RESUMEN

Graphene nanoplatelets (UGZ-1004) are emerging as a promising biomaterial in regenerative medicine. This study comprehensively evaluates UGZ-1004, focusing on its physical properties, cytotoxicity, intracellular interactions, and, notably, its effects on mesenchymal stem cells (MSCs). UGZ-1004 was characterized by lateral dimensions and layer counts consistent with ISO standards and demonstrated a high carbon purity of 0.08%. Cytotoxicity assessments revealed that UGZ-1004 is non-toxic to various cell lines, including 3T3 fibroblasts, VERO kidney epithelial cells, BV-2 microglia, and MSCs, in accordance with ISO 10993-5:2020/2023 guidelines. The study focused on MSCs and revealed that UGZ-1004 supports their gene expression alterations related to self-renewal and proliferation. MSCs exposed to UGZ-1004 maintained their characteristic surface markers. Importantly, UGZ-1004 promoted significant upregulation of genes crucial for cell cycle regulation and DNA repair, such as CDK1, CDK2, and MDM2. This gene expression profile suggests that UGZ-1004 can enhance MSC self-renewal capabilities, ensuring robust cellular function and longevity. Moreover, UGZ-1004 exposure led to the downregulation of genes associated with tumor development, including CCND1 and TFDP1, mitigating potential tumorigenic risks. These findings underscore the potential of UGZ-1004 to not only bolster MSC proliferation but also enhance their self-renewal processes, which are critical for effective regenerative therapies. The study highlights the need for continued research into the long-term impacts of graphene nanoplatelets and their application in MSC-based regenerative medicine.

Asunto(s)

Proliferación Celular , Grafito , Células Madre Mesenquimatosas , Proliferación Celular/efectos de los fármacos , Células Madre Mesenquimatosas/efectos de los fármacos , Células Madre Mesenquimatosas/metabolismo , Células Madre Mesenquimatosas/citología , Animales , Grafito/química , Grafito/farmacología , Ratones , Chlorocebus aethiops , Autorrenovación de las Células/efectos de los fármacos , Autorrenovación de las Células/genética , Células Vero , Regulación de la Expresión Génica/efectos de los fármacos , Nanopartículas/química , Línea Celular , Nanoestructuras/química

RESUMEN

Prostate cancer (PCa) is the second most diagnosed type of cancer in men worldwide. Advanced PCa is resistant to conventional therapies and high recurrence has been associated with high rates of metastasis. Cancer stem cells (CSCs) have been proposed to be responsible for this, due to their ability of self­renewal and differentiation into other cell types. Zinc finger E­box­binding homeobox 1 (ZEB1), a transcription factor involved in the regulation of epithelial­mesenchymal transition (EMT), has been associated with the activation of several mechanisms that lead to resistance to treatment. As recent evidence has shown that CSCs may originate from non­CSCs during EMT, it was hypothesized that knocking down ZEB1 expression in PCa cell lines could revert some properties associated with CSCs. Using lentiviraltransduction, ZEB1 expression was silenced in the PCa DU145 and LNCaP cell lines. The mRNA and protein expression levels of key canonical CSC markers (Krüppel­like factor 4, SOX2, CD44 and CD133) were determined using reverse transcription­-quantitative PCR and western blot analysis, respectively. In addition, the colony forming ability of the ZEB1­knockdown cells was evaluated, and the type of colonies formed (holoclones, paraclones and meroclones) was also characterized. Finally, the ability to form prostatospheres was evaluated in vitro. It was found that in ZEB1­knockdown DU145 cells, the expression levels of CSC phenotype markers (CD44, CD133 and SOX2) were decreased compared with those in the control group. Furthermore, ZEB1­knockdown cells exhibited a lower ability to form prostatospheres and to generate colonies. In conclusion, stable silencing of ZEB1 reversed CSC properties in PCa cell lines. Since ZEB1 is associated with malignancy, therapy resistance and a CSC phenotype in PCa cell lines, targeting ZEB1 may be a key factor to eradicate CSCs and improve the prognosis of patients with advanced PCa.

Asunto(s)

Autorrenovación de las Células/genética , Regulación Neoplásica de la Expresión Génica/genética , Células Madre Neoplásicas/patología , Neoplasias de la Próstata/genética , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/metabolismo , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Autorrenovación de las Células/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Transición Epitelial-Mesenquimal/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Humanos , Masculino , Próstata/citología , Próstata/patología , Neoplasias de la Próstata/patología , Ensayo de Tumor de Célula Madre , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/antagonistas & inhibidores , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/genética

RESUMEN

The aim of this study was to investigate carnitine action against negative effects of etoposide on stem/progenitor spermatogonia and on sperm production. Carnitine (250 mg/kg body weight/day) and etoposide (5 mg/kg body weight/day) were administered from 25-days postpartum to 32-days postpartum. Testes were collected at 32-days postpartum, 64-days postpartum, and 127-days postpartum, and submitted to the immuno-labeling of UTF1, SOX2, and PLZF proteins to identify undifferentiated spermatogonia populations. At 127-days postpartum, sperm were collected for analysis. Carnitine+etoposide group showed a higher numerical density of spermatogonia labeled for all studied proteins at 64-days postpartum (critical age) compared to the etoposide group. Moreover, there was an improvement of spermatic parameters and sperm DNA integrity in rats of the carnitine+etoposide group in comparison with rats of the etoposide group. The results suggest that carnitine improves the self-renewal of undifferentiated spermatogonia and promotes a partial protection on them, alleviating the etoposide harmful late effects and leading to an enhancement of the sperm parameters in adulthood.

Asunto(s)

Carnitina/farmacología , Autorrenovación de las Células/efectos de los fármacos , Etopósido/toxicidad , Espermatogonias/citología , Espermatogonias/efectos de los fármacos , Animales , Daño del ADN , Relación Dosis-Respuesta a Droga , Masculino , Tamaño de los Órganos/efectos de los fármacos , Proteína de la Leucemia Promielocítica con Dedos de Zinc/metabolismo , Ratas , Factores de Transcripción SOXB1/metabolismo , Epitelio Seminífero/efectos de los fármacos , Epitelio Seminífero/crecimiento & desarrollo , Espermatogénesis/efectos de los fármacos , Espermatogonias/metabolismo , Testículo/efectos de los fármacos , Testículo/crecimiento & desarrollo , Factores de Transcripción/metabolismo

RESUMEN

BACKGROUND: Tamoxifen (Tam) is the most frequent treatment for estrogen receptor (ER) positive breast cancer. We recently showed that fibronectin (FN) leads to Tam resistance and selection of breast cancer stem cells. With the aim of developing a nanoformulation that would simultaneously tackle ER and FN/ß1 integrin interactions, we designed polyethylene glycol-polycaprolactone polymersomes polymersomes (PS) that carry Tam and are functionalized with the tumor-penetrating iRGD peptide (iRGD-PS-Tam). RESULTS: Polyethylene glycol-polycaprolactone PS were assembled and loaded with Tam using the hydration film method. The loading of encapsulated Tam, measured by UPLC, was 2.4 ± 0.5 mol Tam/mol polymer. Physicochemical characterization of the PS demonstrated that iRGD functionalization had no effect on morphology, and a minimal effect on the PS size and polydispersity (176 nm and Pdi 0.37 for iRGD-TAM-PS and 171 nm and Pdi 0.36 for TAM-PS). iRGD-PS-Tam were taken up by ER+ breast carcinoma cells in 2D-culture and exhibited increased penetration of 3D-spheroids. Treatment with iRGD-PS-Tam inhibited proliferation and sensitized cells cultured on FN to Tam. Mechanistically, treatment with iRGD-PS-Tam resulted in inhibition ER transcriptional activity as evaluated by a luciferase reporter assay. iRGD-PS-Tam reduced the number of cells with self-renewing capacity, a characteristic of breast cancer stem cells. In vivo, systemic iRGD-PS-Tam showed selective accumulation at the tumor site. CONCLUSIONS: Our study suggests iRGD-guided delivery of PS-Tam as a potential novel therapeutic strategy for the management of breast tumors that express high levels of FN. Future studies in pre-clinical in vivo models are warranted.

Asunto(s)

Antineoplásicos Hormonales/administración & dosificación , Neoplasias de la Mama/tratamiento farmacológico , Portadores de Fármacos/química , Oligopéptidos/química , Receptores de Estrógenos/metabolismo , Tamoxifeno/administración & dosificación , Animales , Antineoplásicos Hormonales/farmacocinética , Antineoplásicos Hormonales/farmacología , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Autorrenovación de las Células/efectos de los fármacos , Femenino , Humanos , Células MCF-7 , Ratones Desnudos , Poliésteres/química , Polietilenglicoles/química , Tamoxifeno/farmacocinética , Tamoxifeno/farmacología , Activación Transcripcional/efectos de los fármacos

RESUMEN

Uncovering the cellular and molecular mechanisms by which hematopoietic stem cell (HSC) self-renewal is regulated can lead to the development of new strategies for promoting ex vivo HSC expansion. Here, we report the discovery that alternative (M2)-polarized macrophages (M2-MΦs) promote, but classical (M1)-polarized macrophages (M1-MΦs) inhibit, the self-renewal and expansion of HSCs from mouse bone marrow (BM) in vitro. The opposite effects of M1-MΦs and M2-MΦs on mouse BM HSCs were attributed to their differential expression of nitric oxide synthase 2 (NOS2) and arginase 1 (Arg1), because genetic knockout of Nos2 and Arg1 or inhibition of these enzymes with a specific inhibitor abrogated the differential effects of M1-MΦs and M2-MΦs. The opposite effects of M1-MΦs and M2-MΦs on HSCs from human umbilical cord blood (hUCB) were also observed when hUCB CD34+ cells were cocultured with M1-MΦs and M2-MΦs generated from hUCB CD34- cells. Importantly, coculture of hUCB CD34+ cells with human M2-MΦs for 8 days resulted in 28.7- and 6.6-fold increases in the number of CD34+ cells and long-term SCID mice-repopulating cells, respectively, compared with uncultured hUCB CD34+ cells. Our findings could lead to the development of new strategies to promote ex vivo hUCB HSC expansion to improve the clinical utility and outcome of hUCB HSC transplantation and may provide new insights into the pathogenesis of hematological dysfunctions associated with infection and inflammation that can lead to differential macrophage polarization.

Asunto(s)

Proliferación Celular/efectos de los fármacos , Autorrenovación de las Células/efectos de los fármacos , Células Madre Hematopoyéticas/citología , Macrófagos/fisiología , Animales , Arginasa/metabolismo , Técnicas de Cocultivo , Sangre Fetal/citología , Humanos , Masculino , Ratones , Óxido Nítrico Sintasa de Tipo II/metabolismo

RESUMEN

Photobiomodulation (PBM) therapy displays relevant properties for tissue healing and regeneration, which may be of interest for the tissue engineering field. Here, we show that PBM is able to improve cell survival and to interact with recombinant human Bone Morphogenetic Protein 4 (rhBMP4) to direct and accelerate odonto/osteogenic differentiation of dental derived mesenchymal stem cells (MSCs). MSCs were encapsulated in an injectable and thermo-responsive cell carrier (Pluronic® F-127) loaded with rhBMP4 and then photoactivated. PBM improved MSCs self-renewal and survival upon encapsulation in the Pluronic® F-127. In the presence of rhBMP4, cell odonto/osteogenic differentiation was premature and markedly improved in the photoactivated MSCs. An in vivo calvarial critical sized defect model demonstrated significant increase in bone formation after PBM treatment. Finally, a balance in the reactive oxygen species levels may be related to the favorable results of PBM and rhBMP4 association. PBM may act in synergism with rhBMP4 and is a promise candidate to direct and accelerate hard tissue bioengineering.

Asunto(s)

Proteína Morfogenética Ósea 4/administración & dosificación , Portadores de Fármacos , Terapia por Luz de Baja Intensidad/métodos , Células Madre Mesenquimatosas/efectos de los fármacos , Células Madre Mesenquimatosas/efectos de la radiación , Poloxámero/química , Ingeniería de Tejidos/métodos , Andamios del Tejido , Adolescente , Adulto , Animales , Proteína Morfogenética Ósea 4/química , Regeneración Ósea , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/efectos de la radiación , Proliferación Celular/efectos de los fármacos , Proliferación Celular/efectos de la radiación , Autorrenovación de las Células/efectos de los fármacos , Autorrenovación de las Células/efectos de la radiación , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Células Cultivadas , Humanos , Hidrogeles , Inyecciones , Láseres de Semiconductores , Terapia por Luz de Baja Intensidad/instrumentación , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/metabolismo , Ratones Desnudos , FN-kappa B/metabolismo , Osteogénesis/efectos de los fármacos , Osteogénesis/efectos de la radiación , Hueso Parietal/lesiones , Hueso Parietal/patología , Hueso Parietal/cirugía , Especies Reactivas de Oxígeno/metabolismo , Factores de Tiempo , Adulto Joven

RESUMEN

The aim of the present study was to demonstrate that ivermectin preferentially inhibited cancer stem­like cells (CSC) in breast cancer cells and downregulated the expression of 'stemness' genes. Computational searching of DrugBank, a database of approved drugs, was performed using the principles of two­dimensional similarity searching; the chemical structure of salinomycin was used as a query. Growth inhibition of the breast cancer cell lin e MDA­MB­231 by ivermectin was investigated in the total cell population, in cell spheroids and in sorted cells that expressed cluster of differentiation (CD)44+/CD24­. The effects of ivermectin treatment on the expression of pluripotency and self­renewal transcription factors, such as homeobox protein nanog (nanog), octamer­binding protein 4 (oct­4) and SRY­box 2 (sox­2), were evaluated by reverse transcription­quantitative polymerase chain reaction and western blotting. Ivermectin exhibited a similarity value of 0.78 in reference to salinomycin. Ivermectin demonstrated an inhibitory effect upon the growth of MDA­MB­231 cells in the range of 0.2­8 µM. Ivermectin preferentially inhibits the viability of CSC­enriched populations (CD44+/CD24­ and cells growing in spheroids) compared with the total cell population. The opposite pattern was observed with paclitaxel treatment. Ivermectin exposure reduced the expression of nanog, oct­4 and sox­2 at the mRNA and protein levels. Ivermectin preferentially inhibited the CSC subpopulation in the MDA­MB­231 cells and downregulated the expression of genes involved in the maintenance of pluripotency and self­renewal.

Asunto(s)

Neoplasias de la Mama/tratamiento farmacológico , Proliferación Celular/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Ivermectina/farmacología , Células Madre Neoplásicas/efectos de los fármacos , Plaguicidas/farmacología , Antiparasitarios/farmacología , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Línea Celular Tumoral , Autorrenovación de las Células/efectos de los fármacos , Femenino , Humanos , Células Madre Neoplásicas/citología , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología