Thermorresponsive magnetic nanoparticles as target drug delivery for cancer treatment

In this research, temperature sensitive microgels with magnetic core for controlled release of 5-fluoruracil was synthesized. Magnetic nanoparticles (Fe3O4) were prepared by coprecipitation method and the surface was functionalized by acrylic acid. Polymer poly(N-isopropylacrylamide) (PNIPAM) were grown by free radical polymerization in presence of cross-liker and initiator. The size of the polymer was manipulated by changing the mole percent of the crosslinker and evaluated for their morphology (TEM), particle size, zeta potential, loading efficiency, drug content and drug release. Furthermore, microgels were tagged with FITC, a fluorochrome which could be applied for cell imaging. Cytotoxicity studies revealed that the microgels were not toxic. These complex nanoparticles (Fe3O4/pNIPAM/FITC/5-Fu) appear to be a great promise to be used in controlled drug delivery and tumor targeting.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Regression of established subcutaneous B16-F10 murine melanoma tumors after gef gene therapy via the mitochondrial apoptotic pathway

ovel treatment modalities, including gene therapy, are needed for patients with advanced melanoma. We evaluated whether the gef gene, a suicide gene from Escherichia coli, had a significant cytotoxic impact on melanoma in vivo. First, we used a non-viral gene delivery approach (pcDNA3.1/gef) to study the inhibition of melanoma cells (B16-F10) proliferation in vitro. Secondly, we used direct intra-tumoral injection of pcDNA3.1/gef complexed with jetPEI to deliver gef cDNA to rapidly growing murine melanomas. We demonstrated that gef gene not only has an antiproliferative effect on B16-F10 cells in vitro, but also induces an important decrease in melanoma tumor volume (77.7% in 8 days) in vivo. Interestingly, after gef gene treatment, melanoma showed apoptosis activation associated with the mitochondrial pathway, suggesting that the induction of this death mechanism may be an effective strategy for its treatment. Our in vivo results indicate that gef gene might become a suitable therapeutic strategy for patients with advanced melanoma

Microenvironmental Modulation of Decorin and Lumican in Temozolomide-Resistant Glioblastoma and Neuroblastoma Cancer Stem-Like Cells

The presence of cancer stem cells (CSCs) or tumor-initiating cells can lead to cancer recurrence in a permissive cell–microenvironment interplay, promoting invasion in glioblastoma (GBM) and neuroblastoma (NB). Extracellular matrix (ECM) small leucine-rich proteoglycans (SLRPs) play multiple roles in tissue homeostasis by remodeling the extracellular matrix (ECM) components and modulating intracellular signaling pathways. Due to their pan-inhibitory properties against receptor tyrosine kinases (RTKs), SLRPs are reported to exert anticancer effects in vitro and in vivo. However, their roles seem to be tissue-specific and they are also involved in cancer cell migration and drug resistance, paving the way to complex different scenarios. The aim of this study was to determine whether the SLRPs decorin (DCN) and lumican (LUM) are recruited in cell plasticity and microenvironmental adaptation of differentiated cancer cells induced towards stem-like phenotype. Floating neurospheres were generated by applying CSC enrichment medium (neural stem cell serum-free medium, NSC SFM) to the established SF-268 and SK-N-SH cancer cell lines, cellular models of GBM and NB, respectively. In both models, the time-dependent synergistic activation of DCN and LUM was observed. The highest DCN and LUM mRNA/protein expression was detected after cell exposure to NSC SFM for 8/12 days, considering these cells as SLRP-expressing (SLRP+) CSC-like. Ultrastructural imaging showed the cellular heterogeneity of both the GBM and NB neurospheres and identified the inner living cells. Parental cell lines of both GBM and NB grew only in soft agar + NSC SFM, whereas the secondary neurospheres (originated from SLRP+ t8 CSC-like) showed lower proliferation rates than primary neurospheres. Interestingly, the SLRP+ CSC-like from the GBM and NB neurospheres were resistant to temozolomide (TMZ) at concentrations >750 μM. Our results suggest that GBM and NB CSC-like promote the activation of huge quantities of SLRP in response to CSC enrichment, simultaneously acquiring TMZ resistance, cellular heterogeneity, and a quiescent phenotype, suggesting a novel pivotal role for SLRP in drug resistance and cell plasticity of CSC-like, allowing cell survival and ECM/niche modulation potential.This study was supported by Fundació la Marató TV3, Project n° 111431

Reversion of neuronal differentiation induced in human adipose-derived stem cells

Adipose-derived mesenchymal stem cells are a great alternative to other types of stem cells obtained from other tissues, since they are found in large numbers and are easy to obtain by liposuction and do not have ethical connotations. These cells can differentiate into neuronal lineage using induction media, although the efficacy of these media is determined by their composition. In previous studies, we have demonstrated the differentiation of human adipose-derived mesenchymal stem cells to neuronal lineage by using three induction media, Neu1, Neu2 and Neu3, each one showing a series of neuronal markers in the treated cells. In the present study, a further step is taken, since once a neuronal differentiation is obtained with these three induction media, they are removed from the medium and both morphology and neuronal markers and the ability to maintain neurosphere formation are analyzed. The results obtained show that when these induction media are withdrawn in the three cases, both the neuronal morphological characteristics and the neuronal markers are lost at different times depending on the medium used. In the case of neurospheres, the ability to maintain this shape is also lost when they are not in contact with neuronal induction factors. Therefore, although the neuronal differentiation mechanism is very promising in this type of cells, it is necessary to carry out more studies to elucidate an induction medium that allows cells differentiated into neurons to maintain neuronal characteristics over time without the need to continue applying neuronal differentiation factors

Cancer Stem Cells in Sarcomas: In Vitro Isolation and Role as Prognostic Markers: A Systematic Review

Sarcomas are a diverse group of neoplasms with an incidence rate of 15% of childhood cancers. They exhibit a high tendency to develop early metastases and are often resistant to available treatments, resulting in poor prognosis and survival. In this context, cancer stem cells (CSCs) have been implicated in recurrence, metastasis, and drug resistance, making the search for diagnostic and prognostic biomarkers of the disease crucial. The objective of this systematic review was to analyze the expression of CSC biomarkers both after isolation from in vitro cell lines and from the complete cell population of patient tumor samples. A total of 228 publications from January 2011 to June 2021 was retrieved from different databases, of which 35 articles were included for analysis. The studies demonstrated significant heterogeneity in both the markers detected and the CSC isolation techniques used. ALDH was identified as a common marker in various types of sarcomas. In conclusion, the identification of CSC markers in sarcomas may facilitate the development of personalized medicine and improve treatment outcomes

E phage gene transfection associated to chemotherapeutic agents increases apoptosis in lung and colon cancer cells

The limited ability of conventional therapies to achieve the long-term survival of metastatic lung and colon cancer patients suggests the need for new treatment options. In this respect, genes encoding cytotoxic proteins have been proposed as a new strategy to enhance the activity of drugs, and combined therapies involving such genes and classical antitumoral drugs have been studied intensively. The E gene from phiX174 encodes a membrane protein with a toxic domain that leads to a decrease in tumour cell growth rates. Therefore, in order to improve the anti-tumour effects of currently used chemotherapeutic drugs on cancer cells, we investigated the association of the E suicide gene with these antineoplastic drugs. The E gene has antitumoral effects in both lung and colon cancer cells. In addition, expression of this gene induces ultrastructural changes in lung cancer transfected cells (A-549), although the significance of these changes remains unknown. The effect of combined therapy (gene and cytotoxic therapy) enhances the inhibition of tumour cell proliferation in comparison to single treatments. Indeed, our in vitro results indicate that an experimental therapeutic strategy based on this combination of E gene therapy and cytotoxic drugs may result in a new treatment strategy for patients with advanced lung and colon cancer

Modulation of HLA class I expression in multidrug-resistant human rhabdomyosarcoma cells

An abnormal HLA expression has been detected in some tumors including rhabdomyosarcoma (RMS). Classical cytotoxic treatment of these tumors, the most common childhood soft tissue malignancy, may induce multidrug resistance (MDR) associated with the expression of a 170-kDa membrane-associated glycoprotein (P-glycoprotein). In order to analyse the connection between modulation of HLA expression and the development of the MDR phenotype mediated by P-glycoprotein in RMS, we used three resistant RMS cell lines; two of these resistant cell lines (TE.32.7.DAC and RD-DAC) were established by in vitro exposure to actinomycin D, a drug of choice in the treatment of RMS; the resistant RMS- GR cell line was established from an embryonal RMS tumor after polychemotherapy. Our results showed that all the resistant cell lines showed a significant increase in the expression of HLA class I surface antigens in comparison to drug-sensitive cells. Blockade of P-glycoprotein with verapamil led to a decrease in HLA class I expression in RMS resistant cell lines. However, no modulation of HLA class II expression was observed in any of the three analyzed cell lines. These findings support the hypothesis that the development of resistance mediated by mdr 1/P-glycoprotein, directly influences the expression of HLA class I in RMS cells, inducing to upregulation. This effect may be relevant to the application in RMS of immunotherapy against tumor-associated antigens presented by HLA class I molecules

Evaluation of poly (lactic-co-glycolic acid) nanoparticles to improve the therapeutic efficacy of paclitaxel in breast cancer

Introduction: Paclitaxel (PTX) is a cornerstone in the treatment of breast cancer, the most common type of cancer in women. However, this drug has serious limitations, including lack of tissue-specificity, poor water solubility, and the development of drug resistance. The transport of PTX in a polymeric nanoformulation could overcome these limitations.Methods: In this study, PLGA-PTX nanoparticles (NPs) were assayed in breast cancer cell lines, breast cancer stem cells (CSCs) and multicellular tumor spheroids (MTSs) analyzing cell cycle, cell uptake (Nile Red-NR-) and alpha-tubulin expression. In addition, PLGA-PTX NPs were tested in vivo using C57BL/6 mice, including a biodistribution assay.Results: PTX-PLGA NPs induced a significant decrease in the PTX IC50 of cancer cell lines (1.31 and 3.03-fold reduction in MDA-MB-231 and E0771 cells, respectively) and CSCs. In addition, MISs treated with PTX-PLGA exhibited a more disorganized surface and significantly higher cell death rates compared to free PTX (27.9% and 16.3% less in MRCSs from MCF-7 and E0771, respectively). PTX-PLGA nanoformulation preserved PTX's mechanism of action and increased its cell internalization. Interestingly, PTX-PLG A NI's not only reduced the tumor volume of treated mice hut also increased the antineoplastic drug accumulation in their lungs, liver, and spleen. In addition, mice treated with PTX-loaded NPs showed blood parameters similar to the control mice, in contrast with free PTX.Conclusion: These results suggest that our PTX-PLGA NPs could be a suitable strategy for breast cancer therapy, improving antitumor drug efficiency and reducing systemic toxicity without altering its mechanism of action

Contractile regulatory proteins tropomyosin and troponin-T as indicators of the modulatory role of retinoic acid

Retinoic acid (RA), the active metabolite of vitamin A, plays a significant role in regulating cardiac form and function throughout the life of the organism. Both cardiac morphogenesis and myocardial differentiation are affected by alterations in RA homeostasis. In order to test the effect of all-trans RA and 13-cis RA on cardiomyocyte differentiation, we studied the level and the subcellular compartmentalization of α-tropomyosin and troponin-T proteins in cultures of chick embryo cardiomyocytes obtained from Hamburger and Hamilton’s (HH) stage 22, 32 and 40 embryos. The retinoids increased the levels of α-tropomyosin and troponin-T in the cytoplasmic and cytoskeletal fractions of cells at all three stages of development. The greatest increases in α-tropomyosin occurred in the cytoplasmic fraction in HH22 cells cultured for 24 h with all-trans RA or 13-cis RA, whereas the greatest increases in troponin-T were found in the cytoplasmic fraction of HH32 cells exposed to retinoids for 24 h. In cultures treated for 48 h with retinoids, the levels of α-tropomyosin and troponin-T showed significant increases in the cytoplasmic compartment of cells treated in HH32-with respect to the control values. These findings are further evidence that RA plays a modulating role in the formation and reorganization of sarcomeric proteins during the process of cardiomyocyte maturation

DCN and LUM mRNA/protein expression in GBM and NB CSC-like enrichments.

<p>(A) Neurosphere generation from SF-268 and SK-N-SH cell lines. Bar, 50 μm. (B) Expression of DCN and LUM mRNAs in CSC enrichment cultures. qRT–PCR results were normalized to ACTB and graphed as relative fold changes (FCs) between neurospheres at different time points (t4, t8, and t12) and adherent t₀ cells with the 2^–ΔΔCt method. All DCN and LUM FC values for the neurospheres were statistically significant (*p < 0.01). (C) Western blotting analysis of DCN and LUM. β-Actin was used as the loading control. Total DCN and LUM levels were higher in the neurospheres than in the adherent cells of both cell lines. A 70-kDa LUM isoform was upregulated in the neurospheres of both cell lines, with the highest expression in the t8 neurospheres. Adherent SF-268 cells showed basal expression of the 37-kDa LUM core protein.</p