Guardians and Mediators of Metastasis: Exploring T Lymphocytes, Myeloid-Derived Suppressor Cells, and Tumor-Associated Macrophages in the Breast Cancer Microenvironment.

Breast cancers (BCs) are solid tumors composed of heterogeneous tissues consisting of cancer cells and an ever-changing tumor microenvironment (TME). The TME includes, among other non-cancer cell types, immune cells influencing the immune context of cancer tissues. In particular, the cross talk of immune cells and their interactions with cancer cells dramatically influence BC dissemination, immunoediting, and the outcomes of cancer therapies. Tumor-infiltrating lymphocytes (TILs), tumor-associated macrophages (TAMs), and myeloid-derived suppressor cells (MDSCs) represent prominent immune cell populations of breast TMEs, and they have important roles in cancer immunoescape and dissemination. Therefore, in this article we review the features of TILs, TAMs, and MDSCs in BCs. Moreover, we highlight the mechanisms by which these immune cells remodel the immune TME and lead to breast cancer metastasis.

Generation and Characterization of a Tumor Stromal Microenvironment and Analysis of Its Interplay with Breast Cancer Cells: An In Vitro Model to Study Breast Cancer-Associated Fibroblast Inactivation.

Breast cancer-associated fibroblasts (BCAFs), the most abundant non-cancer stromal cells of the breast tumor microenvironment (TME), dramatically sustain breast cancer (BC) progression by interacting with BC cells. BCAFs, as well as myofibroblasts, display an up regulation of activation and inflammation markers represented by α-smooth muscle actin (α-SMA) and cyclooxygenase 2 (COX-2). BCAF aggregates have been identified in the peripheral blood of metastatic BC patients. We generated an in vitro stromal model consisting of human primary BCAFs grown as monolayers or 3D cell aggregates, namely spheroids and reverted BCAFs, obtained from BCAF spheroids reverted to 2D cell adhesion growth after 216 h of 3D culture. We firstly evaluated the state of activation and inflammation and the mesenchymal status of the BCAF monolayers, BCAF spheroids and reverted BCAFs. Then, we analyzed the MCF-7 cell viability and migration following treatment with conditioned media from the different BCAF cultures. After 216 h of 3D culture, the BCAFs acquired an inactivated phenotype, associated with a significant reduction in α-SMA and COX-2 protein expression. The deactivation of the BCAF spheroids at 216 h was further confirmed by the cytostatic effect exerted by their conditioned medium on MCF-7 cells. Interestingly, the reverted BCAFs also retained a less activated phenotype as indicated by α-SMA protein expression reduction. Furthermore, the reverted BCAFs exhibited a reduced pro-tumor phenotype as indicated by the anti-migratory effect exerted by their conditioned medium on MCF-7 cells. The deactivation of BCAFs without drug treatment is possible and leads to a reduced capability of BCAFs to sustain BC progression in vitro. Consequently, this study could be a starting point to develop new therapeutic strategies targeting BCAFs and their interactions with cancer cells.

Influence of Tumor Microenvironment and Fibroblast Population Plasticity on Melanoma Growth, Therapy Resistance and Immunoescape.

Cutaneous melanoma (CM) tissue represents a network constituted by cancer cells and tumor microenvironment (TME). A key feature of CM is the high structural and cellular plasticity of TME, allowing its evolution with disease and adaptation to cancer cell and environmental alterations. In particular, during melanoma development and progression each component of TME by interacting with each other and with cancer cells is subjected to dramatic structural and cellular modifications. These alterations affect extracellular matrix (ECM) remodelling, phenotypic profile of stromal cells, cancer growth and therapeutic response. The stromal fibroblast populations of the TME include normal fibroblasts and melanoma-associated fibroblasts (MAFs) that are highly abundant and flexible cell types interacting with melanoma and stromal cells and differently influencing CM outcomes. The shift from the normal microenvironment to TME and from normal fibroblasts to MAFs deeply sustains CM growth. Hence, in this article we review the features of the normal microenvironment and TME and describe the phenotypic plasticity of normal dermal fibroblasts and MAFs, highlighting their roles in normal skin homeostasis and TME regulation. Moreover, we discuss the influence of MAFs and their secretory profiles on TME remodelling, melanoma progression, targeted therapy resistance and immunosurveillance, highlighting the cellular interactions, the signalling pathways and molecules involved in these processes.

Metabolic flexibility in melanoma: A potential therapeutic target.

Cutaneous melanoma (CM) represents one of the most metastasizing and drug resistant solid tumors. CM is characterized by a remarkable metabolic plasticity and an important connection between oncogenic activation and energetic metabolism. In fact, melanoma cells can use both cytosolic and mitochondrial compartments to produce adenosine triphosphate (ATP) during cancer progression. However, the CM energetic demand mainly depends on glycolysis, whose upregulation is strictly linked to constitutive activation of BRAF/MAPK pathway affected by BRAFV600E kinase mutant. Furthermore, the impressive metabolic plasticity of melanoma allows the development of resistance mechanisms to BRAF/MEK inhibitors (BRAFi/MEKi) and the adaptation to microenvironmental changes. The metabolic interaction between melanoma cells and tumor microenvironment affects the immune response and CM growth. In this review article, we describe the regulation of melanoma metabolic alterations and the metabolic interactions between cancer cells and microenvironment that influence melanoma progression and immune response. Finally, we summarize the hallmarks of melanoma therapies and we report BRAF/MEK pathway targeted therapy and mechanisms of metabolic resistance.

Inhibition mechanism of naphthylphenylamine derivatives acting on the CDC25B dual phosphatase and analysis of the molecular processes involved in the high cytotoxicity exerted by one selected derivative in melanoma cells.

The dual phosphatases CDC25 are involved in cell cycle regulation and overexpressed in many tumours, including melanoma. CDC25 is a promising target for discovering anticancer drugs, and several studies focussed on characterisation of quinonoid CDC25 inhibitors, frequently causing undesired side toxic effects. Previous work described an optimisation of the inhibition properties by naphthylphenylamine (NPA) derivatives of NSC28620, a nonquinonoid CDC25 inhibitor. Now, the CDC25B•inhibitor interaction was investigated through fluorescence studies, shedding light on the different inhibition mechanism exerted by NPA derivatives. Among the molecular processes, mediating the specific and high cytotoxicity of one NPA derivative in melanoma cells, we observed decrease of phosphoAkt, increase of p53, reduction of CDC25 forms, cytochrome c cytosolic translocation and increase of caspase activity, that lead to the activation of an apoptotic programme. A basic knowledge on CDC25 inhibitors is relevant for discovering potent bioactive molecules, to be used as anticancer agents against the highly aggressive melanoma.

Structural and denaturation studies of two mutants of a cold adapted superoxide dismutase point to the importance of electrostatic interactions in protein stability.

A peculiar feature of the psychrophilic iron superoxide dismutase from Pseudoalteromonas haloplanktis (PhSOD) is the presence in its amino acid sequence of a reactive cysteine (Cys57). To define the role of this residue, a structural characterization of the effect of two PhSOD mutations, C57S and C57R, was performed. Thermal and denaturant-induced unfolding of wild type and mutant PhSOD followed by circular dichroism and fluorescence studies revealed that C→R substitution alters the thermal stability and the resistance against denaturants of the enzyme, whereas C57S only alters the stability of the protein against urea. The crystallographic data on the C57R mutation suggest an involvement of the Arg side chain in the formation of salt bridges on protein surface. These findings support the hypothesis that the thermal resistance of PhSOD relies on optimization of charge-charge interactions on its surface. Our study contributes to a deeper understanding of the denaturation mechanism of superoxide dismutases, suggesting the presence of a structural dimeric intermediate between the native state and the unfolded state. This hypothesis is supported by the crystalline and solution data on the reduced form of the enzyme.

Enhanced pro-apoptotic activity of rituximab through IBTK silencing in non-Hodgkin lymphoma B-cells.

Rituximab is a commonly used chemotherapeutic drug for patients with aggressive lymphomas, such as non-Hodgkin's lymphoma (NHL). Currently, the combination of Rituximab and chemotherapy (R-CHOP) stands as the most prevalent first-line therapy for NHL. Nevertheless, the development of new therapeutic approaches remains imperative. An increasing body of evidence highlights a novel role for IBTK in tumorigenesis and cancer growth. In this study, we aim to broaden our understanding of IBTK's function in B-lymphoma, with a particular focus on its impact on the expression of the oncogene MYC. Here, we assessed the effects of combining Rituximab with IBTK silencing on cell viability through cell cycle analysis and Annexin V assays in vitro. Furthermore, we leveraged the transplantability of Eµ-myc lymphomas to investigate whether the inhibition of IBTK could elicit anti-tumor effects in the treatment of lymphomas in vivo. Our data suggests that IBTK silencing may serve as an effective anti-tumor agent for aggressive B-Lymphomas, underscoring its role in promoting apoptosis when used in combination with Rituximab, both in in vitro and in vivo settings.

Celecoxib, a Non-Steroidal Anti-Inflammatory Drug, Exerts a Toxic Effect on Human Melanoma Cells Grown as 2D and 3D Cell Cultures.

Cutaneous melanoma (CM) remains one of the leading causes of tumor mortality due to its high metastatic spread. CM growth is influenced by inflammation regulated by prostaglandins (PGs) whose synthesis is catalyzed by cyclooxygenases (COXs). COX inhibitors, including non-steroidal anti-inflammatory drugs (NSAIDs), can inhibit tumor development and growth. In particular, in vitro experiments have shown that celecoxib, a NSAID, inhibits the growth of some tumor cell lines. However, two-dimensional (2D) cell cultures, used in traditional in vitro anticancer assays, often show poor efficacy due to a lack of an in vivo like cellular environment. Three-dimensional (3D) cell cultures, such as spheroids, are better models because they can mimic the common features displayed by human solid tumors. Hence, in this study, we evaluated the anti-neoplastic potential of celecoxib, in both 2D and 3D cell cultures of A2058 and SAN melanoma cell lines. In particular, celecoxib reduced the cell viability and migratory capability and triggered the apoptosis of melanoma cells grown as 2D cultures. When celecoxib was tested on 3D melanoma cell cultures, the drug exerted an inhibitory effect on cell outgrowth from spheroids and reduced the invasiveness of melanoma cell spheroids into the hydrogel matrix. This work suggests that celecoxib could represent a new potential therapeutic approach in melanoma therapy.

Crystallographic and spectroscopic characterizations of Sulfolobus solfataricus TrxA1 provide insights into the determinants of thioredoxin fold stability.

Structural characterizations of thioredoxins (Trxs) are important for their involvement in severe pathologies and for their stable scaffold. Here we report a combined structural and spectroscopic characterization of a Trx isolated from the hyperthermophilic archaeon Sulfolobus solfataricus (SsTrxA1). Thermal denaturation unveils that SsTrxA1 is endowed with a remarkable stability in the explored temperature range 50-105°C. The structure of the oxidized form of SsTrxA1 determined at 1.9Å resolution presents a number of peculiar features. Although the protein was crystallized in a slightly acid medium (pH 6.5) as many as ten intramolecular/intermolecular carboxyl-carboxylate interactions involving glutamic and aspartic acid side chains are found in three independent SsTrxA1 molecules present in the asymmetric unit. Surprisingly for a hyperthermostable protein, the structure of SsTrxA1 is characterized by the presence (a) of a very limited number of intramolecular salt bridges and (b) of a cavity nearby Cys52, a residue that is frequently a phenylananine in other members of the family. Chemical denaturation investigations carried out on SsTrxA1 and SsTrxA2 show that both proteins present a significant stability against guanidine hydrochloride, thus indicating that ionic interactions play a minor role in their stabilization. Compared to Trxs from mesophilic sources, SsTrxA1 displays a longer α-helix 1 and a shorter loop connecting this α-helix with ß-strand 2. As these features are shared with Trxs isolated from thermophilic sources, the shortening of this loop may be a general strategy adopted to stabilize this fold. This feature may be exploited for the design of hyperthermostable Trx scaffolds.

Thyroid Cancer and Fibroblasts.

Thyroid cancer is the most common type of endocrine cancer, and its prevalence continue to rise. Non-metastatic thyroid cancer patients are successfully treated. However, looking for new therapeutic strategies is of great importance for metastatic thyroid cancers that still lead to death. With respect to this, the tumor microenvironment (TME), which plays a key role in tumor progression, should be considered as a new promising therapeutic target to hamper thyroid cancer progression. Indeed, thyroid tumors consist of cancer cells and a heterogeneous and ever-changing niche, represented by the TME, which contributes to establishing most of the features of cancer cells. The TME consists of extracellular matrix (ECM) molecules, soluble factors, metabolites, blood and lymphatic tumor vessels and several stromal cell types that, by interacting with each other and with tumor cells, affect TME remodeling, cancer growth and progression. Among the thyroid TME components, cancer-associated fibroblasts (CAFs) have gained more attention in the last years. Indeed, recent important evidence showed that thyroid CAFs strongly sustain thyroid cancer growth and progression by producing soluble factors and ECM proteins, which, in turn, deeply affect thyroid cancer cell behavior and aggressiveness. Hence, in this article, we describe the thyroid TME, focusing on the desmoplastic stromal reaction, which is a powerful indicator of thyroid cancer progression and an invasive growth pattern. In addition, we discuss the origins and features of the thyroid CAFs, their influence on thyroid cancer growth and progression, their role in remodeling the ECM and their immune-modulating functions. We finally debate therapeutic perspectives targeting CAFs.

Reactive oxygen species, Ki-Ras, and mitochondrial superoxide dismutase cooperate in nerve growth factor-induced differentiation of PC12 cells.

Nerve growth factor (NGF) induces terminal differentiation in PC12, a pheochromocytoma-derived cell line. NGF binds a specific receptor on the membrane and triggers the ERK1/2 cascade, which stimulates the transcription of neural genes. We report that NGF significantly affects mitochondrial metabolism by reducing mitochondrial-produced reactive oxygen species and stabilizing the electrochemical gradient. This is accomplished by stimulation of mitochondrial manganese superoxide dismutase (MnSOD) both transcriptionally and post-transcriptionally via Ki-Ras and ERK1/2. Activation of MnSOD is essential for completion of neuronal differentiation because 1) expression of MnSOD induces the transcription of a neuronal specific promoter and neurite outgrowth, 2) silencing of endogenous MnSOD by small interfering RNA significantly reduces transcription induced by NGF, and 3) a Ki-Ras mutant in the polylysine stretch at the COOH terminus, unable to stimulate MnSOD, fails to induce complete differentiation. Overexpression of MnSOD restores differentiation in cells expressing this mutant. ERK1/2 is also downstream of MnSOD, as a SOD mimetic drug stimulates ERK1/2 with the same kinetics of NGF and silencing of MnSOD reduces NGF-induced late ERK1/2. Long term activation of ERK1/2 by NGF requires SOD activation, low levels of hydrogen peroxide, and the integrity of the microtubular cytoskeleton. Confocal immunofluorescence shows that NGF stimulates the formation of a complex containing membrane-bound Ki-Ras, microtubules, and mitochondria. We propose that active NGF receptor induces association of mitochondria with plasma membrane. Local activation of ERK1/2 by Ki-Ras stimulates mitochondrial SOD, which reduces reactive oxygen species and produces H(2)O(2). Low and spatially restricted levels of H(2)O(2) induce and maintain long term ERK1/2 activity and ultimately differentiation of PC12 cells.

Quinoa as source of type 1 ribosome inactivating proteins: A novel knowledge for a revision of its consumption.

This study investigates on the presence of toxic proteins in quinoa seeds. To this aim, a plethora of biochemical approaches were adopted for the purification and characterization of quinoin, a type 1 ribosome-inactivating protein (RIP) contained in quinoa seeds. We determined its melting temperature (68.2 ± 0.6 °C) and thermostability (loss of activity after 10-min incubation at 70 °C). Considering that quinoa seeds are used as a food, we found that quinoin is cytotoxic against BJ-5ta (human fibroblasts) and HaCaT (human keratinocytes) in a dose- and time-dependent manner. Moreover, in an in vitro digestive pepsin-trypsin treatment, 30% of quinoin is resistant to enzymatic cleavage. This toxin was found in seeds (0.23 mg/g of seeds) and in sprouted seeds obtained after 24-h (0.12 mg/g of sprout) and 48-h (0.09 mg/g of sprout). We suggest a thermal treatment of quinoa seeds before consumption in order to inactivate the toxin, particularly in sprouts, generally consumed raw.

Structure and stability of a thioredoxin reductase from Sulfolobus solfataricus: a thermostable protein with two functions.

Recent investigations have demonstrated that disulfide bridges may play a crucial role in the stabilization of proteins in hyperthermophilic organisms. A major role in the process of disulfide formation is played by ubiquitous proteins belonging to the thioredoxin superfamily, which includes thioredoxins (Trx), thioredoxin reductases (TrxR), and disulfide oxidases/isomerases (PDO/PDI). Here we report a characterization of the structure and stability of the TrxR (SsTrxRB3) isolated from the archaeon Sulfolobus solfataricus. This protein is particularly interesting since it is able to process different substrates (Trxs and PDO) and it is endowed with an additional NADH oxidase activity. The crystal structure of the wild-type enzyme, of its complex with NADP and of the C147A mutant provides interesting clues on the enzyme function. In contrast to what is observed for class II TrxRs, in the structure of the oxidized enzyme, the FAD binding site is occupied by a partially disordered NAD molecule. In the active site of the C147A mutant, which exhibits a marginal NADH oxidase activity, the FAD is canonically bound to the enzyme. Molecular modeling indicates that a FAD molecule can be accommodated in the site of the reduced SsTrxRB3. Depending on the oxidation state, SsTrxRB3 can bind a different cofactor in its active site. This peculiar feature has been related to its dual activity. Denaturation experiments followed by circular dichroism indicate that electrostatic interactions play an important role in the stabilization of this thermostable protein. The analysis of the enzyme 3D-structure has also provided insights into the bases of SsTrxRB3 stability.

Diclofenac-induced apoptosis in the neuroblastoma cell line SH-SY5Y: possible involvement of the mitochondrial superoxide dismutase.

Diclofenac, a nonsteroidal anti-inflammatory drug, induces apoptosis on the neuroblastoma cell line SH-SY5Y through a mitochondrial dysfunction, affecting some antioxidant mechanisms. Indeed, the time- and dose-dependent increase of apoptosis is associated to an early enhancement of the reactive oxygen species (ROS). Mitochondrial superoxide dismutase (SOD2) plays a crucial role in the defence against ROS, thus protecting against several apoptotic stimuli. Diclofenac decreased the protein levels and the enzymatic activity of SOD2, without any significant impairment of the corresponding mRNA levels in the SH-SY5Y extracts. When cells were incubated with an archaeal exogenous thioredoxin, an attenuation of the diclofenac-induced apoptosis was observed, together with an increase of SOD2 protein levels. Furthermore, diclofenac impaired the mitochondrial membrane potential, leading to a release of cytochrome c. These data suggest that mitochondria are involved in the diclofenac-induced apoptosis of SH-SY5Y cells and point to a possible role of SOD2 in this process.

Metabolic Plasticity of Melanoma Cells and Their Crosstalk With Tumor Microenvironment.

Cutaneous melanoma (CM) is a highly aggressive and drug resistant solid tumor, showing an impressive metabolic plasticity modulated by oncogenic activation. In particular, melanoma cells can generate adenosine triphosphate (ATP) during cancer progression by both cytosolic and mitochondrial compartments, although CM energetic request mostly relies on glycolysis. The upregulation of glycolysis is associated with constitutive activation of BRAF/MAPK signaling sustained by BRAFV600E kinase mutant. In this scenario, the growth and progression of CM are strongly affected by melanoma metabolic changes and interplay with tumor microenvironment (TME) that sustain tumor development and immune escape. Furthermore, CM metabolic plasticity can induce a metabolic adaptive response to BRAF/MEK inhibitors (BRAFi/MEKi), associated with the shift from glycolysis toward oxidative phosphorylation (OXPHOS). Therefore, in this review article we survey the metabolic alterations and plasticity of CM, its crosstalk with TME that regulates melanoma progression, drug resistance and immunosurveillance. Finally, we describe hallmarks of melanoma therapeutic strategies targeting the shift from glycolysis toward OXPHOS.

Influence of Fibroblasts on Mammary Gland Development, Breast Cancer Microenvironment Remodeling, and Cancer Cell Dissemination.

The stromal microenvironment regulates mammary gland development and tumorigenesis. In normal mammary glands, the stromal microenvironment encompasses the ducts and contains fibroblasts, the main regulators of branching morphogenesis. Understanding the way fibroblast signaling pathways regulate mammary gland development may offer insights into the mechanisms of breast cancer (BC) biology. In fact, the unregulated mammary fibroblast signaling pathways, associated with alterations in extracellular matrix (ECM) remodeling and branching morphogenesis, drive breast cancer microenvironment (BCM) remodeling and cancer growth. The BCM comprises a very heterogeneous tissue containing non-cancer stromal cells, namely, breast cancer-associated fibroblasts (BCAFs), which represent most of the tumor mass. Moreover, the different components of the BCM highly interact with cancer cells, thereby generating a tightly intertwined network. In particular, BC cells activate recruited normal fibroblasts in BCAFs, which, in turn, promote BCM remodeling and metastasis. Thus, comparing the roles of normal fibroblasts and BCAFs in the physiological and metastatic processes, could provide a deeper understanding of the signaling pathways regulating BC dissemination. Here, we review the latest literature describing the structure of the mammary gland and the BCM and summarize the influence of epithelial-mesenchymal transition (EpMT) and autophagy in BC dissemination. Finally, we discuss the roles of fibroblasts and BCAFs in mammary gland development and BCM remodeling, respectively.

Glutathionylation of the iron superoxide dismutase from the psychrophilic eubacterium Pseudoalteromonas haloplanktis.

Our previous work showed that the adduct between beta-mercaptoethanol and the single cysteine residue (Cys57) in superoxide dismutase from the psychrophilic eubacterium Pseudoalteromonas haloplanktis (PhSOD) reduces the enzyme inactivation by peroxynitrite. In this work, immunoblotting experiments prove that peroxynitrite inactivation of PhSOD involves formation of nitrotyrosine residue(s). In order to study the role of Cys57 as a redox-sensor residue modifiable by cellular thiols, a recombinant PhSOD and two Cys57 mutants were produced and characterized. Recombinant and mutant enzymes share similar activity and peroxynitrite inactivation, but different reactivity towards three glutathione forms. Indeed, oxidized glutathione and S-nitrosoglutathione, but reduced glutathione, lead to S-glutathionylation of recombinant PhSOD. This new covalent modification for a Fe-SOD does not occur in both Cys57 mutants, thus indicating that its target is Cys57. Moreover, mass spectrometry analysis confirmed that S-glutathionylation of Cys57 takes place also with endogenous PhSOD. Formation of this mixed disulfide in PhSOD protects the enzyme from tyrosine nitration and peroxynitrite inactivation. PhSOD undergoes S-glutathionylation during its overproduction in E. coli cells and in a growing culture of P. haloplanktis. In both cases the extent of glutathionylated PhSOD is enhanced upon cell exposure to oxidative agents. We suggest that S-glutathionylation of PhSOD could represent a further cold-adaptation strategy to improve the antioxidant cellular defence mechanism.

Crystallization and preliminary X-ray crystallographic analysis of two dimeric hyperthermostable thioredoxins isolated from Sulfolobus solfataricus.

The thioredoxin system of the archaeon Sulfolobus solfataricus involves a number of different proteins: two thioredoxin reductases (SsTrxRB2 and SsTrxRB3), two distinct thioredoxins (SsTrxA1 and SsTrxA2) and a disulfide oxidoreductase (SsPDO). Here, the crystallization and preliminary crystallographic analyses of SsTrxA1 and SsTrxA2, two dimeric proteins endowed with extraordinary thermal stability, are reported. In addition to the functional thioredoxin domain, both SsTrxA1 and SsTrxA2 present an extra N-terminal fragment of approximately 30 residues. Although crystallization trials have been conducted on both forms of the proteins, crystals that were suitable for X-ray crystallographic analyses have only been obtained for their truncated variants. The crystals of SsTrxA2 belonged to space group P2, with unit-cell parameters a = 28.27, b = 27.88, c = 62.06 A, beta = 92.34 degrees , and diffracted to 1.83 A resolution, whereas the crystals of SsTrxA1 belonged to space group P2(1), with unit-cell parameters a = 51.76, b = 75.09, c = 55.35 A, beta = 112.64 degrees , and diffracted to 1.90 A resolution. The structures of the two proteins have been solved by molecular replacement.

Mitochondrial Flexibility of Breast Cancers: A Growth Advantage and a Therapeutic Opportunity.

Breast cancers are very heterogeneous tissues with several cell types and metabolic pathways together sustaining the initiation and progression of disease and contributing to evasion from cancer therapies. Furthermore, breast cancer cells have an impressive metabolic plasticity that is regulated by the heterogeneous tumour microenvironment through bidirectional interactions. The structure and accessibility of nutrients within this unstable microenvironment influence the metabolism of cancer cells that shift between glycolysis and mitochondrial oxidative phosphorylation (OXPHOS) to produce adenosine triphosphate (ATP). In this scenario, the mitochondrial energetic pathways of cancer cells can be reprogrammed to modulate breast cancer's progression and aggressiveness. Moreover, mitochondrial alterations can lead to crosstalk between the mitochondria and the nucleus, and subsequently affect cancer tissue properties. This article reviewed the metabolic plasticity of breast cancer cells, focussing mainly on breast cancer mitochondrial metabolic reprogramming and the mitochondrial alterations influencing nuclear pathways. Finally, the therapeutic strategies targeting molecules and pathways regulating cancer mitochondrial alterations are highlighted.

Ageritin from poplar mushrooms: scale-up purification and cytotoxicity towards undifferentiated and differentiated SH-SY5Y cells.

Ageritin is the first reported ribotoxin-like protein from basidiomycetes fungi. It can induce ribosomal integrity damage and translation block, and interferes with mitochondrial redox activity of some glioma and neuroblastoma cell lines. Herein, Ageritin has been investigated as a valuable neurotoxin towards either undifferentiated or retinoic acid (RA)-differentiated SH-SY5Y neuroblastoma cells showing a selective cell toxicity against undifferentiated cells. MTT and sulforhodamine B (SRB) assays highlighted that Ageritin markedly decreases the mitochondrial redox activity and viability of undifferentiated cells, meanwhile inducing evident morphological changes eliciting neuronal-like appearance in these cells. Data from lactate dehydrogenase release assay, cytofluorimetric analysis and caspase-3 enzymatic activity measurement suggest that Ageritin promotes cell death through a caspase-dependent apoptotic pathway. The Z-VAD-FMK caspase inhibitor was able to prevent this apoptotic pathway activation. Based on the interesting behaviour of Ageritin vs. SH-SY5Y cells, the development of a scale-up procedure to obtain the purified protein in larger amounts (yield 2.5 mg per 100 g) has been optimized.