular components of ICRAC, Orai1 and stromal interaction molecule 1 (STIM1), have been extensively investigated. Several regulatory mechanisms and proteins contribute to alterations in SOCE and cellular malfunctions in cancer, immune and neurodegenerative diseases, inflammation, and neuronal disorders. This review summarizes these regulatory mechanisms, including glycosylation, pH sensing, and the regulatory proteins golli, α-SNAP, SARAF, ORMDL3, CRACR2A, and TRPM4 channels.
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Calcio/metabolismo , Inflamación/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Enfermedades Neurodegenerativas/metabolismo , Proteína ORAI1/metabolismo , Molécula de Interacción Estromal 1/metabolismo , Animales , Señalización del Calcio , Humanos , Inflamación/patología , Neoplasias/patología , Enfermedades Neurodegenerativas/patologíaRESUMEN
BACKGROUND AND PURPOSE: TRPM4 is a calcium-activated non-selective cation channel expressed in many tissues and implicated in several diseases, and has not yet been validated as a therapeutic target due to the lack of potent and selective inhibitors. We sought to discover a novel series of small-molecule inhibitors by combining in silico methods and cell-based screening assay, with sub-micromolar potency and improved selectivity from previously reported TRPM4 inhibitors. EXPERIMENTAL APPROACH: Here, we developed a high throughput screening compatible assay to record TRPM4-mediated Na+ influx in cells using a Na+ -sensitive dye and used this assay to screen a small set of compounds selected by ligand-based virtual screening using previously known weakly active and non-selective TRPM4 inhibitors as seed molecules. Conventional electrophysiological methods were used to validate the potency and selectivity of the hit compounds in HEK293 cells overexpressing TRPM4 and in endogenously expressing prostate cancer cell line LNCaP. Chemical chaperone property of compound 5 was studied using Western blots and electrophysiology experiments. KEY RESULTS: A series of halogenated anthranilic amides were identified with TRPM4 inhibitory properties with sub-micromolar potency and adequate selectivity. We also showed for the first time that a naturally occurring variant of TRPM4, which displays loss-of-expression and function, is rescued by the most promising compound 5 identified in this study. CONCLUSIONS AND IMPLICATIONS: The discovery of compound 5, a potent and selective inhibitor of TRPM4 with an additional chemical chaperone feature, revealed new opportunities for studying the role of TRPM4 in human diseases and developing clinical drug candidates.
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Amidas/farmacología , Bibliotecas de Moléculas Pequeñas/farmacología , Canales Catiónicos TRPM/antagonistas & inhibidores , Amidas/química , Animales , Relación Dosis-Respuesta a Droga , Descubrimiento de Drogas , Células HEK293 , Células HeLa , Ensayos Analíticos de Alto Rendimiento , Humanos , Ligandos , Ratones , Células RAW 264.7 , Bibliotecas de Moléculas Pequeñas/química , Relación Estructura-Actividad , Canales Catiónicos TRPM/metabolismoRESUMEN
[This corrects the article DOI: 10.1007/s40610-017-0072-8.].
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PURPOSE OF REVIEW: Store-operated calcium entry (SOCE) is dysregulated in prostate cancer, contributing to increased cellular migration and proliferation and preventing cancer cell apoptosis. We here summarize findings on gene expression levels and functions of SOCE components, stromal interaction molecules (STIM1 and STIM2), and members of the Orai protein family (Orai1, 2, and 3) in prostate cancer. Moreover, we introduce new research models that promise to provide insights into whether dysregulated SOCE signaling has clinically relevant implications in terms of increasing the migration and invasion of prostate cancer cells. RECENT FINDINGS: Recent reports on Orai1 and Orai3 expression levels and function were in part controversial probably due to the heterogeneous nature of prostate cancer. Lately, in prostate cancer cells, transient receptor melastatin 4 channel was shown to alter SOCE and play a role in migration and proliferation. We specifically highlight new cancer research models: a subpopulation of cells that show tumor initiation and metastatic potential in mice and zebrafish models. SUMMARY: This review focuses on SOCE component dysregulation in prostate cancer and analyzes several preclinical, cellular, and animal cancer research models.
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N-glycosylation of cell surface proteins affects protein function, stability, and interaction with other proteins. Orai channels, which mediate store-operated Ca(2+) entry (SOCE), are composed of N-glycosylated subunits. Upon activation by Ca(2+) sensor proteins (stromal interaction molecules STIM1 or STIM2) in the endoplasmic reticulum, Orai Ca(2+) channels in the plasma membrane mediate Ca(2+) influx. Lectins are carbohydrate-binding proteins, and Siglecs are a family of sialic acid-binding lectins with immunoglobulin-like repeats. Using Western blot analysis and lectin-binding assays from various primary human cells and cancer cell lines, we found that glycosylation of Orai1 is cell type-specific. Ca(2+) imaging experiments and patch-clamp experiments revealed that mutation of the only glycosylation site of Orai1 (Orai1N223A) enhanced SOCE in Jurkat T cells. Knockdown of the sialyltransferase ST6GAL1 reduced α-2,6-linked sialic acids in the glycan structure of Orai1 and was associated with increased Ca(2+) entry in Jurkat T cells. In human mast cells, inhibition of sialyl sulfation altered the N-glycan of Orai1 (and other proteins) and increased SOCE. These data suggest that cell type-specific glycosylation influences the interaction of Orai1 with specific lectins, such as Siglecs, which then attenuates SOCE. In summary, the glycosylation state of Orai1 influences SOCE-mediated Ca(2+) signaling and, thus, may contribute to pathophysiological Ca(2+) signaling observed in immune disease and cancer.
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Señalización del Calcio/fisiología , Calcio/metabolismo , Proteína ORAI1/metabolismo , Antígenos CD/genética , Antígenos CD/metabolismo , Retículo Endoplásmico/genética , Retículo Endoplásmico/metabolismo , Técnicas de Silenciamiento del Gen , Glicosilación , Humanos , Células Jurkat , Proteína ORAI1/genética , Sialiltransferasas/genética , Sialiltransferasas/metabolismoRESUMEN
Impaired Ca2+ signaling in prostate cancer contributes to several cancer hallmarks, such as enhanced proliferation and migration and a decreased ability to induce apoptosis. Na+ influx via transient receptor potential melastatin 4 channel (TRPM4) can reduce store-operated Ca2+ entry (SOCE) by decreasing the driving force for Ca2+. In patients with prostate cancer, gene expression of TRPM4 is elevated. Recently, TRPM4 was identified as a cancer driver gene in androgen-insensitive prostate cancer.We investigated TRPM4 protein expression in cancer tissue samples from 20 patients with prostate cancer. We found elevated TRPM4 protein levels in prostatic intraepithelial neoplasia (PIN) and prostate cancer tissue compared to healthy tissue. In primary human prostate epithelial cells (hPEC) from healthy tissue and in the androgen-insensitive prostate cancer cell lines DU145 and PC3, TRPM4 mediated large Na+ currents. We demonstrated significantly increased SOCE after siRNA targeting of TRPM4 in hPEC and DU145 cells. In addition, knockdown of TRPM4 reduced migration but not proliferation of DU145 and PC3 cells. Taken together, our data identify TRPM4 as a regulator of SOCE in hPEC and DU145 cells, demonstrate a role for TRPM4 in cancer cell migration and suggest that TRPM4 is a promising potential therapeutic target.
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Movimiento Celular , Neoplasia Intraepitelial Prostática/metabolismo , Neoplasias de la Próstata Resistentes a la Castración/metabolismo , Neoplasias de la Próstata/metabolismo , Canales Catiónicos TRPM/metabolismo , Señalización del Calcio , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Potenciales de la Membrana , Invasividad Neoplásica , Neoplasia Intraepitelial Prostática/genética , Neoplasia Intraepitelial Prostática/patología , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Neoplasias de la Próstata Resistentes a la Castración/genética , Neoplasias de la Próstata Resistentes a la Castración/patología , Interferencia de ARN , Sodio/metabolismo , Canales Catiónicos TRPM/genética , Factores de Tiempo , Transfección , Regulación hacia ArribaRESUMEN
In prostate cancer, reactive oxygen species (ROS) are elevated and Ca(2+) signaling is impaired. Thus, several novel therapeutic strategies have been developed to target altered ROS and Ca(2+) signaling pathways in prostate cancer. Here, we investigate alterations of intracellular Ca(2+) and inhibition of cell viability caused by ROS in primary human prostate epithelial cells (hPECs) from healthy tissue and prostate cancer cell lines (LNCaP, DU145, and PC3). In hPECs, LNCaP and DU145 H2O2 induces an initial Ca(2+) increase, which in prostate cancer cells is blocked at high concentrations of H2O2. Upon depletion of intracellular Ca(2+) stores, store-operated Ca(2+) entry (SOCE) is activated. SOCE channels can be formed by hexameric Orai1 channels; however, Orai1 can form heteromultimers with its homolog, Orai3. Since the redox sensor of Orai1 (Cys-195) is absent in Orai3, the Orai1/Orai3 ratio in T cells determines the redox sensitivity of SOCE and cell viability. In prostate cancer cells, SOCE is blocked at lower concentrations of H2O2 compared with hPECs. An analysis of data from hPECs, LNCaP, DU145, and PC3, as well as previously published data from naive and effector TH cells, demonstrates a strong correlation between the Orai1/Orai3 ratio and the SOCE redox sensitivity and cell viability. Therefore, our data support the concept that store-operated Ca(2+) channels in hPECs and prostate cancer cells are heteromeric Orai1/Orai3 channels with an increased Orai1/Orai3 ratio in cells derived from prostate cancer tumors. In addition, ROS-induced alterations in Ca(2+) signaling in prostate cancer cells may contribute to the higher sensitivity of these cells to ROS.
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Señalización del Calcio/fisiología , Células Epiteliales/fisiología , Peróxido de Hidrógeno/metabolismo , Próstata/fisiología , Neoplasias de la Próstata/fisiopatología , Calcio/metabolismo , Canales de Calcio/genética , Canales de Calcio/metabolismo , Línea Celular Tumoral , Supervivencia Celular/fisiología , Técnicas de Silenciamiento del Gen , Humanos , Peróxido de Hidrógeno/toxicidad , Espacio Intracelular/metabolismo , Masculino , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteína ORAI1 , Oxidación-Reducción , Técnicas de Placa-Clamp , Interferencia de ARN , Especies Reactivas de Oxígeno/metabolismo , Molécula de Interacción Estromal 1RESUMEN
Labelled 5α-dihydrotestosterone (DHT) binding experiments have shown that expression levels of (yet unidentified) membrane androgen receptors (mAR) are elevated in prostate cancer and correlate with a negative prognosis. However, activation of these receptors which mediate a rapid androgen response can counteract several cancer hallmark functions such as unlimited proliferation, enhanced migration, adhesion and invasion and the inability to induce apoptosis. Here, we investigate the downstream signaling pathways of mAR and identify rapid DHT induced activation of store-operated Ca2+ entry (SOCE) in primary cultures of human prostate epithelial cells (hPEC) from non-tumorous tissue. Consequently, down-regulation of Orai1, the main molecular component of Ca2+ release-activated Ca2+ (CRAC) channels results in an almost complete loss of DHT induced SOCE. We demonstrate that this DHT induced Ca2+ influx via Orai1 is important for rapid androgen triggered prostate specific antigen (PSA) release. We furthermore identified alterations of the molecular components of CRAC channels in prostate cancer. Three lines of evidence indicate that prostate cancer cells down-regulate expression of the Orai1 homolog Orai3: First, Orai3 mRNA expression levels are significantly reduced in tumorous tissue when compared to non-tumorous tissue from prostate cancer patients. Second, mRNA expression levels of Orai3 are decreased in prostate cancer cell lines LNCaP and DU145 when compared to hPEC from healthy tissue. Third, the pharmacological profile of CRAC channels in prostate cancer cell lines and hPEC differ and siRNA based knock-down experiments indicate changed Orai3 levels are underlying the altered pharmacological profile. The cancer-specific composition and pharmacology of CRAC channels identifies CRAC channels as putative targets in prostate cancer therapy.
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Canales de Calcio/metabolismo , Señalización del Calcio/genética , Próstata/metabolismo , Neoplasias de la Próstata/metabolismo , Receptores Androgénicos/metabolismo , Calcio/metabolismo , Línea Celular , Línea Celular Tumoral , Dihidrotestosterona/farmacología , Células Epiteliales/metabolismo , Humanos , Masculino , Próstata/citología , Neoplasias de la Próstata/genética , Receptores Androgénicos/genética , Transducción de SeñalRESUMEN
High attrition rates of novel anti-cancer drugs highlight the need for improved models to predict toxicity. Although polo-like kinase 1 (Plk1) inhibitors are attractive candidates for drug development, the role of Plk1 in primary cells remains widely unexplored. Therefore, we evaluated the utility of an RNA interference-based model to assess responses to an inducible knockdown (iKD) of Plk1 in adult mice. Here we show that Plk1 silencing can be achieved in several organs, although adverse events are rare. We compared responses in Plk1-iKD mice with those in primary cells kept under controlled culture conditions. In contrast to the addiction of many cancer cell lines to the non-oncogene Plk1, the primary cells' proliferation, spindle assembly and apoptosis exhibit only a low dependency on Plk1. Responses to Plk1-depletion, both in cultured primary cells and in our iKD-mouse model, correspond well and thus provide the basis for using validated iKD mice in predicting responses to therapeutic interventions.
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Antineoplásicos/toxicidad , Proteínas de Ciclo Celular/antagonistas & inhibidores , Neoplasias/tratamiento farmacológico , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Interferencia de ARN/efectos de los fármacos , Pruebas de Toxicidad/métodos , Animales , Apoptosis/genética , Northern Blotting , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Células Cultivadas , Cartilla de ADN/genética , Evaluación Preclínica de Medicamentos , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Dosificación de Gen/genética , Técnicas de Silenciamiento del Gen , Ingeniería Genética/métodos , Humanos , Ratones , Ratones Transgénicos , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transfección , Quinasa Tipo Polo 1RESUMEN
Cell volume is an important parameter in many physiological processes, and is closely regulated in many cell types. In those cells, swelling induced by hypotonic media is followed by an ion-driven regulatory volume decrease. In many cell types, this regulatory volume decrease requires an intact actin cytoskeleton. Therefore, we investigated the changes in the structure and polymerization state of the actin cytoskeleton in HaCaT keratinocytes during cell swelling and regulatory volume decrease. Disruption of the actin cytoskeleton by 2microM cytochalasin D inhibits regulatory volume decrease in HaCaT cells. Cells swollen in the presence of low concentrations of cytochalasin D (0.8microM, 305-250mosM) keep the elevated volume even after cytochalasin D removal. A further decrease of tonicity (250-200mosM) is again counteracted by regulatory volume decrease reaching the volume, which has been established at 250mosM. In contrast, no visible changes occurred in actin cytoskeleton morphology of EGFP-actin-transfected HaCaT cells during swelling or regulatory volume decrease. However, biochemical analysis showed an increase in total F-actin levels 90s after the onset of hypotonicity. The ratio of Triton-soluble to -insoluble actin also increased after hypotonic shock, suggesting that the measured increase in F-actin is primarily due to de novo polymerization and formation of short actin filaments, i.e., actin oligomers. These results show that a rapid reorganization of the actin cytoskeleton takes place after hypotonic treatment. This reorganization can influence signaling in response to hypotonicity either indirectly by means of sequestering or releasing actin-associated proteins, or directly by the interaction of short actin filaments with plasma membrane ion channels, and may be involved in determining a new volume set point.
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Citoesqueleto de Actina/metabolismo , Tamaño de la Célula , Queratinocitos/citología , Citoesqueleto de Actina/efectos de los fármacos , Actinas/metabolismo , Línea Celular , Tamaño de la Célula/efectos de los fármacos , Citocalasina D/farmacología , Detergentes/farmacología , Humanos , Soluciones Hipotónicas/farmacología , Queratinocitos/efectos de los fármacos , Solubilidad/efectos de los fármacosRESUMEN
Conditional gene silencing in mammalian cells, via the controlled expression of short hairpin RNAs (shRNAs), is an effective method for studying gene function, particularly if the gene is essential for cell survival or development. Here we describe a simple and rapid protocol for the generation of tetracycline (Tet)-inducible vectors that express shRNAs in a time- and dosage-dependent manner. Tet-operator (TetO) sequences responsive to occupation by the Tet-repressor (TetR) were inserted at alternative positions within the wild-type H1 promoter and cloned into a eukaryotic expression vector. Additional cloning sites downstream of the promoter enable the insertion of shRNA sequences. This Tet-inducible shRNA expression system can be used for both transient and stable RNA interference (RNAi) approaches to control gene function in a spatiotemporal fashion. The entire protocol (preparation of constructs, generation of stable cell lines and functional analysis) can be completed in 3 months.