MiR-182-5p Is Upregulated in Hepatic Tissues from a Diet-Induced NAFLD/NASH/HCC C57BL/6J Mouse Model and Modulates Cyld and Foxo1 Expression.

Non-alcoholic fatty liver disease (NAFLD) is considered a relevant liver chronic disease. Variable percentages of NAFLD cases progress from steatosis to steatohepatitis (NASH), cirrhosis and, eventually, hepatocellular carcinoma (HCC). In this study, we aimed to deepen our understanding of expression levels and functional relationships between miR-182-5p and Cyld-Foxo1 in hepatic tissues from C57BL/6J mouse models of diet-induced NAFL/NASH/HCC progression. A miR-182-5p increase was detected early in livers as NAFLD damage progressed, and in tumors compared to peritumor normal tissues. An in vitro assay on HepG2 cells confirmed Cyld and Foxo1, both tumor-suppressor, as miR-182-5p target genes. According to miR-182-5p expression, decreased protein levels were observed in tumors compared to peritumor tissues. Analysis of miR-182-5p, Cyld and Foxo1 expression levels, based on datasets from human HCC samples, showed results consistent with those from our mouse models, and also highlighted the ability of miR-182-5p to distinguish between normal and tumor tissues (AUC 0.83). Overall, this study shows, for the first time, miR-182-5p overexpression and Cyld-Foxo1 downregulation in hepatic tissues and tumors from a diet-induced NAFLD/HCC mouse model. These data were confirmed by the analysis of datasets from human HCC samples, highlighting miR-182-5p diagnostic accuracy and demonstrating the need for further studies to assess its potential role as a biomarker or therapeutic target.

Novel pyrazolo[3,4-d]pyrimidines as dual Src/Bcr-Abl kinase inhibitors: Synthesis and biological evaluation for chronic myeloid leukemia treatment.

The Bcr-Abl tyrosine kinase (TK) is the molecular hallmark of chronic myeloid leukemia (CML). Src is another TK kinase whose involvement in CML was widely demonstrated. Small molecules active as dual Src/Bcr-Abl inhibitors emerged as effective targeted therapies for CML and a few compounds are currently in clinical use. In this study, we applied a target-oriented approach to identify a family of pyrazolo[3,4-d]pyrimidines as dual Src/Bcr-Abl inhibitors as anti-leukemia agents. Considering the high homology between Src and Bcr-Abl, in-house Src inhibitors 8a-l and new analogue compounds 9a-n were screened as dual Src/Bcr-Abl inhibitors. The antiproliferative activity on K562 CML cells and the ADME profile were determined for the most promising compounds. Molecular modeling studies elucidated the binding mode of the inhibitors into the Bcr-Abl (wt) catalytic pocket. Compounds 8j and 8k showed nanomolar activities in enzymatic and cellular assays, together with favorable ADME properties, emerging as promising candidates for CML therapy. Finally, derivatives 9j and 9k, emerging as valuable inhibitors of the most aggressive Bcr-Abl mutation, T315I, constitute a good starting point in the search for compounds able to treat drug-resistant forms of CML. Overall, this study allowed us to identify more potent compounds than those previously reported by the group, marking a step forward in searching for new antileukemic agents.

The Importance of Tumor Stem Cells in Glioblastoma Resistance to Therapy.

Glioblastoma (GBM) is known to be the most common and lethal primary malignant brain tumor. Therapies against this neoplasia have a high percentage of failure, associated with the survival of self-renewing glioblastoma stem cells (GSCs), which repopulate treated tumors. In addition, despite new radical surgery protocols and the introduction of new anticancer drugs, protocols for treatment, and technical advances in radiotherapy, no significant improvement in the survival rate for GBMs has been realized. Thus, novel antitarget therapies could be used in conjunction with standard radiochemotherapy approaches. Targeted therapy, indeed, may address specific targets that play an essential role in the proliferation, survival, and invasiveness of GBM cells, including numerous molecules involved in signal transduction pathways. Significant cellular heterogeneity and the hierarchy with GSCs showing a therapy-resistant phenotype could explain tumor recurrence and local invasiveness and, therefore, may be a target for new therapies. Therefore, the forced differentiation of GSCs may be a promising new approach in GBM treatment. This article provides an updated review of the current standard and experimental therapies for GBM, as well as an overview of the molecular characteristics of GSCs, the mechanisms that activate resistance to current treatments, and a new antitumor strategy for treating GSCs for use as therapy.

Leptin in Tumor Microenvironment.

Leptin is a hormone that plays a major role as mediator of long-term regulation of energy balance, suppressing food intake, and stimulating weight loss. More recently, important physiological roles other than controlling appetite and energy expenditure have been suggested for leptin, including neuroendocrine, reparative, reproductive, and immune functions. These emerging peripheral roles let hypothesize that leptin can modulate also cancer progression. Indeed, many studies have demonstrated that elevated chronic serum concentrations of leptin, frequently seen in obese subjects, represent a stimulatory signal for tumor growth. Current knowledge indicates that also different non-tumoral cells resident in tumor microenvironment may respond to leptin creating a favorable soil for cancer cells. In addition, leptin is produced also within the tumor microenvironment creating the possibility for paracrine and autocrine action. In this review, we describe the main mechanisms that regulate peripheral leptin availability and how leptin can shape tumor microenvironment.

Extracellular Vesicles: New Endogenous Shuttles for miRNAs in Cancer Diagnosis and Therapy?

Extracellular Vesicles (EVs) represent a heterogeneous population of membranous cell-derived structures, including cargo-oriented exosomes and microvesicles. EVs are functionally associated with intercellular communication and play an essential role in multiple physiopathological conditions. Shedding of EVs is frequently increased in malignancies and their content, including proteins and nucleic acids, altered during carcinogenesis and cancer progression. EVs-mediated intercellular communication between tumor cells and between tumor and stromal cells can modulate, through cargo miRNA, the survival, progression, and drug resistance in cancer conditions. These consolidated suggestions and EVs' stability in bodily fluids have led to extensive investigations on the potential employment of circulating EVs-derived miRNAs as tumor biomarkers and potential therapeutic vehicles. In this review, we highlight the current knowledge about circulating EVs-miRNAs in human cancer and the application limits of these tools, discussing their clinical utility and challenges in functions such as in biomarkers and instruments for diagnosis, prognosis, and therapy.

Crocetin Extracted from Saffron Shows Antitumor Effects in Models of Human Glioblastoma.

Over recent years, many authors discussed the effects of different natural compounds on glioblastoma (GBM). Due to its capacity to impair survival and progression of different cancer types, saffron extract (SE), named crocetin (CCT), is particularly noteworthy. In this work, we elucidated the antitumor properties of crocetin in glioma in vivo and in vitro models for the first time. The in vitro results showed that the four tumor cell lines observed in this study (U251, U87, U138, and U373), which were treated with increasing doses of crocetin, showed antiproliferative and pro-differentiative effects as demonstrated by a significant reduction in the number of viable cells, deep changes in cell morphology, and the modulation of mesenchymal and neuronal markers. Indeed, crocetin decreased the expression of Cluster of Differentiation CD44, CD90, CXCR4, and OCT3/4 mesenchymal markers, but increased the expression of ßIII-Tubulin and neurofilaments (NFH) neuronal linage-related markers. Epigenetic mechanisms may modulate these changes, since Histone Deacetylase, HDAC1 and HDAC3 were downmodulated in U251 and U87 cells, whereas HDAC1 expression was downmodulated in U138 and U373 cells. Western blotting analyses of Fatty Acid Synthase, FASN, and CD44 resulted in effective inhibition of these markers after CCT treatment, which was associated with important activation of the apoptosis program and reduced glioma cell movement and wound repair. The in vivo studies aligned with the results obtained in vitro. Indeed, crocetin was demonstrated to inhibit the growth of U251 and U87 cells that were subcutaneously injected into animal models. In particular, the Tumor To Progression or TTP values and Kaplan-Meier curves indicated that crocetin had more major effects than radiotherapy alone, but similar effects to temozolomide (TMZ). An intra-brain cell inoculation of a small number of luciferase-transfected U251 cells provided a model that was able to recapitulate recurrence after surgical tumor removal. The results obtained from the orthotopic intra-brain model indicated that CCT treatment increased the disease-free survival (DFS) and overall survival (OS) rates, inducing a delay in appearance of a detectable bioluminescent lesion. CCT showed greater efficacy than Radio Therapy (RT) but comparable efficacy to temozolomide in xenograft models. Therefore, we aimed to continue the study of crocetin's effects in glioma disease, focusing our attention on the radiosensitizing properties of the natural compound and highlighting the ways in which this was realized.

Chemically stable inhibitors of 14-3-3 protein-protein interactions derived from BV02.

14-3-3 are regulatory proteins that through protein-protein interactions (PPI) with numerous binding partners could be involved in several human diseases, including cancer, neurodegenerative disorders, and pathogens infections. Following our research interest in the development of 14-3-3 PPI inhibitors, here we exploited the privileged 4-aminoantipyrine scaffold in the design and synthesis of some derivatives endowed with antiproliferative activity against K-562 cells, and capable of binding to recombinant 14-3-3σ as evidenced by NMR spectroscopy. The binding mode was further explored by molecular modelling, while coupling confocal microscopy with intensitometric analysis showed that compound 1 was able to promote the nuclear translocation of c-Abl at low micromolar concentrations. Overall, 1 is chemically stable compared to parent 14-3-3 PPI inhibitors, and thus emerged as a confirmed hit for further development.

Cellular and Molecular Mechanisms Mediated by recPrPC Involved in the Neuronal Differentiation Process of Mesenchymal Stem Cells.

Human Dental Pulp Stem Cells (hDPSCs) represent a type of adult mesenchymal stem cells that have the ability to differentiate in vitro in several lineages such as odontoblasts, osteoblasts, chondrocytes, adipocytes and neurons. In the current work, we used hDPSCs as the experimental model to study the role of recombinant prion protein 23⁻231 (recPrPC) in the neuronal differentiation process, and in the signal pathway activation of ERK 1/2 and Akt. We demonstrated that recPrPC was able to activate an intracellular signal pathway mediated by extracellular-signal-regulated kinase 1 and 2 (ERK 1/2) and protein kinase B (Akt). Moreover, in order to understand whether endogenous prion protein (PrPC) was necessary to mediate the signaling induced by recPrPC, we silenced PrPC, demonstrating that the presence of endogenous PrPC was essential for ERK 1/2 and Akt phosphorylation. Since endogenous PrPC is a well-known lipid rafts component, we evaluated the role of these structures in the signal pathway induced by recPrPC. Our results suggest that lipid rafts integrity play a key role in recPrPC activity. In fact, lipid rafts inhibitors, such as fumonisin B1 and MßCD, significantly prevented ERK 1/2 and Akt phosphorylation induced by recPrPC. In addition, we investigated the capacity of recPrPC to induce hDPSCs neuronal differentiation process after long-term stimulation through the evaluation of typical neuronal markers expression such as B3-Tubulin, neurofilament-H (NFH) and growth associated protein 43 (GAP43). Accordingly, when we silenced endogenous PrPC, we observed the inhibition of neuronal differentiation induced by recPrPC. The combined data suggest that recPrPC plays a key role in the neuronal differentiation process and in the activation of specific intracellular signal pathways in hDPSCs.

Dual PI3 K/mTOR inhibition reduces prostate cancer bone engraftment altering tumor-induced bone remodeling.

Morbidity in advanced prostate cancer patients is largely associated with bone metastatic events. The development of novel therapeutic strategies is imperative in order to effectively treat this incurable stage of the malignancy. In this context, Akt signaling pathway represents a promising therapeutic target able to counteract biochemical recurrence and metastatic progression in prostate cancer. We explored the therapeutic potential of a novel dual PI3 K/mTOR inhibitor, X480, to inhibit tumor growth and bone colonization using different in vivo prostate cancer models including the subcutaneous injection of aggressive and bone metastatic (PC3) and non-bone metastatic (22rv1) cell lines and preclinical models known to generate bone lesions. We observed that X480 both inhibited the primary growth of subcutaneous tumors generated by PC3 and 22rv1 cells and reduced bone spreading of PCb2, a high osteotropic PC3 cell derivative. In metastatic bone, X480 inhibited significantly the growth and osteolytic activity of PC3 cells as observed by intratibial injection model. X480 also increased the bone disease-free survival compared to untreated animals. In vitro experiments demonstrated that X480 was effective in counteracting osteoclastogenesis whereas it stimulated osteoblast activity. Our report provides novel information on the potential activity of PI3 K/Akt inhibitors on the formation and progression of prostate cancer bone metastases and supports a biological rationale for the use of these inhibitors in castrate-resistant prostate cancer patients at high risk of developing clinically evident bone lesions.

Expression of Peroxisome Proliferator-Activated Receptor Alpha (PPARα) in Non-Somatotroph Pituitary Tumours and the Effects of PPARα Agonists on MMQ Cells.

Peroxisome proliferator-activated receptor alpha (PPARα) has been involved in the regulation of somatotroph tumour cells and may be targeted by different drugs, some of them are in current clinical use. The aim of this study was to investigate the expression of PPARα in additional phenotypes of pituitary adenomas (PA), the relationship between PPARα and its potential molecular partner aryl hydrocarbon receptor interacting protein (AIP) in these tumours, and the effects of PPARα agonists on lactotroph cells. Seventy-five human PA - 57 non-functioning (NFPA) and 18 prolactinomas (PRL-PA) - were characterised for PPARα and AIP expression by real time RT-PCR and/or immunohistochemistry (IHC), and the effects of fenofibrate and WY 14 643 on MMQ cells were studied in vitro. PPARα was expressed in a majority of PA. PPARα immunostaining was observed in 93.7% PRL-PA vs. 60.6% NFPA (p=0.016), the opposite being found for AIP (83.3% in NFPA vs. 43.7% in PRL-PA, p=0.003). PPARα expression was unrelated to gonadotroph differentiation in NFPA, but positively correlated with tumour volume in PRL-PA. Both drugs significantly reduced MMQ cell growth at high concentrations (100-200 µM). At the same time, despite modest stimulating effects on PRL secretion were observed, these were overcome by the reduction in cell number. In conclusion, PPARα is commonly expressed by PRL-PA and NFPA, regardless of AIP, and may represent a new target of PPARα agonists.

Efficient optimization of pyrazolo[3,4-d]pyrimidines derivatives as c-Src kinase inhibitors in neuroblastoma treatment.

The proto-oncogene c-Src is a non-receptor tyrosine kinase which is involved in the regulation of many cellular processes, such as differentiation, adhesion and survival. c-Src hyperactivation has been detected in many tumors, including neuroblastoma (NB), one of the major causes of death from neoplasia in infancy. We already reported a large family of pyrazolo[3,4-d]pyrimidines active as c-Src inhibitors. Interestingly, some of these derivatives resulted also active on SH-SY5Y NB cell line. Herein, starting from our previous Free Energy Perturbation/Monte Carlo calculations, we report an optimization study which led to the identification of a new series of derivatives endowed with nanomolar Ki values against c-Src, interesting antiproliferative activity on SH-SY5Y cells and a suitable ADME profile.

"Vessels in the Storm": Searching for Prognostic and Predictive Angiogenic Factors in Colorectal Cancer.

High expectations are placed upon anti-angiogenic compounds for metastatic colorectal cancer (mCRC), the first malignancy for which such type of treatment has been approved. Indeed, clinical trials have confirmed that targeting the formation of new vessels can improve in many cases clinical outcomes of mCRC patients. However, current anti-angiogenic drugs are far from obtaining the desirable or expected curative results. Many are the factors probably involved in such disappointing results, but particular attention is currently focused on the validation of biomarkers able to improve the direction of treatment protocols. Because clinical studies have clearly demonstrated that serum or tissue concentration of some angiogenic factors is associated with the evolution of the disease of mCRC patients, they are currently explored as potential biomarkers of prognosis and of tumor response to therapy. However, the complex biology underlying CRC -induced angiogenesis is a hurdle in finding rapid solutions. The aim of this review was to explore molecular mechanisms that determine the formation of tumor-associated vessels during CRC progression, and to discuss the potential role of angiogenic factors as diagnostic, prognostic and predictive biomarkers in CRC.

Optimization of Aminoimidazole Derivatives as Src Family Kinase Inhibitors.

Protein kinases have emerged as crucial targets for cancer therapy over the last decades. Since 2001, 40 and 39 kinase inhibitors have been approved by FDA and EMA, respectively, and the majority are antineoplastic drugs. Morevoer, many candidates are currently in clinical trials. We previously reported a small library of 4-aminoimidazole and 2-aminothiazole derivatives active as Src family kinase (SFK) inhibitors. Starting from these results, we decided to perform an optimization study applying a mix and match strategy to identify a more potent generation of 4-aminoimidazoles. Firstly, a computational study has been performed, then compounds showing the best predicted docking scores were synthesized and screened in a cell-free assay for their SFK inhibitory activity. All the new chemical entities showed IC50s in the nanomolar range, with 2⁻130 fold increased activities compared to the previously reported inhibitors. Finally, the most active compounds have been tested on three cancer cell lines characterized by Src hyperactivation. Compounds 4k and 4l showed an interesting antiproliferative activity on SH-SY5Y neuroblastoma (NB) cell line. In this assay, the compounds resulted more potent than dasatinib, a tyrosine kinase inhibitor approved for the treatment of leukemias and in clinical trials for NB.

Non-conventional role of haemoglobin beta in breast malignancy.

BACKGROUND: Besides its role as oxygen transporter, recent findings suggest that haemoglobin beta (HBB) may have roles in other contexts. METHODS: We evaluated the impact of HBB expression in primary human breast cancers, and in breast cancer cell lines overexpressing HBB by in vitro and in vivo studies. Publicly available microarray databases were used to perform multivariate survival analyses. RESULTS: A significantly higher expression of HBB was observed in invasive carcinoma histotypes vs in situ counterparts, along with a positive correlation between HBB and the Ki67 proliferation marker. HBB-overexpressing breast cancer cells migrate and invade more, show HIF-1α upregulation and their conditioned media enhances angiogenesis. Blocking the oxygen-binding site of HBB reverts the increase of migration and HIF-1α upregulation observed in HBB-overexpressing breast cancer cells. Orthotopically implanted MDA-MB-231 overexpressing HBB (MDA-HBB) generated tumours with faster growth rate and increased neoangiogenesis. Moreover, local recurrence and visceral metastases were observed only in MDA-HBB-implanted mice. Similar results were observed with 4T1 mouse breast cancer cells. Finally, bioinformatics analyses of public data sets correlated high HBB expression with lower overall survival. CONCLUSIONS: HBB expression increases breast cancer cells aggressiveness and associates with poor prognosis, pointing to HBB as a novel biomarker for breast cancer progression.

Adipose-derived stem cells sustain prolonged angiogenesis through leptin secretion.

Recent studies suggest that adipose-derived stem cells (ASCs) play a role in tissue remodeling through the release of cytokines and growth factors. We compared the secreted cytokine profile of hypoxia-conditioned ASCs (hASCs) with normoxic ASCs (nASCs) and we analyzed the effect of ASCs conditioned medium (CM) on endothelial cells. We found that hypoxia induced a transient upregulation of VEGF in ASCs and a notable and enduring upregulation of leptin mRNA expression 30-fold greater than control after 24 h and up to 60-fold greater than control at day 7. CM from hASC stimulated EC tube formation to a significantly greater extent than CM from nASC. This might be due to leptin-secreted factor. Indeed, exogenous leptin stimulated the expression of HIF2-α, but not HIF1-α, and upregulated the expression of Flt-1 and Tie-1 proangiogenic receptors. In conclusion, hASCs may be particularly efficient in sustaining angiogenesis through the release of leptin.

Src inhibition potentiates antitumoral effect of paclitaxel by blocking tumor-induced angiogenesis.

The protein kinase Src is frequently over-activated in advanced cancers where it modulates the signaling transduction cascade of several growth factors. The feasibility of combination treatment of Src inhibitors with chemotherapy is currently under investigation. We evaluated the anti-tumoral effect of paclitaxel (PTX) in combination with S13, a tyrosine kinase inhibitor with a prevalent specificity for Src, in a hormone-insensible prostate cancer (PCa) cell model. In vivo, combination treatment with PTX and S13 reduced dramatically PCa tumor growth with a relevant difference in the density of new blood vessels with respect to control and single treatments. This reduction was determined by a concomitant impairment of endothelial cell migration and of VEGF release by cancer cells. In fact, S13, when used alone, was sufficient to reduce tubule formation in vivo, and to inhibit VEGFR2 activation and FAK expression in endothelial cells. In addition, the combination treatment determined a significant reduction in ROS production and HIF-1 stabilization in PCa cells respect to single treatments with S13 or PTX. In conclusion, Src-inhibition could be an effective therapeutic strategy aimed at supporting the anti-angiogenic action of PTX in aggressive PCa.

Tumor-stroma metabolic relationship based on lactate shuttle can sustain prostate cancer progression.

BACKGROUND: Cancer cell adopts peculiar metabolic strategies aimed to sustain the continuous proliferation in an environment characterized by relevant fluctuations in oxygen and nutrient levels. Monocarboxylate transporters MCT1 and MCT4 can drive such adaptation permitting the transport across plasma membrane of different monocarboxylic acids involved in energy metabolism. METHODS: Role of MCTs in tumor-stroma metabolic relationship was investigated in vitro and in vivo using transformed prostate epithelial cells, carcinoma cell lines and normal fibroblasts. Moreover prostate tissues from carcinoma and benign hypertrophy cases were analyzed for individuating clinical-pathological implications of MCT1 and MCT4 expression. RESULTS: Transformed prostate epithelial (TPE) and prostate cancer (PCa) cells express both MCT1 and MCT4 and demonstrated variable dependence on aerobic glycolysis for maintaining their proliferative rate. In glucose-restriction the presence of L-lactate determined, after 24 h of treatment, in PCa cells the up-regulation of MCT1 and of cytochrome c oxidase subunit I (COX1), and reduced the activation of AMP-activated protein kinase respect to untreated cells. The blockade of MCT1 function, performed by si RNA silencing, determined an appreciable antiproliferative effect when L-lactate was utilized as energetic fuel. Accordingly L-lactate released by high glycolytic human diploid fibroblasts WI-38 sustained survival and growth of TPE and PCa cells in low glucose culture medium. In parallel, the treatment with conditioned medium from PCa cells was sufficient to induce glycolytic metabolism in WI-38 cells, with upregulation of HIF-1a and MCT4. Co-injection of PCa cells with high glycolytic WI-38 fibroblasts determined an impressive increase in tumor growth rate in a xenograft model that was abrogated by MCT1 silencing in PCa cells. The possible interplay based on L-lactate shuttle between tumor and stroma was confirmed also in human PCa tissue where we observed a positive correlation between stromal MCT4 and tumor MCT1 expression. CONCLUSIONS: Our data demonstrated that PCa progression may benefit of MCT1 expression in tumor cells and of MCT4 in tumor-associated stromal cells. Therefore, MCTs may result promising therapeutic targets in different phases of neoplastic transformation according to a strategy aimed to contrast the energy metabolic adaptation of PCa cells to stressful environments.

In-vitro Approaches to Investigate the Detrimental Effect of Light on Dopaminergic Neurons.

Our recent study revealed that fluorescent lamp light can penetrate deep into the brain of mice and rats leading to the development of typical histological characteristics associated with Parkinson's disease such as the loss of dopamine neurons in the substantia nigra. Monochromatic LED lights were thus used in this work to deepen our knowledge on the effects of the major wavelength peaks of fluorescent light on mouse and human dopaminergic cells. In particular, we exposed immortalized dopaminergic MN9D neuronal cells, primary cultures of mouse mesencephalic dopaminergic cells and human dopaminergic neurons differentiated from induced pluripotent stem cells (hiPSC) to different LED light wavelengths. We found that chronic exposure to LED light reduced overall undifferentiated MN9D cell number, with the most significant effects observed at wavelengths of 485 nm and 610 nm. Moreover, LED light especially at 610 nm was able to negatively impact on the survival of mouse mesencephalic dopaminergic cells and of human dopaminergic neurons derived from hiPSC. Notably, differentiated MN9D dopaminergic cells, which closely resemble mature dopamine neuronal phenotype, acutely exposed for 3 h at 610 nm, showed a clear increase in ROS production and cytotoxicity compared to controls undifferentiated MN9D cells. These increases were even more pronounced by the co-treatment with the oxidative agent H2O2. Collectively, these findings suggest that specific wavelengths, particularly those capable of penetrating deep into the brain, could potentially pose an environmental hazard in relation to Parkinson's disease.

Comparative Analysis of Dasatinib Effect between 2D and 3D Tumor Cell Cultures.

Three-dimensional cell culture methods are able to confer new predictive relevance to in vitro tumor models. In particular, the 3D multicellular tumor spheroids model is considered to better resemble tumor complexity associated with drug resistance compared to the 2D monolayer model. Recent advances in 3D printing techniques and suitable biomaterials have offered new promises in developing 3D tissue cultures at increased reproducibility and with high-throughput characteristics. In our study, we compared the sensitivity to dasatinib treatment in two different cancer cell lines, prostate cancer cells DU145 and glioblastoma cells U87, cultured in the 3D spheroids model and in the 3D bioprinting model. DU145 and U87 cells were able to proliferate in 3D alginate/gelatin bioprinted structures for two weeks, forming spheroid aggregates. The treatment with dasatinib demonstrated that bioprinted cells were considerably more resistant to drug toxicity than corresponding cells cultured in monolayer, in a way that was comparable to behavior observed in the 3D spheroids model. Recovery and analysis of cells from 3D bioprinted structures led us to hypothesize that dasatinib resistance was dependent on a scarce penetrance of the drug, a phenomenon commonly reported also in spheroids. In conclusion, the 3D bioprinted model utilizing alginate/gelatin hydrogel was demonstrated to be a suitable model in drug screening when spheroid growth is required, offering advantages in feasibility, reproducibility, and scalability compared to the classical 3D spheroids model.

Mitotic phosphorylation of Tau/MAPT modulates cell cycle progression in prostate cancer cells.

PURPOSE: Tau/MAPT (microtubule associated protein tau) protein is actively studied for the pathologic consequences of its aberrant proteostasis in central nervous system leading to neurodegenerative diseases. Besides its ability to generate insoluble toxic oligomers, Tau homeostasis has attracted attention for its involvement in the formation of the mitotic spindle. This evidence, in association with the description of Tau expression in extra-neuronal tissues, and mainly in cancer tissues, constitutes the rationale for a more in-depth investigation of Tau role also in neoplastic diseases. METHODS: In our study, we investigated the expression of phosphorylated Tau in prostate cancer cell lines with particular focus on the residue Thr231 present in microtubule binding domain. RESULTS: The analysis of prostate cancer cells synchronized with nocodazole demonstrated that the expression of Tau protein phosphorylated at residue Thr231 is restricted to G2/M cell cycle phase. The phosphorylated form was unable to bind tubulin and it does not localize on mitotic spindle. As demonstrated by the use of specific inhibitors, the phosphorylation status of Tau is under the direct control of cdk5 and PP2A, while cdk1 activation was able to exert an indirect control. These mechanisms were also active in cells treated with docetaxel, where counteracting the expression of the dephosphorylated form, by kinase inhibition or protein silencing, determined resistance to drug toxicity. CONCLUSIONS: We hypothesize that phosphorylation status of Tau is a key marker for G2/M phase in prostate cancer cells and that the forced modulation of Tau phosphorylation can interfere with the capacity of cell to efficiently progress through G2/M phase.