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1.
Cells ; 13(9)2024 Apr 23.
Article in English | MEDLINE | ID: mdl-38727266

ABSTRACT

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is a deadly disease with high mortality due to early metastatic dissemination and high chemoresistance. All these factors are favored by its extracellular matrix (ECM)-rich microenvironment, which is also highly hypoxic and acidic. Gemcitabine (GEM) is still the first-line therapy in PDAC. However, it is quickly deaminated to its inactive metabolite. Several GEM prodrugs have emerged to improve its cytotoxicity. Here, we analyzed how the acidic/hypoxic tumor microenvironment (TME) affects the response of PDAC cell death and invadopodia-mediated ECM proteolysis to both GEM and its C18 prodrug. METHODS: For this, two PDAC cell lines, PANC-1 and Mia PaCa-2 were adapted to pHe 6.6 or not for 1 month, grown as 3D organotypic cultures and exposed to either GEM or C18 in the presence and absence of acidosis and the hypoxia inducer, deferoxamine. RESULTS: We found that C18 has higher cytotoxic and anti-invadopodia activity than GEM in all culture conditions and especially in acid and hypoxic environments. CONCLUSIONS: We propose C18 as a more effective approach to conventional GEM in developing new therapeutic strategies overcoming PDAC chemoresistance.


Subject(s)
Deoxycytidine , Gemcitabine , Pancreatic Neoplasms , Tumor Microenvironment , Deoxycytidine/analogs & derivatives , Deoxycytidine/pharmacology , Deoxycytidine/therapeutic use , Humans , Tumor Microenvironment/drug effects , Pancreatic Neoplasms/drug therapy , Pancreatic Neoplasms/pathology , Pancreatic Neoplasms/metabolism , Cell Line, Tumor , Carcinoma, Pancreatic Ductal/drug therapy , Carcinoma, Pancreatic Ductal/pathology , Carcinoma, Pancreatic Ductal/metabolism , Podosomes/metabolism , Podosomes/drug effects , Drug Resistance, Neoplasm/drug effects , Prodrugs/pharmacology
2.
EMBO J ; 42(13): e112198, 2023 07 03.
Article in English | MEDLINE | ID: mdl-37278161

ABSTRACT

There is growing evidence that ion channels are critically involved in cancer cell invasiveness and metastasis. However, the molecular mechanisms of ion signaling promoting cancer behavior are poorly understood and the complexity of the underlying remodeling during metastasis remains to be explored. Here, using a variety of in vitro and in vivo techniques, we show that metastatic prostate cancer cells acquire a specific Na+ /Ca2+ signature required for persistent invasion. We identify the Na+ leak channel, NALCN, which is overexpressed in metastatic prostate cancer, as a major initiator and regulator of Ca2+ oscillations required for invadopodia formation. Indeed, NALCN-mediated Na+ influx into cancer cells maintains intracellular Ca2+ oscillations via a specific chain of ion transport proteins including plasmalemmal and mitochondrial Na+ /Ca2+ exchangers, SERCA and store-operated channels. This signaling cascade promotes activity of the NACLN-colocalized proto-oncogene Src kinase, actin remodeling and secretion of proteolytic enzymes, thus increasing cancer cell invasive potential and metastatic lesions in vivo. Overall, our findings provide new insights into an ion signaling pathway specific for metastatic cells where NALCN acts as persistent invasion controller.


Subject(s)
Prostatic Neoplasms , Sodium , Male , Humans , Sodium/metabolism , Ion Channels/metabolism , Ion Transport , Membrane Proteins/genetics , Membrane Proteins/metabolism
3.
J Cell Biol ; 216(7): 2107-2130, 2017 07 03.
Article in English | MEDLINE | ID: mdl-28550110

ABSTRACT

Endothelial cell adhesion and migration are critical steps of the angiogenic process, whose dysfunction is associated with tumor growth and metastasis. The TRPM8 channel has recently been proposed to play a protective role in prostate cancer by impairing cell motility. However, the mechanisms by which it could influence vascular behavior are unknown. Here, we reveal a novel non-channel function for TRPM8 that unexpectedly acts as a Rap1 GTPase inhibitor, thereby inhibiting endothelial cell motility, independently of pore function. TRPM8 retains Rap1 intracellularly through direct protein-protein interaction, thus preventing its cytoplasm-plasma membrane trafficking. In turn, this mechanism impairs the activation of a major inside-out signaling pathway that triggers the conformational activation of integrin and, consequently, cell adhesion, migration, in vitro endothelial tube formation, and spheroid sprouting. Our results bring to light a novel, pore-independent molecular mechanism by which endogenous TRPM8 expression inhibits Rap1 GTPase and thus plays a critical role in the behavior of vascular endothelial cells by inhibiting migration.


Subject(s)
Cell Movement , Endothelial Cells/enzymology , Neovascularization, Physiologic , TRPM Cation Channels/metabolism , rap1 GTP-Binding Proteins/metabolism , Cell Adhesion , HEK293 Cells , Human Umbilical Vein Endothelial Cells/enzymology , Humans , Integrin beta1/metabolism , Microscopy, Confocal , Microscopy, Fluorescence , Microscopy, Video , Models, Cardiovascular , Protein Binding , Protein Transport , RNA Interference , Signal Transduction , TRPM Cation Channels/genetics , Time Factors , Transfection , rap1 GTP-Binding Proteins/genetics
4.
Free Radic Biol Med ; 51(9): 1765-73, 2011 Nov 01.
Article in English | MEDLINE | ID: mdl-21875664

ABSTRACT

Hydrogen sulfide (H(2)S) is a gasotransmitter that plays several roles in various tissues, including the cardiovascular system. Because it has been recently proposed to act as a mediator of angiogenesis progression, here we investigate the effects of H(2)S in a well-established model of tumor angiogenesis: endothelial cells obtained from human breast carcinoma (B-TECs). Ca(2+) imaging and patch-clamp experiments reveal that acute perfusion with NaHS, a widely employed H(2)S donor, activates cytosolic calcium (Ca(c)) increase, as well as potassium and nonselective cationic currents, in B-TECs. Stimulation with NaHS in the same concentration range (1 nM-200 µM) evoked Ca(c) signals also in "normal" human microvascular endothelial cells (HMVECs), but the amplitude was significantly lower. Moreover, although NaHS failed to promote either migration or proliferation on HMVECs, B-TEC migration was enhanced at low-micromolar NaHS concentrations (1-10 µM). Remarkably H(2)S mediates tumor proangiogenic signaling triggered by vascular endothelial growth factor (VEGF). B-TECs pretreated with dl-propargylglycine (5mM, 30 min), an inhibitor of the H(2)S-producing enzyme cystathionine γ-lyase, showed drastically reduced migration and Ca(c) signals induced by VEGF (20 ng/ml). We conclude that H(2)S plays a role in proangiogenic signaling of tumor-derived but not normal human ECs. Furthermore the ability of this gasotransmitter to interfere with B-TEC responsiveness to VEGF suggests that it could be an interesting target for antiangiogenic strategies in tumor treatment.


Subject(s)
Breast Neoplasms/pathology , Calcium/metabolism , Carcinoma, Ductal, Breast/pathology , Endothelial Cells/drug effects , Hydrogen Sulfide/pharmacology , Signal Transduction/drug effects , Breast Neoplasms/metabolism , Carcinoma, Ductal, Breast/metabolism , Cell Movement/drug effects , Cell Proliferation/drug effects , Dose-Response Relationship, Drug , Endothelial Cells/metabolism , Female , Humans , Tumor Cells, Cultured
5.
Mycoses ; 53(6): 481-7, 2010 Nov.
Article in English | MEDLINE | ID: mdl-19549106

ABSTRACT

The antifungal activity and in vitro toxicity toward animal cells of two inhibitors of oxidosqualene cyclase, squalene bis-diethylamine (SBD) and squalene bis-diethylmethylammonium iodide (SBDI) were studied. Minimum inhibitory concentration (MIC) against dermatophytes and other fungi involved in cutaneous and systemic infections (12 isolates from seven species) were determined by the broth microdilution method based on the reference documents M38-A and M27-A2 of Clinical and Laboratory Standards Institute (CLSI). Both compounds exerted fungistatic activities, although with different action. SBDI was the more active compound and displayed low MIC values (in the 3.12-12.5 µg ml(-1) range) against Microsporum canis, Trichophyton mentagrophytes and one isolate of Scopulariopsis brevicaulis, while SBD showed MIC values against these species in the 3.12-25 µg ml(-1) range. Toxicity was tested on Madin-Darby canine kidney (MDCK) epithelial cells and human microvascular endothelial cells (HMEC). SBDI proved the less toxic compound: it inhibited M. canis, T. mentagrophytes and S. brevicaulis at concentrations below those found toxic for MDCK cells. HMEC were the more sensitive cells.


Subject(s)
Antifungal Agents/pharmacology , Fungi/drug effects , Intramolecular Transferases/antagonists & inhibitors , Squalene/analogs & derivatives , Squalene/pharmacology , Animals , Antifungal Agents/chemistry , Antifungal Agents/toxicity , Cell Survival/drug effects , Cells, Cultured , Dogs , Humans , Microbial Sensitivity Tests , Squalene/toxicity
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