16 research outputs found
Desmoglein 3 and keratin 10 expressions are reduced by chronic exposure to cigarette smoke in human keratinized oral mucosa explants
No full textOBJECTIVE: Oral mucosa is a physiological barrier against several exogenous stimuli, among which cigarette smoke represents a source of reactive oxidizing compounds. No morphological evidences exist on the smoke effects induced in the human oral epithelium. In this study we performed a preliminary light and transmission electron microscopy morphological evaluation focussing in particular on keratinocyte intercellular adhesion and terminal differentiation in chronic smokers.
DESIGN: Human biopsies were obtained from healthy young chronic smoker women (n=5) compared with a parallel group of non-smoker healthy volunteers (n=5), as the smoking habit among women is ever more spreading. Samples were processed for light and transmission electron microscopy. On paraffin sections Masson''s and Dane and Herman''s histochemical staining were performed. Biomarker expressions of intercellular adhesion (desmoglein 3, Dsg3), terminal differentiation (keratin 10, K10 and keratin 14, K14), and basal membrane preservation (laminin) were investigated by immunofluorescence.
RESULTS: In both groups the epithelial structural integrity, homeostasis, and the basal membrane were comparable. Dsg3 and K10 expressions were affected in smokers with the former significantly reduced (p<0.05). Ultrastructural analysis showed hypertrophic keratinocytes in the upper spinous layer and morphologically preserved desmosomes throughout the epithelial compartment.
CONCLUSIONS: The reduction of Dsg3 and K10 expressions indicates that the overall process of keratinocyte terminal differentiation was altered. These preliminary results strongly suggest that Dsg3 and K10 can represent valuable immunomarkers to evaluate the tissue attempt to respond to an exogenous stress such as chronic cigarette smoke, but further samples need to be analysed
Cancer Stem Cells: Devil or Savior—Looking behind the Scenes of Immunotherapy Failure
No full textAlthough the introduction of immunotherapy has tremendously improved the prognosis of patients with metastatic cancers of different histological origins, some tumors fail to respond or develop resistance. Broadening the clinical efficacy of currently available immunotherapy strategies requires an improved understanding of the biological mechanisms underlying cancer immune escape. Globally, tumor cells evade immune attack using two main strategies: avoiding recognition by immune cells and instigating an immunosuppressive tumor microenvironment. Emerging data suggest that the clinical efficacy of chemotherapy or molecularly targeted therapy is related to the ability of these therapies to target cancer stem cells (CSCs). However, little is known about the role of CSCs in mediating tumor resistance to immunotherapy. Due to their immunomodulating features and plasticity, CSCs can be especially proficient at evading immune surveillance, thus potentially representing the most prominent malignant cell component implicated in primary or acquired resistance to immunotherapy. The identification of immunomodulatory properties of CSCs that include mechanisms that regulate their interactions with immune cells, such as bidirectional release of particular cytokines/chemokines, fusion of CSCs with fusogenic stromal cells, and cell-to-cell communication exerted by extracellular vesicles, may significantly improve the efficacy of current immunotherapy strategies. The purpose of this review is to discuss the current scientific evidence linking CSC biological, immunological, and epigenetic features to tumor resistance to immunotherapy
Pancreatic cancer cells retain the epithelial-related phenotype and modify mitotic spindle microtubules after the administration of ukrain in vitro
No full textThe aim of this study is to characterize the phenotype of
pancreatic ductal adenocarcinoma (PDAC) cells in relation
to the expression of epithelial-to-mesenchymal transition
(EMT) markers and determine whether ukrain, an
anticancer drug based on the alkaloids extracted from
greater celandine, modulates in vitro the malignant
behavior of PDAC cells in order to extend our
understanding of its therapeutic potential. Three cell lines
(HPAF-II, HPAC, and PL45) were treated with ukrain (5, 10,
and 20 lmol/l) for 48 h or left untreated (control). Cell
proliferation was assessed by growth curves. Apoptosis
was determined by Hoechst nuclear staining and by
cytochrome c and caspase-8 expressions. The EMT
markers E-cadherin, b-catenin, and vimentin, as well as
actin and tubulin cytoskeletons, were analyzed by
immunofluorescence. Interphase and mitotic microtubules
as well as abnormal mitotic figures were studied by
fluorescence microscopy after tubulin immunolabeling.
Ukrain strongly suppressed cell proliferation and induced
apoptosis possibly through an extrinsic pathway as
cytochrome c immunoreactivity suggested that the integrity
of the mitochondria was not affected. Tubulin expression
indicated an antiproliferative effect of ukrain on the basis of
alterations in mitotic spindle microtubule dynamics,
leading to abnormal mitosis. Membranous E-cadherin/
b-catenin immunoreactivity was similarly expressed in
control-treated and ukrain-treated cells, although the drug
upregulated E-cadherin in cell lysates. Our results suggest
that ukrain exerts its chemotherapeutic action on PDAC
cells targeting mitotic spindle microtubules, leading to
abnormal mitosis and apoptosis, and favoring cell
cohesiveness. The differentiated epithelial phenotype of
HPAF-II, HPAC, and PL45 cell lines concomitant with a
highly invasive potential suggests that further experiments
will be necessary to definitively clarify the role of EMT in
PDAC progression
Malignant phenotype of renal cell carcinoma cells is switched by Ukrain administration in vitro
No full textWe investigated whether Ukrain modulates the malignant phenotype of clear cell renal cell carcinoma (ccRCC) cells Caki-1, Caki-2, and ACHN treated with four doses (5, 10, 20, and 40 μmol/l) for 24 and 48 h. The epithelial-to-mesenchymal transition markers E-cadherin, β-catenin, and vimentin were analyzed by immunofluorescence as well as actin and tubulin; matrix metalloproteinase-2 and matrix metalloproteinase-9 activity was analyzed by SDS-zymography, intracellular and secreted SPARC levels by western blot, and cell cycle by flow cytometry. Ukrain did not induce E-cadherin/β-catenin immunoreactivity at the cell-cell boundary, although it determined the actin cortical expression in Caki-2 and ACHN, and did not affect vimentin organization; however, in some Caki-1 and ACHN cells the perinuclear concentration of vimentin was consistent with its downregulation. Matrix metalloproteinase-2 and matrix metalloproteinase-9 activity was significantly downregulated 48 h after 20 μmol/l Ukrain administration. At this time point, Ukrain significantly decreased migration and invasion, and downregulated SPARC levels in cell supernatants at all doses in Caki-2, and at 20 μmol/l in Caki-1 and ACHN cells. Concomitantly, SPARC was upregulated in all ccRCC cells, suggesting that Ukrain could also affect cell proliferation by cell cycle inhibition, as supported by the cell cycle analysis, as SPARC also acts as a cell cycle inhibitor. Our results suggest that Ukrain may switch the epithelial-to-mesenchymal transition-related phenotype of ccRCC cells, and targets the two major aspects involved in RCC progression, such as tumor invasion/microenvironment remodeling and cell proliferation
WNT signaling modulates PD-L1 expression in the stem cell compartment of triple-negative breast cancer
No full textTriple-negative breast cancers (TNBCs) are characterized by a poor prognosis and lack of targeted treatments, and thus, new therapeutic strategies are urgently needed. Inhibitors against programmed death-1 (PD-1)/PD-1 ligand (PD-L1) have shown significant efficacy in various solid cancers, but their activity against TNBCs remains limited. Here, we report that human TNBCs molecularly stratified for high levels of PD-L1 (PD-L1High) showed significantly enriched expression of immune and cancer stemness pathways compared with those with low PD-L1 expression (PD-L1Low). In addition, the PD-L1High cases were significantly associated with a high stemness score (SSHigh) signature. TNBC cell lines gated for aldehyde dehydrogenase (ALDH) and CD44 stemness markers exhibited increased levels of PD-L1 versus their ALDH-negative and CD44Low counterparts, and PD-L1High cells generated significantly more mammospheres than PD-L1Low cells. Murine mammary SCA-1-positive tumor cells with PD-L1High expression generated tumors in vivo with higher efficacy than PD-L1Low cells. Furthermore, treatment of TNBC cells with selective WNT inhibitors or activators downregulated or upregulated PD-L1 expression, respectively, implying a functional cross-talk between WNT activity and PD-L1 expression. Remarkably, human TNBC samples contained tumor elements co-expressing PD-L1 with ALDH1A1 and/or CD44v6. Additionally, both PD-L1-/SCA1-positive and ALDH1A1-positive tumor elements were found in close contact with CD3-, and PD-1-positive T cells in murine and human tumor samples. Overall, our study suggests that PD-L1-positive tumor elements with a stemness phenotype may participate in the complex dynamics of TNBC-related immune evasion, which might be targeted through WNT signaling inhibition
Transcriptomics and metabolomics integration reveals redox-dependent metabolic rewiring in breast cancer cells
No full textRewiring glucose metabolism toward aerobic glycolysis provides cancer cells with a rapid generation of pyruvate, ATP, and NADH, while pyruvate oxidation to lactate guarantees refueling of oxidized NAD+ to sustain glycolysis. CtPB2, an NADH-dependent transcriptional co-regulator, has been proposed to work as an NADH sensor, linking metabolism to epigenetic transcriptional reprogramming. By integrating metabolomics and transcriptomics in a triple-negative human breast cancer cell line, we show that genetic and pharmacological down-regulation of CtBP2 strongly reduces cell proliferation by modulating the redox balance, nucleotide synthesis, ROS generation, and scavenging. Our data highlight the critical role of NADH in controlling the oncogene-dependent crosstalk between metabolism and the epigenetically mediated transcriptional program that sustains energetic and anabolic demands in cancer cells
Molecular basis of telaprevir resistance due to V36 and T54 mutations in the NS3-4A protease of the hepatitis C virus
No full textBackground The inhibitor telaprevir (VX-950) of the hepatitis C virus (HCV) protease NS3-4A has been tested in a recent phase 1b clinical trial in patients infected with HCV genotype 1. This trial revealed residue mutations that confer varying degrees of drug resistance. In particular, two protease positions with the mutations V36A/G/L/M and T54A/S were associated with low to medium levels of drug resistance during viral breakthrough, together with only an intermediate reduction of viral replication fitness. These mutations are located in the protein interior and far away from the ligand binding pocket. Results Based on the available experimental structures of NS3-4A, we analyze the binding mode of different ligands. We also investigate the binding mode of VX-950 by protein-ligand docking. A network of non-covalent interactions between amino acids of the protease structure and the interacting ligands is analyzed to discover possible mechanisms of drug resistance. We describe the potential impact of V36 and T54 mutants on the side chain and backbone conformations and on the non-covalent residue interactions. We propose possible explanations for their effects on the antiviral efficacy of drugs and viral fitness. Molecular dynamics simulations of T54A/S mutants and rotamer analysis of V36A/G/L/M side chains support our interpretations. Experimental data using an HCV V36G replicon assay corroborate our findings. Conclusion T54 mutants are expected to interfere with the catalytic triad and with the ligand binding site of the protease. Thus, the T54 mutants are assumed to affect the viral replication efficacy to a larger degree than V36 mutants. Mutations at V36 and/or T54 result in impaired interaction of the protease residues with the VX-950 cyclopropyl group, which explains the development of viral breakthrough variants
Anticancer innovative therapy: Highlights from the ninth annual meeting
No full textThe Ninth Annual Conference of “Anticancer Innovative Therapy”, organized by Fondazione IRCCS Istituto Nazionale dei Tumori di Milano (Fondazione IRCCS INT) and hosted by Hotel Michelangelo, was held in Milan on 25 January 2019. Cutting-edge science was presented in two main scientific sessions: i) pre-clinical evidences and new targets, and ii) clinical translation. The Keynote lecture entitled “Cancer stem cells (CSCs): metabolic strategies for their identification and eradication” presented by M. Lisanti, was one of the highlights of the conference. One key concept of the meeting was how the continuous advances in our knowledge about molecular mechanisms in various fields of research (cancer metabolism reprogramming, epigenetic regulation, transformation/invasiveness, and immunology, among others) are driving cancer research towards more effective personalized antineoplastic strategies. Specifically, recent preclinical data on the following topics were discussed: 1. Polycomb group proteins in cancer; 2. A d16HER2 splice variant is a flag of HER2 addiction across HER2-positive cancers; 3. Studying chromatin as a nexus between translational and basic research; 4. Metabolomic analysis in cancer patients; 5. CDK4-6 cyclin inhibitors: clinical activity and future perspectives as immunotherapy adjuvant; and 6. Cancer stem cells (CSCs): metabolic strategies for their identification and eradication. In terms of clinical translation, several novel approaches were presented: 1. Developing CAR-T cell therapies: an update of preclinical and clinical development at University of North Carolina; 2. Vγ9Vδ2 T-cell activation and immune suppression in multiple myeloma; 3. Predictive biomarkers for real-world immunotherapy: the cancer immunogram model in the clinical arena; and 4. Mechanisms of resistance to immune checkpoint blockade in solid tumors. Overall, the pre-clinical and clinical findings presented could pave the way to identify novel actionable therapeutic targets to significantly enhance the care of persons with cancer
Modeling of Intracellular Taurine Levels Associated with Ovarian Cancer Reveals Activation of p53, ERK, mTOR and DNA-damage-sensing-dependent Cell Protection
No full textTaurine, a non-proteogenic amino acid, and commonly used nutritional supplement can protect various tissues from degeneration associated with the action of the DNA-damaging chemotherapeutic agent cisplatin. Whether and how taurine protects human ovarian cancer (OC) cells from DNA damage caused by cisplatin is not well understood. We have found that OC ascites-derived cells contained significantly more intracellular taurine than cell cultures modeling OC. In culture, elevation of intracellular taurine concentration to OC ascites-cells-associated levels suppressed proliferation of various OC cell lines and patient-derived organoids, reduced glycolysis, and induced cell protection from cisplatin. Taurine cell protection was associated with decreased DNA damage in response to cisplatin. A combination of RNA sequencing, reverse phase protein arrays, live-cell microscopy, flow cytometry, and biochemical validation experiments provided evidence for taurine-mediated induction of mutant- or wild-type p53 binding to DNA, and activation of p53 effectors involved in negative regulation of the cell cycle (p21), and glycolysis (TIGAR). Paradoxically, taurine's suppression of cell proliferation was associated with activation of pro-mitogenic signal transduction including ERK, mTOR, and increased mRNA expression of major DNA damage sensing molecules such as DNAPK, ATM and ATR. While inhibition of ERK or p53 did not interfere with taurine's ability to protect cells from cisplatin, suppression of mTOR with Torin2, a clinically relevant inhibitor that also targets DNAPK and ATM/ATR, broke taurine's cell protection. Our studies implicate that elevation of intracellular taurine could suppress cell growth, metabolism, and activate cell protective mechanisms involving mTOR and DNA damage sensing signal transduction
