Arginine vasopressin regulates the renal Na+-Cl- and Na+-K+-Cl- cotransporters through with-no-lysine kinase 4 and inhibitor 1 phosphorylation.

Vasopressin regulates water homeostasis via the V2 receptor in the kidney at least in part through protein kinase A (PKA) activation. Vasopressin, through an unknown pathway, upregulates the activity and phosphorylation of Na+-Cl- cotransporter (NCC) and Na+-K+-2Cl- cotransporter 2 (NKCC2) by Ste20-related proline/alanine-rich kinase (SPAK) and oxidative stress-responsive kinase 1 (OSR1), which are regulated by the with-no-lysine kinase (WNK) family. Phosphorylation of WNK4 at PKA consensus motifs may be involved. Inhibitor 1 (I1), a protein phosphatase 1 (PP1) inhibitor, may also play a role. In human embryonic kidney (HEK)-293 cells, we assessed the phosphorylation of WNK4, SPAK, NCC, or NKCC2 in response to forskolin or desmopressin. WNK4 and cotransporter phosphorylation were studied in desmopressin-infused WNK4-/- mice and in tubule suspensions. In HEK-293 cells, only wild-type WNK4 but not WNK1, WNK3, or a WNK4 mutant lacking PKA phosphorylation motifs could upregulate SPAK or cotransporter phosphorylation in response to forskolin or desmopressin. I1 transfection maximized SPAK phosphorylation in response to forskolin in the presence of WNK4 but not of mutant WNK4 lacking PP1 regulation. We observed direct PP1 regulation of NKCC2 dephosphorylation but not of NCC or SPAK in the absence of WNK4. WNK4-/- mice with desmopressin treatment did not increase SPAK/OSR1, NCC, or NKCC2 phosphorylation. In stimulated tubule suspensions from WNK4-/- mice, upregulation of pNKCC2 was reduced, whereas upregulation of SPAK phosphorylation was absent. These findings suggest that WNK4 is a central node in which kinase and phosphatase signaling converge to connect cAMP signaling to the SPAK/OSR1-NCC/NKCC2 pathway.NEW & NOTEWORTHY With-no-lysine kinases regulate the phosphorylation and activity of the Na+-Cl- and Na+-K+-2Cl- cotransporters. This pathway is modulated by arginine vasopressin (AVP). However, the link between AVP and WNK signaling remains unknown. Here, we show that AVP activates WNK4 through increased phosphorylation at putative protein kinase A-regulated sites and decreases its dephosphorylation by protein phosphatase 1. This work increases our understanding of the signaling pathways mediating AVP actions in the kidney.

Association of SLC12A1 and GLUR4 Ion Transporters with Neoadjuvant Chemoresistance in Luminal Locally Advanced Breast Cancer.

Chemoresistance to standard neoadjuvant treatment commonly occurs in locally advanced breast cancer, particularly in the luminal subtype, which is hormone receptor-positive and represents the most common subtype of breast cancer associated with the worst outcomes. Identifying the genes associated with chemoresistance is crucial for understanding the underlying mechanisms and discovering effective treatments. In this study, we aimed to identify genes linked to neoadjuvant chemotherapy resistance in 62 retrospectively included patients with luminal breast cancer. Whole RNA sequencing of 12 patient biopsies revealed 269 differentially expressed genes in chemoresistant patients. We further validated eight highly correlated genes associated with resistance. Among these, solute carrier family 12 member 1 (SLC12A1) and glutamate ionotropic AMPA type subunit 4 (GRIA4), both implicated in ion transport, showed the strongest association with chemoresistance. Notably, SLC12A1 expression was downregulated, while protein levels of glutamate receptor 4 (GLUR4), encoded by GRIA4, were elevated in patients with a worse prognosis. Our results suggest a potential link between SLC12A1 gene expression and GLUR4 protein levels with chemoresistance in luminal breast cancer. In particular, GLUR4 protein could serve as a potential target for drug intervention to overcome chemoresistance.

Radiomics Analyses to Predict Histopathology in Patients with Metastatic Testicular Germ Cell Tumors before Post-Chemotherapy Retroperitoneal Lymph Node Dissection.

BACKGROUND: The identification of histopathology in metastatic non-seminomatous testicular germ cell tumors (TGCT) before post-chemotherapy retroperitoneal lymph node dissection (PC-RPLND) holds significant potential to reduce treatment-related morbidity in young patients, addressing an important survivorship concern. AIM: To explore this possibility, we conducted a study investigating the role of computed tomography (CT) radiomics models that integrate clinical predictors, enabling personalized prediction of histopathology in metastatic non-seminomatous TGCT patients prior to PC-RPLND. In this retrospective study, we included a cohort of 122 patients. METHODS: Using dedicated radiomics software, we segmented the targets and extracted quantitative features from the CT images. Subsequently, we employed feature selection techniques and developed radiomics-based machine learning models to predict histological subtypes. To ensure the robustness of our procedure, we implemented a 5-fold cross-validation approach. When evaluating the models' performance, we measured metrics such as the area under the receiver operating characteristic curve (AUC), sensitivity, specificity, precision, and F-score. RESULT: Our radiomics model based on the Support Vector Machine achieved an optimal average AUC of 0.945. CONCLUSIONS: The presented CT-based radiomics model can potentially serve as a non-invasive tool to predict histopathological outcomes, differentiating among fibrosis/necrosis, teratoma, and viable tumor in metastatic non-seminomatous TGCT before PC-RPLND. It has the potential to be considered a promising tool to mitigate the risk of over- or under-treatment in young patients, although multi-center validation is critical to confirm the clinical utility of the proposed radiomics workflow.

HPV16 E6 and E7 Oncoproteins Stimulate the Glutamine Pathway Maintaining Cell Proliferation in a SNAT1-Dependent Fashion.

Persistent high-risk human papillomavirus infection is the main risk factor for cervical cancer establishment, where the viral oncogenes E6 and E7 promote a cancerous phenotype. Metabolic reprogramming in cancer involves alterations in glutamine metabolism, also named glutaminolysis, to provide energy for supporting cancer processes including migration, proliferation, and production of reactive oxygen species, among others. The aim of this work was to analyze the effect of HPV16 E6 and E7 oncoproteins on the regulation of glutaminolysis and its contribution to cell proliferation. We found that the E6 and E7 oncoproteins exacerbate cell proliferation in a glutamine-dependent manner. Both oncoproteins increased the levels of transporter SNAT1, as well as GLS2 and GS enzymes; E6 also increased LAT1 transporter protein levels, while E7 increased ASCT2 and xCT. Some of these alterations are also regulated at a transcriptional level. Consistently, the amount of SNAT1 protein decreased in Ca Ski cells when E6 and E7 expression was knocked down. In addition, we demonstrated that cell proliferation was partially dependent on SNAT1 in the presence of glutamine. Interestingly, SNAT1 expression was higher in cervical cancer compared with normal cervical cells. The high expression of SNAT1 was associated with poor overall survival of cervical cancer patients. Our results indicate that HPV oncoproteins exacerbate glutaminolysis supporting the malignant phenotype.

The Roles of Histone Post-Translational Modifications in the Formation and Function of a Mitotic Chromosome.

During mitosis, many cellular structures are organized to segregate the replicated genome to the daughter cells. Chromatin is condensed to shape a mitotic chromosome. A multiprotein complex known as kinetochore is organized on a specific region of each chromosome, the centromere, which is defined by the presence of a histone H3 variant called CENP-A. The cytoskeleton is re-arranged to give rise to the mitotic spindle that binds to kinetochores and leads to the movement of chromosomes. How chromatin regulates different activities during mitosis is not well known. The role of histone post-translational modifications (HPTMs) in mitosis has been recently revealed. Specific HPTMs participate in local compaction during chromosome condensation. On the other hand, HPTMs are involved in CENP-A incorporation in the centromere region, an essential activity to maintain centromere identity. HPTMs also participate in the formation of regulatory protein complexes, such as the chromosomal passenger complex (CPC) and the spindle assembly checkpoint (SAC). Finally, we discuss how HPTMs can be modified by environmental factors and the possible consequences on chromosome segregation and genome stability.

Evaluating the effect of curcumin on the metacestode of Taenia crassiceps.

Curcumin, a curcuminoid present in the rhizome of the plant Curcuma longa has multiple pharmacological effects including anticarcinogenic and anti-inflammatory properties. This work evaluates the anthelmintic effect of the curcumin molecule (98% pure) on Taenia crassiceps cysticerci viability in vitro. Cysticerci incubated in the presence of increasing concentrations of curcumin showed a dose-dependent mortality correlated with a significant increase in the production of reactive oxygen species and a partial inhibition of thioredoxin-glutathione reductase, the only disulfide reductase present in these parasites. At 500 µM curcumin, a 100% of cysticerci lethality was obtained after 2 h of treatment. These results suggest the curcumin-induced oxidative stress could be in the origin of the anthelminthic effect of curcumin. Mice with cysticerci were injected intraperitoneally with 20, 40, or 60 mM curcumin daily for 30 days. A decrease in the burden of cysticerci (46%) was observed with a 60 mM dose of curcumin, supporting this compound as a potential anthelmintic drug.

Multiple molecular mechanisms are involved in the activation of the kidney sodium-chloride cotransporter by hypokalemia.

Low potassium intake activates the kidney sodium-chloride cotransporter (NCC) whose phosphorylation and activity depend on the With-No-Lysine kinase 4 (WNK4) that is inhibited by chloride binding to its kinase domain. Low extracellular potassium activates NCC by decreasing intracellular chloride thereby promoting chloride dissociation from WNK4 where residue L319 of WNK4 participates in chloride coordination. Since the WNK4-L319F mutant is constitutively active and chloride-insensitive in vitro, we generated mice harboring this mutation that displayed slightly increased phosphorylated NCC and mild hyperkalemia when on a 129/sv genetic background. On a low potassium diet, upregulation of phosphorylated NCC was observed, suggesting that in addition to chloride sensing by WNK4, other mechanisms participate which may include modulation of WNK4 activity and degradation by phosphorylation of the RRxS motif in regulatory domains present in WNK4 and KLHL3, respectively. Increased levels of WNK4 and kidney-specific WNK1 and phospho-WNK4-RRxS were observed in wild-type and WNK4L319F/L319F mice on a low potassium diet. Decreased extracellular potassium promoted WNK4-RRxS phosphorylation in vitro and ex vivo as well. These effects might be secondary to intracellular chloride depletion, as reduction of intracellular chloride in HEK293 cells increased phospho-WNK4-RRxS. Phospho-WNK4-RRxS levels were increased in mice lacking the Kir5.1 potassium channel, which presumably have decreased distal convoluted tubule intracellular chloride. Similarly, phospho-KLHL3 was modulated by changes in intracellular chloride in HEK293 cells. Thus, our data suggest that multiple chloride-regulated mechanisms are responsible for NCC upregulation by low extracellular potassium.

Titanium Dioxide (E171) Induces Toxicity in H9c2 Rat Cardiomyoblasts and Ex Vivo Rat Hearts.

Cardiovascular diseases are the leading cause of death worldwide. Food-grade TiO2 (E171) is the most widely used additive in the food industry. Existing evidence shows TiO2 nanoparticles reach systemic circulation through biological barriers, penetrate cell membranes, accumulate in cells of different organs, and cause damage; however, their effects on cardiac cells and the development of heart diseases are still unexplored. Therefore, in this work, we tested E171 toxicity in rat cardiomyoblasts and hearts. E171 internalization and impact on cell viability, proliferation, mitochondria, lysosomes, F-actin distribution, and cell morphology were evaluated in H9c2 cells. Additionally, effects of E171 were measured on cardiac function in ex vivo rat hearts. E171 was uptaken by cells and translocated into the cytoplasm. E171 particles changed cell morphology reducing proliferation and metabolic activity. Higher caspase-3 and caspase-9 expression as well as Tunel-positive cells induced by E171 exposure indicate apoptotic death. Mitochondrial and lysosome alterations resulting from mitophagy were detected after 24 and 48 h exposure, respectively. Additionally, high E171 concentrations caused rearrangements of the F-actin cytoskeleton. Finally, hearts exposed to E171 showed impaired cardiac function. These results support E171 toxicity in cardiac cells in vitro altering cardiac function in an ex vivo model, indicating that consumption of this food additive could be toxic and may lead to the development of cardiovascular disease.

Proteasome inhibition alters mitotic progression through the upregulation of centromeric α-Satellite RNAs.

Cell cycle progression requires control of the abundance of several proteins and RNAs over space and time to properly transit from one phase to the next and to ensure faithful genomic inheritance in daughter cells. The proteasome, the main protein degradation system of the cell, facilitates the establishment of a proteome specific to each phase of the cell cycle. Its activity also strongly influences transcription. Here, we detected the upregulation of repetitive RNAs upon proteasome inhibition in human cancer cells using RNA-seq. The effect of proteasome inhibition on centromeres was remarkable, especially on α-Satellite RNAs. We showed that α-Satellite RNAs fluctuate along the cell cycle and interact with members of the cohesin ring, suggesting that these transcripts may take part in the regulation of mitotic progression. Next, we forced exogenous overexpression and used gapmer oligonucleotide targeting to demonstrate that α-Sat RNAs have regulatory roles in mitosis. Finally, we explored the transcriptional regulation of α-Satellite DNA. Through in silico analyses, we detected the presence of CCAAT transcription factor-binding motifs within α-Satellite centromeric arrays. Using high-resolution three-dimensional immuno-FISH and ChIP-qPCR, we showed an association between the α-Satellite upregulation and the recruitment of the transcription factor NFY-A to the centromere upon MG132-induced proteasome inhibition. Together, our results show that the proteasome controls α-Satellite RNAs associated with the regulation of mitosis.

Large-scale topological disruption of chromosome territories 9 and 22 is associated with nonresponse to treatment in CML.

Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm defined by the presence of t(9;22) translocation whose origin has been associated with the tridimensional genome organization. This rearrangement leads to the fusion of BCR and ABL1 genes giving rise to a chimeric protein with constitutive kinase activity. Imatinib, a tyrosine kinase inhibitor (TKI), is used as a first-line treatment for CML, though ~40% of CML patients do not respond. Here, using structured illumination microscopy (SIM) and 3D reconstruction, we studied the 3D organization patterns of the ABL1 and BCR genes, and their chromosome territories (CTs) CT9 and CT22, in CD34+ cells from CML patients that responded or not to TKI. We found that TKI resistance in CML is associated with high levels of structural disruption of CT9 and CT22 in CD34+ cells, increased CT volumes (especially for CT22), intermingling between CT9 and CT22, and an open-chromatin epigenetic mark in CT22. Altogether our results suggest that large-scale disruption of CT9 and CT22 correlates with the clinical response of CML patients, which could be translated into a potential prognostic marker of response to treatment in this disease and provide novel insights into the mechanisms underlying resistance to TKI in CML.

p21-Activated Kinase 1 Promotes Breast Tumorigenesis via Phosphorylation and Activation of the Calcium/Calmodulin-Dependent Protein Kinase II.

p21-Activated kinase-1 (Pak1) is frequently overexpressed and/or amplified in human breast cancer and is necessary for transformation of mammary epithelial cells. Here, we show that Pak1 interacts with and phosphorylates the Calcium/Calmodulin-dependent Protein Kinase II (CaMKII), and that pharmacological inhibition or depletion of Pak1 leads to diminished activity of CaMKII. We found a strong correlation between Pak1 and CaMKII expression in human breast cancer samples, and combined inhibition of Pak1 and CaMKII with small-molecule inhibitors was synergistic and induced apoptosis more potently in Her2 positive and triple negative breast cancer (TNBC) cells. Co-adminstration of Pak and CaMKII small-molecule inhibitors resulted in a dramatic reduction of proliferation and an increase in apoptosis in a 3D cell culture setting, as well as an impairment in migration and invasion of TNBC cells. Finally, mice bearing xenografts of TNBC cells showed a significant delay in tumor growth when treated with small-molecule inhibitors of Pak and CaMKII. These data delineate a signaling pathway from Pak1 to CaMKII that is required for efficient proliferation, migration and invasion of mammary epithelial cells, and suggest new therapeutic strategies in breast cancer.

Simvastatin Enhances the Immune Response Against Mycobacterium tuberculosis.

Tuberculosis remains a serious threat worldwide. For this reason, it is necessary to identify agents that shorten the duration of treatment, strengthen the host immune system, and/or decrease the damage caused by the infection. Statins are drugs that reduce plasma cholesterol levels and have immunomodulatory, anti-inflammatory and antimicrobial effects. Although there is evidence that statins may contribute to the containment of Mycobacterium tuberculosis infection, their effects on peripheral blood mononuclear cells (PBMCs) involved in the immune response have not been previously described. Using PBMCs from 10 healthy subjects infected with M. tuberculosis H37Rv, we analyzed the effects of simvastatin on the treatment of the infections in an in vitro experimental model. Direct quantification of M. tuberculosis growth (in CFU/mL) was performed. Phenotypes and cell activation were assessed via multi-color flow cytometry. Culture supernatant cytokine levels were determined via cytokine bead arrays. The induction of apoptosis and autophagy was evaluated via flow cytometry and confocal microscopy. Simvastatin decreased the growth of M. tuberculosis in PBMCs, increased the proportion of NKT cells in culture, increased the expression of co-stimulatory molecules in monocytes, promoted the secretion of the cytokines IL-1ß and IL-12p70, and activated apoptosis and autophagy in monocytes, resulting in a significant reduction in bacterial load. We also observed an increase in IL-10 production. We did not observe any direct antimycobacterial activity. This study provides new insight into the mechanism through which simvastatin reduces the mycobacterial load in infected PBMCs. These results demonstrate that simvastatin activates several immune mechanisms that favor the containment of M. tuberculosis infection, providing relevant evidence to consider statins as candidates for host-directed therapy. They also suggest that future studies are needed to define the roles of statin-induced anti-inflammatory mechanisms in tuberculosis treatment.

Internalization of Titanium Dioxide Nanoparticles Is Mediated by Actin-Dependent Reorganization and Clathrin- and Dynamin-Mediated Endocytosis in H9c2 Rat Cardiomyoblasts.

Titanium dioxide nanoparticles (TiO2 NPs) are widely used for industrial and commercial applications. Once inside the body, they translocate into the bloodstream and reach different areas of the cardiovascular system including the heart, increasing the risk of developing cardiovascular diseases; consequently, the investigation of their interaction with cardiac cells is required. We previously showed that TiO2 NPs are internalized by H9c2 rat cardiomyoblasts, and here, we examined the molecular mechanisms underlying this process. TiO2 NPs internalization was evaluated by transmission electron microscopy, time-lapse microscopy, and flow cytometry. Changes in the actin cytoskeleton were studied by phalloidin staining. Endocytic uptake mechanisms for nanoparticles were probed with chemical inhibitors, whereas clathrin and dynamin expression was measured by Western blot. Cellular uptake of TiO2 NPs occurred early after 30 min exposure, and large aggregates were observed after 1 h. Actin cytoskeleton reorganization included cell elongation plus lower density and stability of actin fibers. Cytochalasin-D inhibited TiO2 NPs uptake, indicating actin-mediated internalization. Dynamin and clathrin levels increased early after TiO2 NPs exposure, and their inhibition reduced nanoparticle uptake. Therefore, TiO2 NPs internalization by H9c2 rat cardiomyoblasts involves actin cytoskeleton reorganization and clathrin/dynamin-mediated endocytosis.

HPV-18 E6 Oncoprotein and Its Spliced Isoform E6*I Regulate the Wnt/ß-Catenin Cell Signaling Pathway through the TCF-4 Transcriptional Factor.

The Wnt/ß-catenin signaling pathway regulates cell proliferation and differentiation and its aberrant activation in cervical cancer has been described. Persistent infection with high risk human papillomavirus (HR-HPV) is the most important factor for the development of this neoplasia, since E6 and E7 viral oncoproteins alter cellular processes, promoting cervical cancer development. A role of HPV-16 E6 in Wnt/ß-catenin signaling has been proposed, although the participation of HPV-18 E6 has not been previously studied. The aim of this work was to investigate the participation of HPV-18 E6 and E6*I, in the regulation of the Wnt/ß-catenin signaling pathway. Here, we show that E6 proteins up-regulate TCF-4 transcriptional activity and promote overexpression of Wnt target genes. In addition, it was demonstrated that E6 and E6*I bind to the TCF-4 (T cell factor 4) and ß-catenin, impacting TCF-4 stabilization. We found that both E6 and E6*I proteins interact with the promoter of Sp5, in vitro and in vivo. Moreover, although differences in TCF-4 transcriptional activation were found among E6 intratype variants, no changes were observed in the levels of regulated genes. Furthermore, our data support that E6 proteins cooperate with ß-catenin to promote cell proliferation.

Glutathione depletion triggers actin cytoskeleton changes via actin-binding proteins.

The importance of glutathione (GSH) in alternative cellular roles to the canonically proposed, were analyzed in a model unable to synthesize GSH. Gene expression analysis shows that the regulation of the actin cytoskeleton pathway is strongly impacted by the absence of GSH. To test this hypothesis, we evaluate the effect of GSH depletion via buthionine sulfoximine (5 and 12.5 mM) in human neuroblastoma MSN cells. In the present study, 70% of GSH reduction did not induce reactive oxygen species, lipoperoxidation, or cytotoxicity, which enabled us to evaluate the effect of glutathione in the absence of oxidative stress. The cells with decreasing GSH levels acquired morphology changes that depended on the actin cytoskeleton and not on tubulin. We evaluated the expression of three actin-binding proteins: thymosin ß4, profilin and gelsolin, showing a reduced expression, both at gene and protein levels at 24 hours of treatment; however, this suppression disappears after 48 hours of treatment. These changes were sufficient to trigger the co-localization of the three proteins towards cytoplasmic projections. Our data confirm that a decrease in GSH in the absence of oxidative stress can transiently inhibit the actin binding proteins and that this stimulus is sufficient to induce changes in cellular morphology via the actin cytoskeleton.

Airborne particulate matter in vitro exposure induces cytoskeleton remodeling through activation of the ROCK-MYPT1-MLC pathway in A549 epithelial lung cells.

Airborne particulate matter with an aerodynamic diameter ≤10µm (PM10) is considered a risk factor for the development of lung cancer. Little is known about the cellular mechanisms by which PM10 is associated with cancer, but there is evidence that its exposure can lead to an acquired invasive phenotype, apoptosis evasion, inflammasome activation, and cytoskeleton remodeling in lung epithelial cells. Cytoskeleton remodeling occurs through actin stress fiber formation, which is partially regulated through ROCK kinase activation, we aimed to investigate if this protein was activated in response to PM10 exposure in A549 lung epithelial cells. Results showed that 10µg/cm2 of PM10 had no influence on cell viability but increased actin stress fibers, cytoplasmic ROCK expression, and phosphorylation of myosin phosphatase-targeting 1 (MYPT1) and myosin light chain (MLC) proteins, which are targeted by ROCK. The inhibition of ROCK prevented actin stress fiber formation and the phosphorylation of MYPT1 and MLC, suggesting that PM10 activated the ROCK-MYPT1-MLC pathway in lung epithelial cells. The activation of ROCK1 has been involved in the acquisition of malignant phenotypes, and its induction by PM10 exposure could contribute to the understanding of PM10 as a risk factor for cancer development through the mechanisms associated with invasive phenotype.

BUB1 and SURVIVIN proteins are not degraded after a prolonged mitosis and accumulate in the nuclei of HCT116 cells.

Spindle poisons activate the spindle assembly checkpoint and prevent mitotic exit until cells die or override the arrest. Several studies have focused on spindle poison-mediated cell death, but less is known about consequences in cells that survive a mitotic arrest. During mitosis, proteins such as CYCLIN B, SECURIN, BUB1 and SURVIVIN are degraded in order to allow mitotic exit, and these proteins are maintained at low levels in the next interphase. In contrast, exit from a prolonged mitosis depends only on degradation of CYCLIN B; it is not known whether the levels of other proteins decrease or remain high. Here, we analyzed the levels and localization of the BUB1 and SURVIVIN proteins in cells that escaped from a paclitaxel-mediated prolonged mitosis. We compared cells with a short arrest (HCT116 cells) with cells that spent more time in mitosis (HT29 cells) after paclitaxel treatment. BUB1 and SURVIVIN were not degraded and remained localized to the nuclei of HCT116 cells after a mitotic arrest. Moreover, BUB1 nuclear foci were observed; BUB1 did not colocalize with centromere proteins. In HT29 cells, the levels of BUB1 and SURVIVIN decreased during the arrest, and these proteins were not present in cells that reached the next interphase. Using time-lapse imaging, we observed morphological heterogeneity in HCT116 cells that escaped from the arrest; this heterogeneity was due to the cytokinesis-like mechanism by which the cells exited mitosis. Thus, our results show that high levels of BUB1 and SURVIVIN can be maintained after a mitotic arrest, which may promote resistance to cell death.

Secretome derived from breast tumor cell lines alters the morphology of human umbilical vein endothelial cells.

Metastases, responsible for most of the solid tumor associated deaths, require angiogenesis and changes in endothelial cells. In this work, the effect of the secretomes of three breast tumor cell lines (MCF-7, MDA-MB-231 and ZR-75-30) on human umbilical vein endothelial cells (HUVEC) morphology was investigated. HUVEC treated with secretomes from breast cells were analyzed by confocal and time-lapse microscopy. Secretomes from ZR-75-30 and MDA-MB-231 cells modify the morphology and adhesion of HUVEC. These changes may provoke the loss of endothelial monolayer integrity. In consequence, tumor cells could have an increased access to circulation, which would then enhance metastasis.

MAD2γ, a novel MAD2 isoform, reduces mitotic arrest and is associated with resistance in testicular germ cell tumors.

BACKGROUND: Prolonged mitotic arrest in response to anti-cancer chemotherapeutics, such as DNA-damaging agents, induces apoptosis, mitotic catastrophe, and senescence. Disruptions in mitotic checkpoints contribute resistance to DNA-damaging agents in cancer. MAD2 has been associated with checkpoint failure and chemotherapy response. In this study, a novel splice variant of MAD2, designated MAD2γ, was identified, and its association with the DNA damage response was investigated. METHODS: Endogenous expression of MAD2γ and full-length MAD2 (MAD2α) was measured using RT-PCR in cancer cell lines, normal foreskin fibroblasts, and tumor samples collected from patients with testicular germ cell tumors (TGCTs). A plasmid expressing MAD2γ was transfected into HCT116 cells, and its intracellular localization and checkpoint function were evaluated according to immunofluorescence and mitotic index. RESULTS: MAD2γ was expressed in several cancer cell lines and non-cancerous fibroblasts. Ectopically expressed MAD2γ localized to the nucleus and reduced the mitotic index, suggesting checkpoint impairment. In patients with TGCTs, the overexpression of endogenous MAD2γ, but not MAD2α, was associated with resistance to cisplatin-based chemotherapy. Likewise, cisplatin induced the overexpression of endogenous MAD2γ, but not MAD2α, in HCT116 cells. CONCLUSIONS: Overexpression of MAD2γ may play a role in checkpoint disruption and is associated with resistance to cisplatin-based chemotherapy in TGCTs.

Internalization of titanium dioxide nanoparticles by glial cells is given at short times and is mainly mediated by actin reorganization-dependent endocytosis.

Many nanoparticles (NPs) have toxic effects on multiple cell lines. This toxicity is assumed to be related to their accumulation within cells. However, the process of internalization of NPs has not yet been fully characterized. In this study, the cellular uptake, accumulation, and localization of titanium dioxide nanoparticles (TiO2 NPs) in rat (C6) and human (U373) glial cells were analyzed using time-lapse microscopy (TLM) and transmission electron microscopy (TEM). Cytochalasin D (Cyt-D) was used to evaluate whether the internalization process depends of actin reorganization. To determine whether the NP uptake is mediated by phagocytosis or macropinocytosis, nitroblue tetrazolium (NBT) reduction was measured and the 5-(N-ethyl-N-isopropyl)-amiloride was used. Expression of proteins involved with endocytosis and exocytosis such as caveolin-1 (Cav-1) and cysteine string proteins (CSPs) was also determined using flow cytometry. TiO2 NPs were taken up by both cell types, were bound to cellular membranes and were internalized at very short times after exposure (C6, 30 min; U373, 2h). During the uptake process, the formation of pseudopodia and intracellular vesicles was observed, indicating that this process was mediated by endocytosis. No specific localization of TiO2 NPs into particular organelles was found: in contrast, they were primarily localized into large vesicles in the cytoplasm. Internalization of TiO2 NPs was strongly inhibited by Cyt-D in both cells and by amiloride in U373 cells; besides, the observed endocytosis was not associated with NBT reduction in either cell type, indicating that macropinocytosis is the main process of internalization in U373 cells. In addition, increases in the expression of Cav-1 protein and CSPs were observed. In conclusion, glial cells are able to internalize TiO2 NPs by a constitutive endocytic mechanism which may be associated with their strong cytotoxic effect in these cells; therefore, TiO2 NPs internalization and their accumulation in brain cells could be dangerous to human health.