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Tea tree oil is an essential oil distilled from the leaves of Melaleuca alternifolia, a plant native to Australia. It has been used in traditional medicine for its antiseptic and anti-inflammatory properties to treat various skin conditions and infections. It has also been incorporated into many topical formulations to treat cutaneous infections and speed wound healing. In vivo and in vitro studies report antiproliferative effects in skin disorders but the molecular mechanisms underlying this effect remain to be still elucidated. In this study MTT assay, scanning electron microscopy-field emission gun, flow cytometry, cell cycle assays, and laser scanning confocal microscopy were utilized to investigate a novel mechanism underlying the antiproliferative effects of tea tree oil and terpinen-4-ol on transformed skin (melanoma) M14 cells. The analysis of the actin cytoskeleton by laser scanning confocal microscopy evidenced a clear action of both essential oil and its main active component on F-actin, which interfered with bundling of actin microfilaments in stress fibers. As for the microtubular network, both tea tree oil and terpinen-4-ol induced a disorganization of the perinuclear cage with the rupture and collapse of microtubules. Finally, they noticeably changed the intermediate filaments architecture by inducing the formation of large vimentin cables. Results obtained in the present study point to the cytoskeleton as a further target of tea tree oil and terpinen-4-ol and could account for the inhibition of proliferation and invasion of skin transformed M14 cells. In our experimental conditions, vimentin intermediate filaments appear to be the cytoskeletal element more affected by the treatments. Moreover, the role of cross-linker proteins in the mechanism of action of tea tree oil has been discussed.

PURPOSE: The aim of this ex vivo study was to assess the ability to remove oral biofilm by different combinations of mechanical and chemical treatments on smooth and rough titanium surfaces, as well as their impact on osteoconduction. MATERIALS AND METHODS: Forty-eight sandblasted acid-etched (SLA) and 48 machined titanium disks were contaminated with oral bacterial biofilm and exposed to the following treatments: (1) titanium brush (TB), (2) TB + 40% citric acid (CA), (3) TB + 5.25% sodium hypochlorite (NaOCl), (4) air polishing with glycine powder (AP), (5) AP + 40% CA, and (6) AP + 5.25% NaOCl. Residual bacteria and chemical contamination were assessed using viable bacterial count assay, scanning electron microscopy (SEM), and x-ray spectroscopy (XPS). Human primary osteoblast (hOB) adhesion and osteocalcin (OC) release were also evaluated. RESULTS: The microbiologic, SEM, and XPS analysis indicate a higher biofilm removal efficiency of combined mechanical-chemical treatments compared with exclusively mechanical approaches, especially on SLA surfaces. SEM analysis revealed significant alterations of surface microtopography on the disks treated with TB, while no changes were observed after AP treatment. OC release by hOBs was mainly decreased on disks treated with CA and NaOCl. CONCLUSION: The combination of mechanical and chemical treatments provides effective oral biofilm removal on both SLA and machined implant surfaces. NaOCl and CA may have a negative effect on osteoblasts cultured on SLA samples.

Background There is a huge body of literature data on ZnOnanoparticles (ZnO NPs) toxicity. However, the reported results are seen to be increasingly discrepant, and deep comprehension of the ZnO NPs behaviour in relation to the different experimental conditions is still lacking. A recent literature overview emphasizes the screening of the ZnO NPs toxicity with more than one assay, checking the experimental reproducibility also versus time, which is a key factor for the robustness of the results. In this paper we compared high-throughput real-time measurements through Electric Cell-substrate Impedance-Sensing (ECIS®) with endpoint measurements of multiple independent assays. Results ECIS-measurements were compared with traditional cytotoxicity tests such as MTT, Neutral red, Trypan blue, and cloning efficiency assays. ECIS could follow the cell behavior continuously and noninvasively for days, so that certain long-term characteristics of cell proliferation under treatment with ZnO NPs were accessible. This was particularly important in the case of pro-mitogenic activity exerted by low-dose ZnO NPs, an effect not revealed by endpoint independent assays. This result opens new worrisome questions about the potential mitogenic activity exerted by ZnO NPs, or more generally by NPs, on transformed cells. Of importance, impedance curve trends (morphology) allowed to discriminate between different cell death mechanisms (apoptosis vs autophagy) in the absence of specific reagents, as confirmed by cell structural and functional studies by high-resolution microscopy. This could be advantageous in terms of costs and time spent. ZnO NPs-exposed A549 cells showed an unusual pattern of actin and tubulin distribution which might trigger mitotic aberrations leading to genomic instability. Conclusions ZnO NPs toxicity can be determined not only by the intrinsic NPs characteristics, but also by the external conditions like the experimental setting, and this could account for discrepant data from different assays. ECIS has the potential to recapitulate the needs required in the evaluation of nanomaterials by contributing to the reliability of cytotoxicity tests. Moreover, it can overcome some false results and discrepancies in the results obtained by endpoint measurements. Finally, we strongly recommend the comparison of cytotoxicity tests (ECIS, MTT, Trypan Blue, Cloning efficiency) with the ultrastructural cell pathology studies. Graphic Abstract

Though liposome-based drugs are in clinical use, the mechanism of cell internalization of liposomes is yet an object of controversy. The present experimental investigation, carried out on human glioblastoma cells, indicated different internalization routes for two diastereomeric liposomes. Molecular dynamics simulations of the lipid bilayers of the two formulations indicated that the different stereochemistry of a lipid component controls some parameters such as area per lipid molecule and fluidity of lipid membranes, surface potential and water organization at the lipid/water interface, all of which affect the interaction with biomolecules and cell components.

The surgical treatment of peri-implantitis is currently based on the removal of biofilms from the implant surface by primary means of mechanical and physical treatments. However, such approaches often determine some alterations of the implant surface with detrimental effects on re-osseointegration. This study aims to evaluate the effects of four different mechanical and physical treatments on titanium samples with moderately rough surface. Air powder abrasion (AP) with glycine powder, a titanium brush (TB) and a diode laser at 3 W (L3) and 4 W (L4) were tested. Surface morphology, roughness and chemical composition were then assessed by scanning electron microscope (SEM), white light interferometer and X-ray photoelectron spectroscopy (XPS), respectively. The microscopic analysis revealed significant alterations in surface morphology on TB samples, while AP and L3 had only a minor or null impact. L4 samples revealed signs of overheating due to the excessive power. Nevertheless, the overall roughness of the samples was not significantly altered in terms of roughness parameters. Similarly, surface chemical composition was not significantly affected by the treatments. Among the treatments tested in this study, air powder abrasion with glycine powder and 3 W diode laser had the lowest impact on surface physicochemical properties.

Objective. Platelet-rich fibrin (PRF) clots and membranes are autologous blood concentrates widely used in oral surgical procedures; less is known, however, about the liquid formulations of such products. The aim of this in vitro study is to assess the behavior of different implant surfaces when in contact with two liquid leucocyte- and platelet-rich fibrin (L-PRF) products.Methods. Six commercial pure titanium discs, of 9.5 mm diameter and 1.5 mm thickness, were used. Three of these samples had a micro/nano-rough surface; three were machined. Three different protocols were tested. Protocols involved the immersion of the samples in(1)a platelets, lymphocytes, and fibrinogen liquid concentrate (PLyF) for 10 minutes,(2)an exudate obtained from L-PRF clots rich in fibronectin and vitronectin for 5 minutes, and(3)the fibronectin/vitronectin exudate for 2 minutes followed by immersion in the PLyF concentrate for further 8 minutes. After these treatments, the samples were fixed and observed using a scanning electron microscope (SEM).Results. Under microscopic observation,(1)the samples treated with the PLyF concentrate revealed a dense fibrin network in direct contact with the implant surface and a significant number of formed elements of blood;(2)in the samples treated with the fibronectin/vitronectin exudates, only a small number of white and red blood cells were detectable; and(3)in samples exposed to the combined treatment, there was an apparent increase in the thickness of the fibrin layer. When compared to the machined surface, the micro/nano-rough samples showed an overall increased retention of fibrin, leading to a thicker coating.Conclusions. Liquid L-PRF products promote the formation of a dense fibrin clot on micro/nano-rough implant surfaces in vitro. The adjunctive treatment of surfaces with the fibronectin/vitronectin exudate could provide support to contact of the fibrin with the surface, though it is not essential for the clot formation. Further studies are necessary to better elucidate the properties and benefits of liquid L-PRF products.

Several antiseptic agents have been proposed for the treatment of peri-implantitis as a complementary therapeutic strategy in addition to mechanical devices. The aim of this study was to compare six different antiseptics, as well as alternative formulations of the same chemical agent, with respect to their decontamination efficacy and impact on chemical properties of the implant surface. Titanium disks with a micro-rough surface, previously contaminated with Porphyromonas gingivalis and Streptococcus mutans biofilms, were treated for 2 min with different antiseptics (liquid sodium hypochlorite 5.25%, gel sodium hypochlorite 5.25%, liquid chlorhexidine 0.2%, gel chlorhexidine 1%, gel citric acid 40%, and gel orthophosphoric acid 37%) or sterile saline solution (control) and their antibacterial activity as well as their ability to remove biofilm were assessed by viable bacterial count and scanning electron microscopy, respectively. Spectroscopic analysis was also performed on non-contaminated disks after exposure to the antiseptics, in order to detect any change in the elemental composition of the titanium surface. All the antimicrobial formulations examined were effective against P. gingivalis and S. mutans biofilms. SEM analysis revealed however that liquid sodium hypochlorite 5.25% was more effective in dissolving biofilm residues. Spectroscopic analysis detected traces of the antiseptics, probably due to insufficient rinsing of the titanium surfaces. In conclusion, since gel formulations of these antiseptic agents possessed a similar antibacterial activity to the liquid formulations, these may be proposed as alternative treatments given their properties to avoid overflows and increase contact time without significant side effects on the bone.

Liposomes functionalized on their surface with carbohydrates (glycoliposomes) represent an optimal approach for targeting of drugs to diseased tissues in vivo, thanks to biocompatibility, low toxicity and easy manufacturing of these lipid nanoparticles. Here we report on the study of liposomes including a novel glycosylated amphiphile and on the comparison of their features with those of glycosylated analogues described previously. Further, the capability of the different glucosylated formulations to interact with three breast cancer cell lines was investigated. Our results show that the hydrophobic portion of the lipid bilayer strongly influences both the properties and the internalization of glycosylated liposomes.

Achaete-scute homolog 1 gene (ASCL1) is a gene classifier for the proneural (PN) transcriptional subgroup of glioblastoma (GBM) that has a relevant role in the neuronal-like differentiation of GBM cancer stem cells (CSCs) through the activation of a PN gene signature. Besides prototypical ASCL1 PN target genes, the molecular effectors mediating ASCL1 function in regulating GBM differentiation and, most relevantly, subgroup specification are currently unknown. Here we report that ASCL1 not only promotes the acquisition of a PN phenotype in CSCs by inducing a glial-to-neuronal lineage switch but also concomitantly represses mesenchymal (MES) features by directly downregulating the expression of N-Myc downstream-regulated gene 1 (NDRG1), which we propose as a novel gene classifier of MES GBMs. Increasing the expression of ASCL1 in PN CSCs results in suppression of self-renewal, promotion of differentiation and, most significantly, decrease in tumorigenesis, which is also reproduced by NDRG1 silencing. Conversely, both abrogation of ASCL1 expression in PN CSCs and enforcement of NDRG1 expression in either PN or MES CSCs induce proneural-to-mesenchymal transition (PMT) and enhanced mesenchymal features. Surprisingly, ASCL1 overexpression in MES CSCs increases malignant features and gives rise to a neuroendocrine-like secretory phenotype. Altogether, our results propose that the fine interplay between ASCL1 and its target NDRG1 might serve as potential subgroup-specific targetable vulnerability in GBM; enhancing ASCL1 expression in PN GBMs might reduce tumorigenesis, whereas repressing NDRG1 expression might be actionable to hamper the malignancy of GBM belonging to the MES subgroup.

Since their discovery in the 1960s, liposomes have been studied in depth, and they continue to constitute a field of intense research. Liposomes are valued for their biological and technological advantages, and are considered to be the most successful drug-carrier system known to date. Notable progress has been made, and several biomedical applications of liposomes are either in clinical trials, are about to be put on the market, or have already been approved for public use. In this review, we briefly analyze how the efficacy of liposomes depends on the nature of their components and their size, surface charge, and lipidic organization. Moreover, we discuss the influence of the physicochemical properties of liposomes on their interaction with cells, half-life, ability to enter tissues, and final fate in vivo. Finally, we describe some strategies developed to overcome limitations of the "first-generation" liposomes, and liposome-based drugs on the market and in clinical trials.

Each of the steps involved in invasion of tumors requires specific molecular program in which the modulation of adhesive and migratory properties of disseminating cells plays an essential role. The improvement in the knowledge of these mechanisms can lead to discovery of new target candidates in drug development. In this study we focused attention on the product of the human AHI-1 (Abelson helper integration site) gene Jouberin (Jbn).In particular, we explore by in vitro invasion assay, AHI-1 knockdown and electron microscopy, if Jbn is involved in the signaling machinery that regulates tumor invasion. To this purpose tumor cells of different histological derivation (brain, breast, skin) were employed.We found that Jbn expression correlates with the proliferation, invasive potential and invasion strategy of the tested tumor cells, and that its downregulation reduces their capability of migrating and invading the extracellular matrix.The results obtained in this study for the first time point to Jbn as a new candidate involved in the invasion process of tumor cells, and as potential molecular target in anticancer therapy.

Poly(ethylene glycol) (PEG) stabilized liposomes are widely used in drug delivery research because these surface grafted polymers stabilize liposomes in blood stream. However, hydrophilic polymers also affect lipid membrane organization and it is fundamental to investigate their effect on the physicochemical properties of lipid bilayers as well as on their interaction with biological membrane. PEG monolaurate was included in liposomes formulated with dimyristoyl-sn-glycero-3-phosphocholine and one of two cationic gemini surfactants that differ each other for the configuration of a stereogenic center on the polar headgroup. These formulations previously showed good efficiency as drug and DNA delivery systems. The effect of the presence of different amounts of a PEG-lipid and of the liposome preparation protocol was investigated by turbidimetry, dynamic laser light scattering, differential scanning calorimetry and electrophoretic mobility. Flow cytometry and laser scanning confocal microscopy on murine (C6) and on human glioblastoma (LN229) cell lines were also carried out to evaluate the influence of pegylation and of the protocol of preparation on the interaction of liposomes with the biological milieu.

Background The chemokine receptor CXCR4 plays a crucial role in tumors, including glioblastoma multiforme (GBM), the most aggressive glioma. Phosphatidylcholine-specific phospholipase C (PC-PLC), a catabolic enzyme of PC metabolism, is involved in several aspects of cancer biology and its inhibition down-modulates the expression of growth factor membrane receptors interfering with their signaling pathways. In the present work we investigated the possible interplay between CXCR4 and PC-PLC in GBM cells. Methods Confocal microscopy, immunoprecipitation, western blot analyses, and the evaluation of migration and invasion potential were performed on U87MG cells after PC-PLC inhibition with the xanthate D609. The intracellular metabolome was investigated by magnetic resonance spectroscopy; lactate levels and lactate dehydrogenase (LDH) activity were analyzed by colorimetric assay. Results Our studies demonstrated that CXCR4 and PC-PLC co-localize and are associated on U87MG cell membrane. D609 reduced CXCR4 expression, cell proliferation and invasion, interfering with AKT and EGFR activation and expression. Metabolic analyses showed a decrease in intracellular lactate concentration together with a decrement in LDH activity. Conclusions Our data suggest that inhibition of PC-PLC could represent a new molecular approach in glioma biology not only for its ability in modulating cell metabolism, glioma growth and motility, but also for its inhibitory effect on crucial molecules involved in cancer progression.

Background The CXCL12/CXCR4 pathway regulates tumor cell proliferation, metastasis, angiogenesis and the tumor-microenvironment cross-talk in several solid tumors, including glioblastoma (GBM), the most common and fatal brain cancer. In the present study, we evaluated the effects of peptide R, a new specific CXCR4 antagonist that we recently developed by a ligand-based approach, in an in vitro and in vivo model of GBM. The well-characterized CXCR4 antagonist Plerixafor was also included in the study. Methods The effects of peptide R on CXCR4 expression, cell survival and migration were assessed on the human glioblastoma cell line U87MG exposed to CXCL12, by immunofluorescence and western blotting, MTT assay, flow cytometry and transwell chamber migration assay. Peptide R was then tested in vivo, by using U87MG intracranial xenografts in CD1 nude mice. Peptide R was administered for 23 days since cell implantation and tumor volume was assessed by magnetic resonance imaging (MRI) at 4.7 T. Glioma associated microglia/macrophage (GAMs) polarization (anti-tumor M1 versus pro-tumor M2 phenotypes) and expressions of vascular endothelial growth factor (VEGF) and CD31 were assessed by immunohistochemistry and immunofluorescence. Results We found that peptide R impairs the metabolic activity and cell proliferation of human U87MG cells and stably reduces CXCR4 expression and cell migration in response to CXCL12 in vitro. In the orthotopic U87MG model, peptide R reduced tumor cellularity, promoted M1 features of GAMs and astrogliosis, and hindered intra-tumor vasculature. Conclusions Our findings suggest that targeting CXCR4 by peptide R might represent a novel therapeutic approach against GBM, and contribute to the rationale to further explore in more complex pre-clinical settings the therapeutic potential of peptide R, alone or in combination with standard therapies of GBM. Electronic supplementary material The online version of this article (doi:10.1186/s13046-016-0326-y) contains supplementary material, which is available to authorized users.

It has been confirmed that multidrug resistant (MDR) melanoma cells (M14 ADR2) are more sensitive than their wild-type counterparts (M14 WT) to H2O2 and aldehydes, the products of bovine serum amine oxidase (BSAO)-catalyzed oxidation of spermine. The metabolites formed by BSAO and spermine are more toxic, in M14 cells, than exogenous H2O2 and acrolein, even though their concentration is lower during the initial phase of incubation due to their more gradual release than the exogenous products. Binding of BSAO to the cell membrane and release of the reaction products of spermine into the immediate vicinity of the cells, or directly into the cells, may explain the apparently paradoxical phenomenon. Both WT and MDR cells, after pre-treatment for 24 h, or longer, with the lysosomotropic compound chloroquine (CQ), show to be sensitized to subsequent exposure to BSAO/spermine enzymatic system. Evidence of ultrastructural aberrations and acridine orange release from lysosomes is presented in this study that is in favor of the permeabilization of the lysosomal membrane as the major cause of sensitization by CQ. Pre-treatment with CQ amplifies the ability of the metabolites formed from spermine by oxidative deamination to induce cell death. Melanocytes, differently from melanoma cells, were unaffected by the enzymatic system, even when preceded by CQ treatment. Since it is conceivable that combined treatment with a lysosomotropic compound and BSAO/spermine would be effective against tumour cells, it is of interest to search for such novel compounds, which might be promising for application in a therapeutic setting.

Fluorescently-tagged lipids are a powerful tool for investigating the dynamics of lipids in cell biological studies and in biophysical applications. Herein we report the synthesis and the characterization of a new bola amphiphile, PC12, based on a bisimidic perylene moiety: a central perylene unit is symmetrically linked to two aliphatic chains both ending with a quaternary ammonium group. Absorption and fluorescent properties of the newly synthesized compound were investigated in DMSO and in water as a function of concentration and temperature. Further, the entrapment efficiency, the fluorescence behavior in dimyristoyl-sn-glycero-phosphocholine (DMPC) liposomes, and cell uptake on human and murine glioblastoma cell lines were evaluated. When loaded in liposomes, PC12 follows the same destiny of liposomes themselves in the cell cultures: this is an interesting result because PC12 could be used both as an aspecific dye (free form) and as an organelle-specific lipid probe.

Urotensin II (UT-II) is a potent vasoconstrictor peptide and its receptor (UTR) was correlated with human cortico-adrenal carcinoma proliferation. In this study, we have evaluated the correlation between UTR expression and prognosis of human prostate adenocarcinoma and the involvement of this receptor in the regulation of biological properties on both in vivo and in vitro models. UTR mRNA and protein, evaluated by real-time PCR and Western blotting, respectively, were expressed at high levels only in androgen-dependent LNCaP cells. In order to investigate UTR changes occurring in human prostate tumorigenesis, we have also evaluated the expression of UTR in vivo in 195 human prostate tissue samples. UTR was always expressed at low intensity in hyperplastic tissues and at high intensity in well-differentiated carcinomas (Gleason 2-3). Moreover, we have evaluated the effects of an antagonist of UTR, urantide on migration and invasion of LNCaP cells. Urantide induced a dose-dependent decrease of motility and invasion of LNCaP cells whose characteristic ameboid movement seems to be advantageous for their malignancy. These effects were paralleled by down-regulating the autophosphorylation of focal adhesion kinase and the integrin surface expression on LNCaP cells. The effects on cell motility and invasion were likely due to the inhibition of RhoA activity induced by both urantide and shRNA UTR. These data suggest that UTR can be considered a prognostic marker in human prostate adenocarcinoma patients.

In this study we present new data from experiments focused on the antitumor activity of tea tree oil (TTO), an essential oil distilled from Melaleuca alternifolia. TTO proved to be capable of inhibiting the growth of melanoma cells and of overcoming multidrug resistance (MDR), as we reported in our previous study. Moreover, the survival role of the MDR-marker P-glycoprotein appears to be involved in the mechanism of invasion of melanoma cells. The results reported herein indicate that TTO and its main active component, terpinen-4-ol, can also interfere with the migration and invasion processes of drug-sensitive and drug-resistant melanoma cells.

BACKGROUND: Transposable Elements (TEs) comprise nearly 45% of the entire genome and are part of sophisticated regulatory network systems that control developmental processes in normal and pathological conditions. The retroviral/retrotransposon gene machinery consists mainly of Long Interspersed Nuclear Elements (LINEs-1) and Human Endogenous Retroviruses (HERVs) that code for their own endogenous reverse transcriptase (RT). Interestingly, RT is typically expressed at high levels in cancer cells. Recent studies report that RT inhibition by non-nucleoside reverse transcriptase inhibitors (NNRTIs) induces growth arrest and cell differentiation in vitro and antagonizes growth of human tumors in animal model. In the present study we analyze the anticancer activity of Abacavir (ABC), a nucleoside reverse transcription inhibitor (NRTI), on PC3 and LNCaP prostate cancer cell lines. PRINCIPAL FINDINGS: ABC significantly reduces cell growth, migration and invasion processes, considerably slows S phase progression, induces senescence and cell death in prostate cancer cells. Consistent with these observations, microarray analysis on PC3 cells shows that ABC induces specific and dose-dependent changes in gene expression, involving multiple cellular pathways. Notably, by quantitative Real-Time PCR we found that LINE-1 ORF1 and ORF2 mRNA levels were significantly up-regulated by ABC treatment. CONCLUSIONS: Our results demonstrate the potential of ABC as anticancer agent able to induce antiproliferative activity and trigger senescence in prostate cancer cells. Noteworthy, we show that ABC elicits up-regulation of LINE-1 expression, suggesting the involvement of these elements in the observed cellular modifications.

Liposomes formulated with dimyristoyl-sn-glycero-phosphatidylcholine, DMPC, and either one of the cationic gemini surfactants (S,S)-2,3-dimethoxy-1,4-bis(N-hexadecyl-N,N-dimethylammonio)butane bromide, 1a, and (S,R)-2,3-dimethoxy-1,4-bis(N-hexadecyl-N,N-dimethylammonio)butane bromide, 1b, were investigated as vehicles of the photosensitizer m-tetrahydroxyphenylchlorin, m-THPC, to cell models of malignant glioma. The delivery efficiency of DMPC/1a and DMPC/1b liposome formulations were evaluated on the murine glioblastoma cell line C6 and on the human glioblastoma cell line LN229 by flow cytometry and laser scanning confocal microscopy. The stereochemistry of the spacer of the gemini was found to strongly influence the delivery efficiency of m-THPC to cells, the mode of interaction with the cell membrane, and the intracellular distribution of m-THPC. The physicochemical features of liposomes were investigated with the aim of explaining the parameters that control their biological features. Differences that could account for the different biological activity of the formulations concern the values of surface potential and the environment of m-THPC at the water/liposome interface.

Malignant melanoma shows high levels of intrinsic drug resistance associated with a highly invasive phenotype. In this study, we investigated the role of the drug transporter P-glycoprotein (Pgp) in the invasion potential of drug-sensitive (M14 WT, Pgp-negative) and drug-resistant (M14 ADR, Pgp-positive) human melanoma cells. Coimmunoprecipitation experiments assessed the association of Pgp with the adhesion molecule CD44 in multidrug resistant (MDR) melanoma cells, compared with parental ones. In MDR cells, the two proteins colocalized in the plasma membrane as visualized by confocal microscopy and immunoelectron microscopy on ultrathin cryosections. MDR melanoma cells displayed a more invasive phenotype compared with parental cells, as demonstrated by quantitative transwell chamber invasion assay. This was accomplished by a different migration strategy adopted by resistant cells (“chain collective”) previously described in tumor cells with high metastatic capacity. The Pgp molecule, after stimulation with specific antibodies, appeared to cooperate with CD44, through the activation of ERK1/2 and p38 mitogen-activated protein kinase (MAPK) proteins. This activation led to an increase of metalloproteinase (MMP-2, MMP-3, and MMP-9) mRNAs, and proteolytic activities, which are associated with an increased invasive behavior. RNA interference experiments further demonstrated Pgp involvement in migration and invasion of resistant melanoma cells. A link was identified between MDR transporter Pgp, and MAPK signaling and invasion.

Poster presented at the "Supramolecular Chemistry Day – 2nd Edition", 22 October 2020.

Oral presentation at the "Photodynamic Therapy and Photodiagnosis Update E-CONGRESS 2020", 5-6 November 2020.

Introduction Acquisition of mesenchymal characteristics confers to breast cancer (BC) cells the capability of invading tissues different from primary tumor site, allowing cell migration and metastasis. Regulators of the mesenchymal-epithelial transition (MET) may represent targets for anticancer agents. Accruing evidence supports functional implications of choline phospholipid metabolism in oncogene-activated cell signaling and differentiation. We investigated the effects of D609, a xanthate inhibiting phosphatidylcholine-specific phospholipase C (PC-PLC) and sphingomyelin synthase (SMS), as a candidate regulator of cell differentiation and MET in the highly metastatic BC cell line MDA-MB-231. Methods PC-PLC expression and activity were investigated using confocal laser scanning microscopy (CLSM), immunoblotting and enzymatic assay on human MDA-MB-231 compared with MCF-7 and SKBr3 BC cells and a nontumoral immortalized counterpart (MCF-10A). The effects of D609 on PC-PLC and SMS activity, loss of mesenchymal markers and changes in migration and invasion potential were monitored in MDA-MB-231 cells by enzymatic assays, CLSM, immunoblotting and transwell chamber invasion combined with scanning electron microscopy examinations. Cell proliferation, formation and composition of lipid bodies and cell morphology were investigated in D609-treated BC cells by cell count, CLSM, flow-cytometry of BODIPY-stained cells, nuclear magnetic resonance and thin-layer chromatography. Results PC-PLC (but not phospholipase D) showed 2- to 6-fold activation in BC compared with nontumoral cells, the highest activity (up to 0.4 pmol/μg protein/min) being detected in the poorly-differentiated MDA-MB-231 cells. Exposure of the latter cells to D609 (50 μg/mL, 24-72 h) resulted into 60-80% PC-PLC inhibition, while SMS was transiently inhibited by a maximum of 21%. These features were associated with progressive decreases of mesenchymal traits such as vimentin and N-cadherin expression, reduced galectin-3 and milk fat globule EGF-factor 8 levels, β-casein formation and decreased in vitro cell migration and invasion. Moreover, proliferation arrest, changes in cell morphology and formation of cytosolic lipid bodies typical of cell differentiation were induced by D609 in all investigated BC cells. Conclusions These results support a critical involvement of PC-PLC in controlling molecular pathways responsible for maintaining a mesenchymal-like phenotype in metastatic BC cells and suggests PC-PLC deactivation as a means to promote BC cell differentiation and possibly enhance the effectiveness of antitumor treatments.