Targeting BMI-1 as an innovative approach to overcome cancer multi drug resistance

Aging is among the major risk factors for the development of cancer and the main cause of cancer relapse and therapeutic failure is due to the onset of chemoresistance. Since many anticancer therapies cause cell death by inducing oxidative stress, one of the mechanisms activated by cancer cells to adapt and survive to the cytotoxic effect of therapeutic agents, is to stimulate the antioxidant defense. In this regard, previous studies from the research group of my PhD tutor, showed that chronic exposure of cancer cells (HTLA-230) to the chemotherapeutic drug etoposide, at a dose comparable to that used in clinic, leads to the selection of a resistant cell line (HTLA-ER). Interestingly, resistant cells are characterized by efficient oxidative metabolism, display high amounts of glutathione (GSH) and overexpress the oncogenic protein BMI-1, which is capable of regulating GSH levels and is associated with cancer stemness, epithelial-mesenchymal transition (EMT), tumor invasion, metastasis and poor prognosis. Notably, both cell lines exhibit the same p53 mutation and no changes in the expression of MDM2, the endogenous inhibitor of p53. Based on this background, the aim of this thesis is to investigate new strategies that can circumvent cancer chemoresistance, mainly by reducing intracellular GSH content and reactivating mutated p53. Therefore, the effects of sulfasalazine, an xCT inhibitor, of PRIMA-1MET, a compound able to reactivate p53 functions, and of PTC596, a novel BMI-1 inhibitor, were investigated. Firstly, it has been confirmed that HTLA-ER cells are multidrug-resistant, being unaffected by the cytotoxic effects of different chemotherapeutic drugs, and not only by etoposide. Then, it has been found that PRIMA-1MET promotes apoptosis only in parental cells, whereas PTC596 does not trigger apoptosis in both cell populations. In addition, PRIMA-1MET and PTC596 inhibited the clonogenic potential of parental and resistant cells. Since sulfasalazine alone or combined was not effective on resistant cells it was excluded from the subsequent experiments. The evaluation of the expression of p53 and BMI-1, target proteins of the two tested drugs, showed that PRIMA-1MET does not modulate p53 protein levels, while PTC596 is able to significantly reduce the expression of BMI-1. Furthermore, given that EMT is involved in chemoresistance, EMT-related proteins were evaluated. In detail, PRIMA-1MET and, most importantly, PTC596 reduced the expression of n-cadherin, b-catenin and SNAIL, demonstrating an inhibition of EMT process. In addition, both compounds prevented the formation of cancer stem cells. Analysis of cellular redox state showed that treatments with PRIMA-1MET of parental cells and with PTC596 of both cell lines induced overproduction of reactive oxygen species and increased membrane lipid peroxidation. With regard to antioxidant response, it has been observed that i) resistant cells expressed a double GSH amount compared to that of parental cells and ii) PRIMA-1MET and, more markedly, PTC596 were able to deplete the thiol antioxidant content. These promising findings certainly demonstrate the involvement of cellular redox state in chemoresistance, and suggest that PTC596, the most effective compound in resistant cells, can induce ferroptosis, a cell death characterized by membrane lipid peroxidation and reduction of GSH levels. In fact, the co-treatment of PTC596 with compounds capable of inducing or inhibiting ferroptosis, confirmed the induction of this type of cell death. Therefore, an approach targeting BMI-1 and its related pathways could be a winning strategy to overcome cancer resistance to therapy

Synthesis of Polystyrene-Based Cationic Nanomaterials with Pro-Oxidant Cytotoxic Activity on Etoposide-Resistant Neuroblastoma Cells

Drug resistance is a multifactorial phenomenon that limits the action of antibiotics and chemotherapeutics. Therefore, it is essential to develop new therapeutic strategies capable of inducing cytotoxic effects circumventing chemoresistance. In this regard, the employment of natural and synthetic cationic peptides and polymers has given satisfactory results both in microbiology, as antibacterial agents, but also in the oncological field, resulting in effective treatment against several tumors, including neuroblastoma (NB). To this end, two polystyrene-based copolymers (P5, P7), containing primary ammonium groups, were herein synthetized and tested on etoposide-sensitive (HTLA-230) and etoposide-resistant (HTLA-ER) NB cells. Both copolymers were water-soluble and showed a positive surface charge due to nitrogen atoms, which resulted in protonation in the whole physiological pH range. Furthermore, P5 and P7 exhibited stability in solution, excellent buffer capacity, and nanosized particles, and they were able to reduce NB cell viability in a concentration-dependent way. Interestingly, a significant increase in reactive oxygen species (ROS) production was observed in both NB cell populations treated with P5 or P7, establishing for both copolymers an unequivocal correlation between cytotoxicity and ROS generation. Therefore, P5 and P7 could be promising template macromolecules for the development of new chemotherapeutic agents able to fight NB chemoresistance

Physicochemical Characterization of two Cationic Copolymers Effective on Etoposide-Sensitive and Resistant Neuroblastoma Cells

New therapeutic agents as antibiotics and chemotherapeutics capable of inducing cytotoxic effects and employed in the treatment of lethal human diseases as cancer and infections need also to circumvent the increasing chemoresistance, which limits their action. Natural and synthetic cationic peptides and polymers have given appealing results both in microbiology, and in the oncological field, where they resulted effective against several tumors, including human neuroblastoma (NB) [1,2]. To this end, we recently synthetized two polystyrene-based cationic copolymers (P5 and P7), which proved a potent ROS-related cytotoxicity against both etoposide-sensitive (HTLA-230) and -resistant (HTLA-ER) NB cells [3]. Interestingly, the cytotoxic effects of P5 and P7 were even higher on HTLA-ER cells, thus proving that they could be promising template macromolecules for the development of new chemotherapeutic agents able to fight NB chemoresistance.Water-solubility, surface charge, protonation profile in the physiological pH range, Z-potential, polydispersity index, buffer capacity, and particles size are pivotal features for the feasibility of biomedical application of new bioactive macromolecules. In our poster, in addition to show the spectroscopic characterization of P5 and P7, we have reported their complete physicochemical characterization. [1] L.T. Eliassen, et al. Int. J. Cancer. 2006, 119, 493\u2013500. [2] J. Tan, et al. Biomat. 2020, 252, 120078. [3] S. Alfei, et al. Nanomaterials 2021, 11, 977

Cytotoxic Activity of Dendrimer Nanoparticles and Dendrimer Drugs Formulations on Human Neuroblastoma Cells: Our Recent Update

Human neuroblastoma (NB) is a pediatric tumor, which, after an initial response to therapy, usually develops resistance. Etoposide (ETO) which is a drug commonly used to clinically treat NB, exerts anticancer effects by increasing reactive oxygen species (ROS) generation [1,2]. Similarly, gallic acid (GA), although not specifically in NB treatment, exerts pro-oxidant anti-cancer effects associated to low toxicity for healthy cells. Unfortunately, low stability, poor solubility and an unfavorable pharmacokinetic negatively influence ETO and GA efficacy [1, 2]. To address GA and ETO issues, biodegradable dendrimer nanoparticles (DNPs) were prepared for entrapping ETO [2], as well as for encapsulating and covalently binding GA, obtaining the drugs-loaded dendrimers ETOD, GALD and GAD [1, 2]. The cytotoxic activity of DNPs, GA, ETOD, GALD and GAD was tested on ETO-sensitive and ETO-resistant NB cells. Unexpectedly, DNPs were able to exert per se a ROS-mediated cytotoxic activity comparable to ETO, on both cell populations. ETOD, combining DNPs and ETO, showed a synergistic action of the two molecules, a slow release of the drug and a significantly improved protracted bioactivity [2]. Free GA proved a dose-dependent ROS-mediated cytotoxicity on both cell populations, but intriguingly, when administered in dendrimer formulations, at a dose not cytotoxic for NB cells, nullified any pro-oxidant activity of DNPs [1]. Collectively, DNPs could represent a platform to develop novel devices against NB, while ETOD could be a biodegradable device for the efficient delivery of ETO into NB cells. GALD and GAD, due to the presence of GA, were inactive on NB cells, but GA resized in nanoparticles and at very low dose has shown considerable ability in counteracting ROS production induced by DNPs, thereby exerting a possible protective action for healthy cells

Imidazo-Pyrazole-Loaded Palmitic Acid and Polystyrene-Based Nanoparticles: Synthesis, Characterization and Antiproliferative Activity on Chemo-Resistant Human Neuroblastoma Cells

: Neuroblastoma (NB) is a childhood cancer, commonly treated with drugs, such as etopo side (ETO), whose efficacy is limited by the onset of resistance. Here, aiming at identifying new treatments for chemo-resistant NB, the effects of two synthesized imidazo-pyrazoles (IMPs) (4G and 4I) were investigated on ETO-sensitive (HTLA-230) and ETO-resistant (HTLA-ER) NB cells, detecting 4I as the more promising compound, that demonstrated IC50 values lower than those of ETO on HTLA ER. Therefore, to further improve the activity of 4I, we developed 4I-loaded palmitic acid (PA) and polystyrene-based (P5) cationic nanoparticles (P5PA-4I NPs) with high drug loading (21%) and encapsulation efficiency (97%), by a single oil-in-water emulsification technique. Biocompatible PA was adopted as an emulsion stabilizer, while synthesized P5 acted as an encapsulating agent, solu bilizer and hydrophilic–lipophilic balance (HLB) improver. Optic microscopy and cytofluorimetric analyses were performed to investigate the micromorphology, size and complexity distributions of P5PA-4I NPs, which were also structurally characterized by chemometric-assisted Fourier transform infrared spectroscopy (FTIR). Potentiometric titrations allowed us to estimate the milliequivalents of PA and basic nitrogen atoms present in NPs. P5PA-4I NPs afforded dispersions in water with excellent buffer capacity, essential to escape lysosomal degradation and promote long residence time inside cells. They were chemically stable in an aqueous medium for at least 40 days, while in dynamic light scattering (DLS) analyses, P5PA-4I showed a mean hydrodynamic diameter of 541 nm, small polydispersity (0.194), and low positive zeta potentials (+8.39 mV), assuring low haemolytic toxicity. Biological experiments on NB cells, demonstrated that P5PA-4I NPs induced ROS-dependent cytotoxic effects significantly higher than those of pristine 4I, showing a major efficacy compared to ETO in reducing cell viability in HTLA-ER cells. Collectively, this 4I-based nano-formulation could represent a new promising macromolecular platform to develop a new delivery system able to increase the cytotoxicity of the anticancer drugs

D-\u3b1-Tocopherol-Based Micelles for Successful Encapsulation of Retinoic Acid

All-trans-retinoic acid (ATRA) represents the first-choice treatment for several skin diseases, including epithelial skin cancer and acne. However, ATRA\u2019s cutaneous side effects, like redness and peeling, and its high instability limit its efficacy. To address these drawbacks and to improve ATRA solubilization, we prepared ATRA-loaded micelles (ATRA-TPGSs), by its encapsulation in D-\u3b1-tocopheryl-polyethylene-glycol-succinate (TPGS). First, to explore the feasibility of the project, a solubility study based on the equilibrium method was performed; then, six ATRA-TPGS formulations were prepared by the solvent-casting method using different TPGS amounts. ATRA-TPGSs showed small sizes (11\u201320 nm), low polydispersity, slightly negative zeta potential, and proved good encapsulation efficiency, confirmed by a chemometric-assisted Fourier transform infrared spectroscopy (FTIR) investigation. ATRA-TPGS stability was also investigated to choose the most stable formulation. Using Carbopol\uae 980 as gelling agent, ATRA-TPGS-loaded gels were obtained and analyzed for their rheological profiles. Ex vivo release studies from ATRA-TPGSs were performed by Franz cells, demonstrating a permeation after 24 h of 22 \ub1 4 \ub5 cm 122 . ATRA-TPGSs showed enhanced cytotoxic effects on melanoma cells, suggesting that these formulations may represent a valid alternative to improve patient compliance and to achieve more efficacious therapeutic outcomes

Potential Role of miRNAs in the Acquisition of Chemoresistance in Neuroblastoma

Neuroblastoma (NB) accounts for about 8–10% of pediatric cancers, and the main causes of death are the presence of metastases and the acquisition of chemoresistance. Metastatic NB is characterized by MYCN amplification that correlates with changes in the expression of miRNAs, which are small non-coding RNA sequences, playing a crucial role in NB development and chemoresistance. In the present study, miRNA expression was analyzed in two human MYCN-amplified NB cell lines, one sensitive (HTLA-230) and one resistant to Etoposide (ER-HTLA), by microarray and RT-qPCR techniques. These analyses showed that miRNA-15a, -16-1, -19b, -218, and -338 were down-regulated in ER-HTLA cells. In order to validate the presence of this down-regulation in vivo, the expression of these miRNAs was analyzed in primary tumors, metastases, and bone marrow of therapy responder and non-responder pediatric patients. Principal component analysis data showed that the expression of miRNA-19b, -218, and -338 influenced metastases, and that the expression levels of all miRNAs analyzed were higher in therapy responders in respect to non-responders. Collectively, these findings suggest that these miRNAs might be involved in the regulation of the drug response, and could be employed for therapeutic purposes

PKCα Inhibition as a Strategy to Sensitize Neuroblastoma Stem Cells to Etoposide by Stimulating Ferroptosis

Cancer stem cells (CSCs) are a limited cell population inside a tumor bulk characterized by high levels of glutathione (GSH), the most important antioxidant thiol of which cysteine is the limiting amino acid for GSH biosynthesis. In fact, CSCs over-express xCT, a cystine transporter stabilized on cell membrane through interaction with CD44, a stemness marker whose expression is modulated by protein kinase Cα (PKCα). Since many chemotherapeutic drugs, such as Etoposide, exert their cytotoxic action by increasing reactive oxygen species (ROS) production, the presence of high antioxidant defenses confers to CSCs a crucial role in chemoresistance. In this study, Etoposide-sensitive and -resistant neuroblastoma CSCs were chronically treated with Etoposide, given alone or in combination with Sulfasalazine (SSZ) or with an inhibitor of PKCα (C2-4), which target xCT directly or indirectly, respectively. Both combined approaches are able to sensitize CSCs to Etoposide by decreasing intracellular GSH levels, inducing a metabolic switch from OXPHOS to aerobic glycolysis, down-regulating glutathione-peroxidase-4 activity and stimulating lipid peroxidation, thus leading to ferroptosis. Our results suggest, for the first time, that PKCα inhibition inducing ferroptosis might be a useful strategy with which to fight CSC chemoresistance

DataSheet_1_PLX4032 resistance of patient-derived melanoma cells: crucial role of oxidative metabolism.docx

BackgroundMalignant melanoma is the most lethal form of skin cancer which shows BRAF mutation in 50% of patients. In this context, the identification of BRAFV600E mutation led to the development of specific inhibitors like PLX4032. Nevertheless, although its initial success, its clinical efficacy is reduced after six-months of therapy leading to cancer relapse due to the onset of drug resistance. Therefore, investigating the mechanisms underlying PLX4032 resistance is fundamental to improve therapy efficacy. In this context, several models of PLX4032 resistance have been developed, but the discrepancy between in vitro and in vivo results often limits their clinical translation.MethodsThe herein reported model has been realized by treating with PLX4032, for six months, patient-derived BRAF-mutated melanoma cells in order to obtain a reliable model of acquired PLX4032 resistance that could be predictive of patient’s treatment responses. Metabolic analyses were performed by evaluating glucose consumption, ATP synthesis, oxygen consumption rate, P/O ratio, ATP/AMP ratio, lactate release, lactate dehydrogenase activity, NAD+/NADH ratio and pyruvate dehydrogenase activity in parental and drug resistant melanoma cells. The intracellular oxidative state was analyzed in terms of reactive oxygen species production, glutathione levels and NADPH/NADP+ ratio. In addition, a principal component analysis was conducted in order to identify the variables responsible for the acquisition of targeted therapy resistance.ResultsCollectively, our results demonstrate, for the first time in patient-derived melanoma cells, that the rewiring of oxidative phosphorylation and the maintenance of pyruvate dehydrogenase activity and of high glutathione levels contribute to trigger the onset of PLX4032 resistance.ConclusionTherefore, it is possible to hypothesize that inhibitors of glutathione biosynthesis and/or pyruvate dehydrogenase activity could be used in combination with PLX4032 to overcome drug resistance of BRAF-mutated melanoma patients. However, the identification of new adjuvant targets related to drug-induced metabolic reprogramming could be crucial to counteract the failure of targeted therapy in metastatic melanoma.</p

Table_1_PLX4032 resistance of patient-derived melanoma cells: crucial role of oxidative metabolism.docx

BackgroundMalignant melanoma is the most lethal form of skin cancer which shows BRAF mutation in 50% of patients. In this context, the identification of BRAFV600E mutation led to the development of specific inhibitors like PLX4032. Nevertheless, although its initial success, its clinical efficacy is reduced after six-months of therapy leading to cancer relapse due to the onset of drug resistance. Therefore, investigating the mechanisms underlying PLX4032 resistance is fundamental to improve therapy efficacy. In this context, several models of PLX4032 resistance have been developed, but the discrepancy between in vitro and in vivo results often limits their clinical translation.MethodsThe herein reported model has been realized by treating with PLX4032, for six months, patient-derived BRAF-mutated melanoma cells in order to obtain a reliable model of acquired PLX4032 resistance that could be predictive of patient’s treatment responses. Metabolic analyses were performed by evaluating glucose consumption, ATP synthesis, oxygen consumption rate, P/O ratio, ATP/AMP ratio, lactate release, lactate dehydrogenase activity, NAD+/NADH ratio and pyruvate dehydrogenase activity in parental and drug resistant melanoma cells. The intracellular oxidative state was analyzed in terms of reactive oxygen species production, glutathione levels and NADPH/NADP+ ratio. In addition, a principal component analysis was conducted in order to identify the variables responsible for the acquisition of targeted therapy resistance.ResultsCollectively, our results demonstrate, for the first time in patient-derived melanoma cells, that the rewiring of oxidative phosphorylation and the maintenance of pyruvate dehydrogenase activity and of high glutathione levels contribute to trigger the onset of PLX4032 resistance.ConclusionTherefore, it is possible to hypothesize that inhibitors of glutathione biosynthesis and/or pyruvate dehydrogenase activity could be used in combination with PLX4032 to overcome drug resistance of BRAF-mutated melanoma patients. However, the identification of new adjuvant targets related to drug-induced metabolic reprogramming could be crucial to counteract the failure of targeted therapy in metastatic melanoma.</p