Fibronectin and type IV collagen activate ERα AF-1 by c-Src pathway: effect on breast cancer cell motility

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Controlled Release of 5-FU from Chi–DHA Nanoparticles Synthetized with Ionic Gelation Technique: Evaluation of Release Profile Kinetics and Cytotoxicity Effect

The ionic gelation technique allows us to obtain nanoparticles able to function as carriers for hydrophobic anticancer drugs, such as 5-fluoruracil (5-FU). In this study, reticulated chitosan– docosahexaenoic acid (Chi–DHAr) nanoparticles were synthesized by using a chemical reaction between amine groups of chitosan (Chi) and carboxylic acids of docosahexaenoic acid (DHA) and the presence of a link between Chi and DHA was confirmed by FT-IR, while the size and morphology of the obtained Chi-DHAr nanoparticles was evaluated with dynamic light scattering (DLS) and scanning electron microscopy (SEM), respectively. Drug-loading content (DLC) and drug-loading efficiency (DLE) of 5-FU in Chi-DHAr nanoparticles were 33.74 ± 0.19% and 7.9 ± 0.26%, respectively, while in the non-functionalized nanoparticles (Chir + 5FU), DLC, and DLE were in the ranges of 23.73 ± 0.14%, 5.62%, and 0.23%, respectively. The in vitro release profile, performed in phosphate buffer saline (PBS, pH 7.4) at 37 °C, indicated that the synthetized Chi–DHAr nanoparticles provided a sustained release of 5-FU. Based on the obtained regression coefficient value (R2), the first order kinetic model provided the best fit for both Chir and Chi-DHAr nanoparticles. Finally, cytotoxicity studies of chitosan, 5-FU, Chir, Chir + 5-FU, Chi-DHAr, and Chi-DHAr + 5-FU nanoparticles were conducted. Overall, Chi-DHAr nanoparticles proved to be much more biocompatible than Chir nanoparticles while retaining the ability to release the drug with high efficiency, especially towards specific types of cancerous cells

FoxO3a Drives the Metabolic Reprogramming in Tamoxifen-Resistant Breast Cancer Cells Restoring Tamoxifen Sensitivity

Tamoxifen-resistant breast cancer cells (TamR-BCCs) are characterized by an enhanced metabolic phenotype compared to tamoxifen-sensitive cells. FoxO3a is an important modulator of cell metabolism, and its deregulation has been involved in the acquisition of tamoxifen resistance. Therefore, tetracycline-inducible FoxO3a was overexpressed in TamR-BCCs (TamR/TetOn-AAA), which, together with their control cell line (TamR/TetOn-V), were subjected to seahorse metabolic assays and proteomic analysis. FoxO3a was able to counteract the increased oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) observed in TamR by reducing their energetic activity and glycolytic rate. FoxO3a caused glucose accumulation, very likely by reducing LDH activity and mitigated TamR biosynthetic needs by reducing G6PDH activity and hindering NADPH production via the pentose phosphate pathway (PPP). Proteomic analysis revealed a FoxO3a-dependent marked decrease in the expression of LDH as well as of several enzymes involved in carbohydrate metabolism (e.g., Aldolase A, LDHA and phosphofructokinase) and the analysis of cBioPortal datasets of BC patients evidenced a significant inverse correlation of these proteins and FoxO3a. Interestingly, FoxO3a also increased mitochondrial biogenesis despite reducing mitochondrial functionality by triggering ROS production. Based on these findings, FoxO3a inducing/activating drugs could represent promising tools to be exploited in the management of patients who are refractory to antiestrogen therapy

<<The>> estrogen receptor α is the key regulator of the bifunctional role of FoxO3a transcription factor in breast cancer motility and invasiveness

Dottorato di Ricerca in Biochimica Cellulare ed Attività dei Farmaci in Oncologia, XXVI Ciclo,a.a. 2012-2013The role of the Forkhead box class O (FoxO)3a transcription factor in breast cancer migration and invasion is controversial. Here we show that FoxO3a overexpression decreases motility, invasiveness, and anchorage-independent growth in estrogen receptor α-positive (ERα+) cancer cells while eliciting opposite effects in ERα-silenced cells and in ERα-negative (ERα−) cell lines, demonstrating that the nuclear receptor represents a crucial switch in FoxO3a control of breast cancer cell aggressiveness. In ERα+ cells, FoxO3a-mediated events were paralleled by a significant induction of Caveolin-1 (Cav1), an essential constituent of caveolae negatively associated to tumor invasion and metastasis. Cav1 induction occurs at the transcriptional level through FoxO3a binding to a Forkhead responsive core sequence located at position −305/−299 of the Cav1 promoter. 17β-estradiol (E2) strongly emphasized FoxO3a effects on cell migration and invasion, while ERα and Cav1 silencing were able to reverse them, demonstrating that both proteins are pivotal mediators of these FoxO3a controlled processes. In vivo, an immunohistochemical analysis on tissue sections from patients with ERα+ or ERα− invasive breast cancers or in situ ductal carcinoma showed that nuclear FoxO3a inversely (ERα+) or directly (ERα−) correlated with the invasive phenotype of breast tumors. In conclusion, FoxO3a role in breast cancer motility and invasion depends on ERα status, disclosing a novel aspect of the well-established FoxO3a/ERα interplay. Therefore FoxO3a might become a pursuable target to be suitably exploited in combination therapies either in ERα+ or ERα− breast tumors.Università della Calabri

Estrogens modulate the expression of the deleted gene homolog phosphatase and tensin on chromosome 10 (PTEN) in human seminoma cells TCam2

Dottorato di Ricerca in Biochimica Cellulare ed Attività dei Farmaci in Oncologia, XXIII Ciclo,2010-2011Università della Calabri

<<A>> cross- talk between estrogens and IGF1R pathways controls leydig and adrenocortical tumor cell proliferation

Dottorato di Ricerca in Biochimica Cellulare ed Attività dei Farmaci in Oncologia, XXIV Ciclo, a.a. 2010-2011Università della Calabri

PPARγ ligands as novel agents able to inhibit breast tumor growth and progression

Dottorato di Ricerca in “Biochimica Cellulare ed Attività dei Farmaci in Oncologia” , XXIV Ciclo, a.a. 2010-2011Università della Calabri

Study of autophagic and epigenetic mechanisms in experimental models of inflammatory and neuropathic pain for the identification of new pharmacological targets

Dottorato di Ricerca in Biochimica Cellulare ed Attività dei Farmaci in Oncologia, XXVI Ciclo, SSD, a.a. 2013Pain is defined by IASP as “an unpleasant sensory and emotional experience associated with actual or potential tissue damage, or described in terms of such damage” (IASP, 2011). While physiological pain is like a warning system, useful to prevent damage to the organism, pathological pain is an unpleasant sensation, permanent also after damage and it is characterized by an enhanced sensitivity to both innocuous and noxious stimuli (termed allodynia and hyperalgesia, respectively). While acute pain resolves in few days, chronic pain lasts longer than three/six months. Neuropathic pain, a common form of chronic pain, was defined as “pain caused by a lesion or disease of the somatosensory nervous system” (IASP, 2011). Pharmacological treatments available, including tricycles antidepressant and gabapentin, have limited efficacy in most of patients (Childers et al, 2007). Therefore, a better understanding of pain physiopathology and the development of new treatments are very important. Here, we characterised two new molecular mechanisms, autophagy and epigenetic mechanisms, and their role in pain processing. Autophagy is the main mechanisms involved in the degradation of proteins and organelles, in cell remodelling and survival during periods of nutrient deficiency. The decrease in the autophagic activity seems to interfere with the degradation of proteins and with the turnover of nutrients, while a greater activation of this pathway appears to facilitate the clearance of protein aggregates and to promote neuronal survival in various neurodegenerative diseases. On the other hand, too high autophagic activity can be detrimental and lead to cell death, suggesting that the regulation of autophagy has an important role in determining cell fate. However, despite numerous studies on the role of autophagy in neurodegenerative diseases, the role of this process in the pathophysiology of neuropathic pain remains poorly studied. Epigenetic mechanisms are chemical modifications of chromatin that influence gene expression without altering the DNA sequence. Although in recent years scientific research has produced significant results in the epigenetics field, only few studies have focused on the involvement of epigenetic mechanisms in relation to pain states. Experimental evidence suggests that changes in the expression of some genes are involved in the early stages of induction and maintenance of chronic pain states. Among these genes, recent evidence suggests a role for the FKBP5 gene, an important regulator of the glucocorticoid receptor, involved in the regulation system of the stress response. In addition, recent studies show that this gene is under strong epigenetic control. In view of this, the objectives of this research were: • To characterise the autophagic process at spinal cord level in different experimental models of neuropathic and inflammatory pain; • To verify the relevance of spinal autophagy for pain processing; • To identify pain conditions in which the gene FKBP5 plays a role; • To study the role of FKBP5 on pain processing at spinal cord level; • To characterize the enzymes involved in DNA methylation; The results obtained in the first experimental part of this thesis showed a modulation of the main autophagic markers in experimental models of neuropathic pain. In particular, in the model that involves the ligation of the L5 spinal nerve (SNL) and in the model that involves the transection of the tibial nerve and peroneal (SNI), it was observed an increase in the levels of the associated form of the protein LC3 (LC3II ) and of protein p62 , which is involved in the early stages of degradation of the autophagic process. The observed increase in p62 protein levels suggested a possible impairment of autophagic flux. To verify this hypothesis the consequences of a local block of autophagy at spinal level were investigated on pain behaviour. In particular, the treatment of naïve animals with chloroquine, a lisosomal inhibitor, resulted in the establishment of a state of hyperalgesia typically observed after peripheral damage of the spinal nerves. The results obtained in the second experimental part demonstrate an involvement of the gene FKBP5 in the induction and in the maintenance phases of chronic pain. In particular, knockout animals have shown a lower sensitivity to mechanical stimuli following the onset of various chronic pain states. The silencing of the gene at the spinal cord level has allowed us to understand the role of the gene FKBP5 in pain processing after an injury. Finally, the study and characterization of DNMT1, the enzyme involved in DNA methylation, has allowed us to suggest the active involvement of other proteins in the process of DNA demethylation and then in the expression of genes. In conclusion, the data reported in this study indicate an impairment of autophagy in experimental models of neuropathic pain, supporting the neuroprotective role of this process in the spinal cord. It was also demonstrated the involvement of the gene FKBP5 in the induction and in the maintenance phases of chronic pain. Altogether, these data pave the way to further investigations aimed to a better understanding of the mechanisms underling chronic pain and to the identification of potential molecular targets for the development of new therapeutic strategiesUniversità della Calabri

Role of PPAR γ in breast cancer cell death and in adipocyte differentiation

Dottorato di Ricerca in Biochimica Cellulare ed Attività dei Farmaci in Oncologia, XXVI Ciclo,a.a. 2013Università della Calabri