Cytokine Plasma Levels in Breast Cancer Patients, Before and After Surgery.

Studying the levels of cytokines in the plasma of patients could be valuable in guiding immunotherapy policies. We assessed the plasma levels of 4 major cytokines [interferon (IFN)-ß, interleukin-2 (IL-2), tumor necrosis factor alpha (TNF-α), transforming growth factor beta (TGF-ß)] collected from 19 patients with ductal breast cancer (BCa), before surgery (BS) and 5 days after surgery (AS). The ratio AS/BS was also calculated and correlated with histopathological variables and tumor-infiltrating lymphocyte (TIL) density. The IFN-ß and TNF-α levels were significantly higher in BCa patients, BS and AS, than healthy controls (P < 0.02). High IL-2 levels BS were linked with node involvement (P = 0.02), and marginally with HER2 expression (P = 0.08), while high TNF-α levels were linked with high PgR expression (P = 0.02). Increasing IFN-ß, IL-2, and TNF-α levels were noted AS, which was more evident in patients with larger tumors. The TGF-ß levels were significantly lower in BCa patients (P < 0.007). Linear regression analysis showed a direct association of IFN-ß levels AS (P = 0.02, r = 0.52) and of TNF-α AS/BS-ratio (P = 0.001, r = 0.72) with TIL-density. It is suggested that although effector immune response is evident in the majority of early stage BCa patients, removal of the primary tumor further unblocks such responses.

Synthesis of 2-Amino-N'-aroyl(het)arylhydrazides, DNA Photocleavage, Molecular Docking and Cytotoxicity Studies against Melanoma CarB Cell Lines.

Diacylhydrazine bridged anthranilic acids with aryl and heteroaryl domains have been synthesized as the open flexible scaffold of arylamide quinazolinones in order to investigate flexibility versus rigidity towards DNA photocleavage and sensitivity. Most of the compounds have been synthesized via the in situ formation of their anthraniloyl chloride and subsequent reaction with the desired hydrazide and were obtained as precipitates, in moderate yields. All compounds showed high UV-A light absorption and are eligible for DNA photocleavage studies under this "harmless" irradiation. Despite their reduced UV-B light absorption, a first screening indicated the necessity of a halogen at the p-position in relation to the amine group and the lack of an electron-withdrawing group on the aryl group. These characteristics, in general, remained under UV-A light, rendering these compounds as a novel class of UV-A-triggered DNA photocleavers. The best photocleaver, the compound 9, was active at concentrations as low as 2 µΜ. The 5-Nitro-anthranilic derivatives were inactive, giving the opposite results to their related rigid quinazolinones. Molecular docking studies with DNA showed possible interaction sites, whereas cytotoxicity experiments indicated the iodo derivative 17 as a potent cytotoxic agent and the compound 9 as a slight phototoxic compound.

Hypoxia and acidity regulate immune checkpoint molecule and IFN-ß expression in non-small cell lung cancer cell lines.

PURPOSE: Non-small cell lung cancer (NSCLC) is one of the most lethal tumors in humans. Immunotherapy with immune checkpoint inhibitors (ICIs) has revolutionized the treatment of patients with advanced diseases. Tumor microenvironment conditions like hypoxia and low pH may compromise the efficacy of ICIs. MATERIALS AND METHODS: We report the effect of hypoxia and acidity on the expression levels of the major checkpoint molecules, namely PD-L1, CD80, and CD47, in the A549 and H1299 NSCLC cell lines. RESULTS: Hypoxia induces PD-L1 protein and mRNA expression, represses CD80 mRNA levels, and enhances IFNß protein expression. An opposite effect was noticed when cells were exposed to acidic conditions. Hypoxia-induced the CD47 molecule at protein and mRNA levels. It is concluded that hypoxia and acidity are important regulators of the expression of PD-L1 and CD80 immune checkpoint molecules. Acidity contributes to the suppression of the interferon type I pathway. CONCLUSIONS: These findings suggest that hypoxia and acidity assist cancer cells in the escape from immune surveillance through direct effects on cancer cells' ability to present immune checkpoint molecules and release type I interferons. Targeting hypoxia and acidity may enhance the activity of ICIs in NSCLC.

Applications and Advances of Multicellular Tumor Spheroids: Challenges in Their Development and Analysis.

Biomedical research requires both in vitro and in vivo studies in order to explore disease processes or drug interactions. Foundational investigations have been performed at the cellular level using two-dimensional cultures as the gold-standard method since the early 20th century. However, three-dimensional (3D) cultures have emerged as a new tool for tissue modeling over the last few years, bridging the gap between in vitro and animal model studies. Cancer has been a worldwide challenge for the biomedical community due to its high morbidity and mortality rates. Various methods have been developed to produce multicellular tumor spheroids (MCTSs), including scaffold-free and scaffold-based structures, which usually depend on the demands of the cells used and the related biological question. MCTSs are increasingly utilized in studies involving cancer cell metabolism and cell cycle defects. These studies produce massive amounts of data, which demand elaborate and complex tools for thorough analysis. In this review, we discuss the advantages and disadvantages of several up-to-date methods used to construct MCTSs. In addition, we also present advanced methods for analyzing MCTS features. As MCTSs more closely mimic the in vivo tumor environment, compared to 2D monolayers, they can evolve to be an appealing model for in vitro tumor biology studies.

Effect of Arylazo Sulfones on DNA: Binding, Cleavage, Photocleavage, Molecular Docking Studies and Interaction with A375 Melanoma and Non-Cancer Cells.

A set of arylazo sulfones, known to undergo N-S bond cleavage upon light exposure, has been synthesized, and their activity in the dark and upon irradiation towards DNA has been investigated. Their interaction with calf-thymus DNA has been examined, and the significant affinity observed (most probably due to DNA intercalation) was analyzed by means of molecular docking "in silico" calculations that pointed out polar contacts, mainly via the sulfonyl moiety. Incubation with plasmid pBluescript KS II revealed DNA cleavage that has been studied over time and concentration. UV-A irradiation considerably improved DNA damage for most of the compounds, whereas under visible light the effect was slightly lower. Moving to in vitro experiments, irradiation was found to slightly enhance the death of the cells in the majority of the compounds. Naphthylazosulfone 1 showed photo-disruptive effect under UV-A irradiation (IC50 ~13 µΜ) followed by derivatives 14 and 17 (IC50 ~100 µΜ). Those compounds were irradiated in the presence of two non-cancer cell lines and were found equally toxic only upon irradiation and not in the dark. The temporal and spatial control of light, therefore, might provide a chance for these novel scaffolds to be useful for the development of phototoxic pharmaceuticals.

Viral-induced inflammation can lead to adverse pregnancy outcomes.

INTRODUCTION: Parvoviruses are DNA viruses of small size. There have been a number of reports indicating the possible effects of B19 infections during pregnancy. These effects include spontaneous abortions, stillbirth, fetal damage, and quite often, fetal anemia with hydrops fetalis.

Expression of CD47 and SIRPα Macrophage Immune-Checkpoint Pathway in Non-Small-Cell Lung Cancer.

BACKGROUND: Cancer cells escape macrophage phagocytosis by expressing the CD47 integrin-associated protein that binds to the SIRPα ligand (signal regulatory protein alpha) expressed by macrophages. Immunotherapy targeting this pathway is under clinical development. METHODS: We investigated the expression of CD47/SIRPα molecules in a series of 98 NSCLCs, in parallel with the infiltration of tumor stroma by CD68+ macrophages, tumor-infiltrating lymphocytes (TILs), and PD-L1/PD-1 molecules. RESULTS: Extensive membranous CD47 expression by cancer cells characterized 29/98 cases. SIRPα and CD68 were expressed, to a varying extent, by tumor-associated macrophages (Μφ, TAMs). A high CD68Mφ-score in inner tumor areas was linked with improved overall survival (p = 0.005); and this was independent of the stage (p = 0.02, hazard ratio 0.4). In contrast, high SIRPα expression by CD68+ TAMs (SIRPα/CD68-ratio) was linked with CD47 expression by cancer cells, low TIL-score, and poor prognosis (p = 0.02). A direct association of CD47 expression by cancer cells and the % FOXP3+ TILs (p = 0.01, r = 0.25) was also noted. CONCLUSIONS: TAMs play an important role in the prognosis of operable NSCLC. As SIRPα+ macrophages adversely affect prognosis, it is suggested that the CD47/SIRPα axis is a sound target for adjuvant immunotherapy policies, aiming to improve the cure rates in operable NSCLC.

Loss of HLA-class-I expression in non-small-cell lung cancer: Association with prognosis and anaerobic metabolism.

Cancer immuno-editing frequently leads to loss of HLA-class-I molecule (HLA) expression and impaired immune surveillance. We investigated the expression of HLAs in a series of operable non-small-cell lung carcinomas (NSCLCs). Complete loss and extensive loss of expression was noted in 41.5% and 23.4% of cases, respectively. Low CD8 + and FOXP3 + TIL-density was significantly associated with loss of HLAs (p < 0.05 and p = 0.003, respectively). High PD-L1 expression was linked with sustained expression of HLAs. A significant association of loss of HLA-expression with overexpression of LDH5 (p = 0.01) and marginally with HIF1α was recorded. Cell line experiments confirmed that hypoxia and acidity down-regulate the expression of HLAs. A direct association between HLAs and Beclin-1 expression was also noted (p = 0.01). Loss of HLA-expression was linked with poorer survival (p < 0.01), independent of stage. It is concluded that loss of HLA-class-I molecules is frequent in NSCLC and directly linked to micro-environmental hypoxia and acidity.

Suppressed PLIN3 frequently occurs in prostate cancer, promoting docetaxel resistance via intensified autophagy, an event reversed by chloroquine.

Lipid metabolism reprogramming is one of the adaptive events that drive tumor development and survival, and may account for resistance to chemotherapeutic drugs. Perilipins are structural proteins associated with lipophagy and lipid droplet integrity, and their overexpression is associated with tumor aggressiveness. Here, we sought to explore the role of lipid droplet-related protein perilipin-3 (PLIN3) in prostate cancer (PCa) chemotherapy. We investigated the role of PLIN3 suppression in docetaxel cytotoxic activity in PCa cell lines. Additional effects of PLIN3 depletion on autophagy-related proteins and gene expression patterns, apoptotic potential, proliferation rate, and ATP levels were examined. Depletion of PLIN3 resulted in docetaxel resistance, accompanied by enhanced autophagic flux. We further assessed the synergistic effect of autophagy suppression with chloroquine on docetaxel cytotoxicity. Inhibition of autophagy with chloroquine reversed chemoresistance of stably transfected shPLIN3 PCa cell lines, with no effect on the parental ones. The shPLIN3 cell lines also exhibited reduced Caspase-9 related apoptosis initiation. Moreover, we assessed PLIN3 expression in a series of PCa tissue specimens, were complete or partial loss of PLIN3 expression was frequently noted in 70% of the evaluated specimens. Following PLIN3 silencing, PCa cells were characterized by impaired lipophagy and acquired an enhanced autophagic response upon docetaxel-induced cytotoxic stress. Such an adaptation leads to resistance to docetaxel, which could be reversed by the autophagy blocker chloroquine. Given the frequent loss of PLIN3 expression in PCa specimens, we suggest that combination of docetaxel with chloroquine may improve the efficacy of docetaxel treatment in PLIN3-deficient cancer patients.

Prognostic Relevance of the Relative Presence of CD4, CD8 and CD20 Expressing Tumor Infiltrating Lymphocytes in Operable Non-small Cell Lung Cancer Patients.

BACKGROUND/AIM: Non-small cell lung cancer (NSCLC) is one of the most lethal tumors. Given the failure of conventional therapeutic strategies, immunotherapy has emerged as a promising treatment modality that may improve the survival of patients with operable and advanced disease. PATIENTS AND METHODS: We examined the relative presence of CD20+ B-cells, CD8+ cytotoxic, and CD4+ helper/regulatory T-cells in the tumor-infiltrating lymphocyte (TIL)-population in a series of surgically-treated NSCLCs, and assessed their role as prognostic indicators after surgery. RESULTS: A high percent of CD4+ and CD8+ TILs in the tumor stroma was linked with poor (p=0.003) and good prognosis (p=0.01), respectively. High CD4/CD8 ratio defined a significantly worst prognosis [median survival 22 months vs. undefined, p=0.0002, hazard ratio (HR) 0.3 vs. 3.0]. Statistically significant results were also noted when the analysis was focused on the invading tumor front. In a multivariate model, the CD4/CD8-ratio assessed in the tumor stroma and the stage of disease were independent prognostic variables (p=0.0001, HR=4.1 and p=0.001, HR=1.5, respectively). CONCLUSION: The balance between CD4+ and CD8+ lymphocytes infiltrating the tumor stroma is a crucial factor defining anti-tumor immune surveillance, has strong prognostic value, and may be tested as a predictive biomarker for immunotherapy in operable NSCLC.

6-Nitro-Quinazolin-4(3H)-one Exhibits Photodynamic Effects and Photodegrades Human Melanoma Cell Lines. A Study on the Photoreactivity of Simple Quinazolin-4(3H)-ones.

Photochemo and photodynamic therapies are minimally invasive approaches for the treatment of cancers and powerful weapons for competing bacterial resistance to antibiotics. Synthetic and naturally occurring quinazolinones are considered privileged anticancer and antibacterial agents, with several of them to have emerged as commercially available drugs. In the present study, applying a single-step green microwave irradiation mediated protocol we have synthesized eleven quinazolinon-4(3H)-ones, from cheap readily available anthranilic acids, in very good yields and purity. These products were irradiated in the presence of pBR322 plasmid DNA under UVB, UVA and visible light. Four of the compounds proved to be very effective DNA photocleavers, at low concentrations, being time and concentration dependent as well as pH independent. Participation of reactive oxygen species was related to the substitution of quinazolinone derivatives. 6-Nitro-quinazolinone in combination with UVA irradiation was found to be in vitro photodestructive for three cell lines; glioblastoma (U87MG and T98G) and mainly melanoma (A-375). Thus, certain appropriately substituted quinazolinones may serve as new lead photosensitizers for the development of promising biotechnological applications and as novel photochemo and photodynamic therapeutics.

Ectonucleotidase CD73 and CD39 expression in non-small cell lung cancer relates to hypoxia and immunosuppressive pathways.

AIMS: Adenosine triphosphate (ATP) is released at a high concentration in the tumor microenvironment. The overexpression of ectonucleotidases in non-small-cell lung cancer (NSCLC), metabolizing ΑΤP to the immunosuppressive adenosine, is studied. MATERIALS AND METHODS: We examined the expression of the ectonucleotidases CD73 and CD39 in NSCLC in parallel with immunological parameters and markers of hypoxia and anaerobic metabolism. In vitro experiments with A549 and H1299 lung cancer cell lines were also conducted. RESULTS: CD73 and CD39 were not expressed by normal bronchial and alveolar epithelium. In contrast, these were overexpressed by cancer cells, cancer-associated fibroblasts (CAFs), and tumor-infiltrating lymphocytes (TILs). High CD73 cancer cell expression was directly linked with lactate dehydrogenase LDH5 and with hypoxia-inducible factor HIF1α expression by cancer cells. The expression of CD39 by CAFs was directly linked with PD-L1 expression by cancer cells. A significant abundance of FOXP3+ and PD-1+ TILs was noted in tumors with high CD73 and CD39 stroma expression. In in vitro experiments, hypoxia and acidity induced CD73 mRNA and protein levels in cancer cell lines. Exposure of cancer cell lines to adenosine induced the expression of PD-L1 and LDHA mRNA and protein levels. CONCLUSION: Ectonucleotidases are up-regulated in cancer cells, CAFs, and TILs in lung tumors. Such overexpression is linked with regulatory TIL-phenotype and PD-L1 up-regulation by cancer cells. Overexpression of LDH5 is up-regulated by adenosine, creating a vicious cycle, as the high amounts of ATP produced by LDH5-mediated anaerobic glycolysis promote the production of adenosine by a tumor microenvironment rich in ectonucleotidases.

Programmed death-1 receptor (PD-1) and PD-ligand-1 (PD-L1) expression in non-small cell lung cancer and the immune-suppressive effect of anaerobic glycolysis.

The microenvironment of a tumor may regulate the anti-tumor immune response. Intratumoral acidosis and hypoxia may suppress lymphocyte proliferation and migration, and this may have important implications in modern immunotherapy. The expression of PD-L1 by cancer cells and of PD-1 by tumor infiltrating lymphocytes (TILs) was assessed in tissue specimens from 98 operable NSCLC patients. Their prognostic role and their association with makers of glycolysis and anaerobic metabolism were assessed. Strong cytoplasmic/membrane PD-L1 expression was noted in 45/98 cases. Intense presence of TILs was noted in 42/98 cases (high TIL-score), and intense presence of PD-1 expressing TILs (high PIL-score) in 17/98 cases. PD-L1 expression was directly correlated with high PIL-score (p = 0.005). A significant inverse relationship was found between lactate dehydrogenase LDH5 expression and PIL-score (p = 0.008). Similarly, low PIL-score was significantly linked with high-hexokinase HXKII and monocarboxylate transporter MCT2 expression (p < 0.04). Cases with both intense TIL-score and PIL-score had significantly better survival (p < 0.05). For patients with high TIL-score or high PIL-score, PD-L1 overexpression defined significantly poorer survival (p = 0.01 and p = 0.03, respectively). In multivariate analysis, stage (p = 0.002, HR 3.33, 95%CI 1.4-4.5) and TIL-score (p = 0.02, HR 2.12, 95%CI 1.1-4.0) were independent predictive variables of death events. Given the low specificity of PD-L1 as a biomarker for anti-PD-1/PD-L1 immunotherapy, a combined assessment of TIL, PD-L1, PD-1, and LDH5 provides a tool for an immunological/metabolic classification of NSCLC tumors, with a different prognosis and different expected response to anti-PD-1/PD-L1 immunotherapy, which should be considered in relevant clinical trials.

CYP17A1 and Androgen-Receptor Expression in Prostate Carcinoma Tissues and Cancer Cell Lines.

BACKGROUND: CYP17A1 is involved in the steroidogenesis of dehydroepiandrosterone and androstenedione. CYP17A is a target for the hormonal treatment of prostate cancer (PCa). OBJECTIVES: To investigate the role of CYP17A1 as a driver of PCa growth. MATERIALS AND METHODS: We examined the expression of CYP17A1 and of androgen receptors (AR) in PCa specimens and in PCa cell lines. RESULTS: CYP17A1 was strongly expressed in the cytoplasm of PCa cells (median 50% of cancer cells, range 0-100%). The nuclear AR expression in cancer cells was directly related with CYP17A1 (p < 0.0001, r = 0.51). The hormone dependent 22Rv1 cell line expressed the CYP17A1 and AR protein and mRNA, in contrast to the PC3 and DU145 cell lines (p < 0.0001). Testosterone and dexamethasone induced nuclear expression of AR and this effect was abolished by abiraterone. CYP17A1 levels were not affected by the incubation with testosterone, while abiraterone significantly reduced its expression. Abiraterone reduced the growth rate and migration of testosterone stimulated 22Rv1 cells. CONCLUSIONS: CYP17A1 is strongly expressed in half about of human prostate carcinomas, implying an intracellular androgen synthesis by cancer cells. Abiraterone effectively blocked nuclear accumulation of AR and suppressed CYP17A1 expression. CYP17A1 may function as a biomarker to select the best hormonal anticancer therapy.

Increased Soluble PD-L1 Levels in the Plasma of Patients with Epithelial Ovarian Cancer Correlate with Plasma Levels of miR34a and miR200.

BACKGROUND: Recently, programmed cell death protein 1 (PD1) blocking and anti-programmed death-ligand 1 (PD-L1) agents were approved for the treatment of various human malignancies. MATERIALS AND METHODS: Our study examined the expression of PD-L1 in neoplastic tissue (17 patients) and the plasma soluble (s)PD-L1 of 32 patients with ovarian carcinoma, in parallel with the levels of specific microRNAs (miRs), using immunohistochemistry, enzyme-linked immunosorbent assay (ELISA) and real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR), respectively. RESULTS: PD-L1 levels were significantly higher in the plasma of patients with ovarian cancer compared to healthy women (p=0.01). High miR200 levels were related to high sPD-L1 levels (p=0.03), whilst high miR34a levels were associated with low sPD-L1 levels (p=0.02). Immunohistochemical expression of PD-L1 by cancer cells was not related to plasma miR levels, nor to the level of sPD-L1. CONCLUSION: As well as cancer cell expression of PD-L1, a high sPD-L1 level characterizes a subset of patients with ovarian cancer. The value of this latter feature as a biomarker for the administration of anti-PD-L1/PD1 therapy needs further evaluation. Micro-RNAs, such as miR34a and miR200, may have a role in the efficacy of immunotherapy.

Autophagic flux response and glioblastoma sensitivity to radiation.

OBJECTIVE: Glioblastoma is the most common primary brain tumor in adults and one of the most lethal human tumors. It constitutes a unique non-metastasizing human tumor model with high resistance to radiotherapy and chemotherapy. The current study investigates the association between autophagic flux and glioblastoma cell resistance. METHODS: The expression kinetics of autophagy- and lysosome-related proteins following exposure of two glioblastoma cell lines (T98 and U87) to clinically relevant radiation doses was examined. Then, the response of cells resistant to radiotherapy and chemotherapy was investigated after silencing of LC3A, LC3B, and TFEB genes in vitro and in vivo. RESULTS: Following irradiation with 4 Gy, the relatively radioresistant T98 cells exhibited enhanced autophagic flux. The more radiosensitive U87 cell line suffered a blockage of autophagic flux. Silencing of LC3A, LC3B, and TFEB genes in vitro, significantly sensitized cells to radiotherapy and temozolomide (U87: P < 0.01 and < 0.05, respectively; T98: P < 0.01 and < 0.01, respectively). Silencing of the LC3A gene sensitized mouse xenografts to radiation. CONCLUSIONS: Autophagy in cancer cells may be a key factor of radio-resistance and chemo-resistance in glioblastoma cells. Blocking autophagy may improve the efficacy of radiochemotherapy for glioblastoma patients.

SMER28 is a mTOR-independent small molecule enhancer of autophagy that protects mouse bone marrow and liver against radiotherapy.

Effective cytoprotectors that are selective for normal tissues could decrease radiotherapy and chemotherapy sequelae and facilitate the safe administration of higher radiation doses. This could improve the cure rates of radiotherapy for cancer patients. Autophagy is a cytoplasmic cellular process that is necessary for the clearance of damaged or aged proteins and organelles. It is a strong determinant of post-irradiation cell fate. In this study, we investigated the effect of the mTOR-independent small molecule enhancer of autophagy (SMER28) on mouse liver autophagy and post-irradiation recovery of mouse bone marrow and liver. SMER28 enhanced the autophagy flux and improved the survival of normal hepatocytes. This effect was specific for normal cells because SMER28 had no protective effect on hepatoma or other cancer cell line survival in vitro. In vivo subcutaneous administration of SMER28 protected mouse liver and bone marrow against radiation damage and facilitated survival of mice after lethal whole body or abdominal irradiation. These findings open a new field of research on autophagy-targeting radioprotectors with clinical applications in oncology, occupational, and space medicine.

Comparison of the effect of the antiandrogen apalutamide (ARN-509) versus bicalutamide on the androgen receptor pathway in prostate cancer cell lines.

Apalutamide (ARN-509) is an antiandrogen that binds selectively to androgen receptors (AR) and does not show antagonist-to-agonist switch like bicalutamide. We compared the activity of ARN versus bicalutamide on prostate cancer cell lines. The 22Rv1, PC3, and DU145 cell lines were used to study the effect of ARN and bicalutamide on the expression cytoplasmic/nuclear kinetics of AR, AR-V7 variant, phosphorylated AR, as well as the levels of the AR downstream proteins prostate-specific antigen and TMPRSS2, under exposure to testosterone and/or hypoxia. The effects on autophagic flux (LC3A, p62, TFEB, LAMP2a, cathepsin D) and cell metabolism-related enzymes (hypoxia-inducible factor 1α/2α, BNIP3, carbonic anhydrase 9, LDHA, PDH, PDH-kinase) were also studied. The 22Rv1 cell line responded to testosterone by increasing the nuclear entry of AR, AR-V7, and phosphorylated AR and by increasing the levels of prostate-specific antigen and TMPRSS2. This effect was strongly abrogated by ARN and to a clearly lower extent by bicalutamide at 10 µmol/l, both in normoxia and in hypoxia. ARN had a stronger antiproliferative effect than bicalutamide, which was prominent in the 22Rv1 hormone-responsive cell line, and completely repressed cell proliferation at a concentration of 100 µmol/l. No effect of testosterone or of antiandrogens on autophagy flux, hypoxia-related proteins, or metabolism enzyme levels was noted. The PC3 and DU145 cell lines showed poor expression of the proteins and were not responsive to testosterone. On the basis of in-vitro studies, evidence has been reported that ARN is more potent than bicalutamide in blocking the AR pathway in normoxia and in hypoxia. This reflects a more robust, dose-dependent, repressive effect on cell proliferation.

Amifostine Protects Mouse Liver Against Radiation-induced Autophagy Blockage.

BACKGROUND/AIM: Amifostine is the only selective normal tissue cytoprotector, approved for the protection against platinum toxicities and radiotherapy-induced xerostomia. Free radical scavenger and DNA repair activities have been attributed to the drug. MATERIALS AND METHODS: We investigated the effect of amifostine on autophagy, lysosomal biogenesis and lipophagy of normal mouse liver exposed to clinically relevant doses of radiation. RESULTS: The study provides evidence that ionizing radiation blocks autophagy activity and lysosomal biogenesis in normal mouse liver. Amifostine, protects the liver autophagic machinery and induces lysosomal biogenesis. By suppressing autophagy, ionizing radiation induces lipid droplet accumulation, while pre-treatment with amifostine protects lipophagy and up-regulates the TIP47 protein and mRNA levels, showing a maintenance of lipid metabolism in the liver cells. CONCLUSION: It is concluded that amifostine, aside to DNA protection activity, exerts its cytoprotective function by preventing radiation-induced blockage of autophagy, lysosomal biogenesis and lipophagy.

Metabolic cooperation between co-cultured lung cancer cells and lung fibroblasts.

Cooperation of cancer cells with stromal cells, such as cancer-associated fibroblasts (CAFs), has been revealed as a mechanism sustaining cancer cell survival and growth. In the current study, we focus on the metabolic interactions of MRC5 lung fibroblasts with lung cancer cells (A549 and H1299) using co-culture experiments and studying changes of the metabolic protein expression profile and of their growth and migration abilities. Using western blotting, confocal microscopy and RT-PCR, we observed that in co-cultures MRC5 respond by upregulating pyruvate dehydrogenase (PDH) and the monocarboxylate transporter MCT1. In contrast, cancer cells increase the expression of glucose transporters (GLUT1), LDH5, PDH kinase and the levels of phosphorylated/inactivated pPDH. H1299 cells growing in the same culture medium with fibroblasts exhibit a 'metastasis-like' phenomenon by forming nests within the fibroblast area. LDH5 and pPDH were drastically upregulated in these nests. The growth rate of both MRC5 and cancer cells increased in co-cultures. Suppression of LDHA or PDK1 in cancer cells abrogates the stimulatory signal from cancer cells to fibroblasts. Incubation of MRC5 fibroblasts with lactate resulted in an increase of LDHB and of PDH expression. Silencing of PDH gene in fibroblasts, or silencing of PDK1 or LDHA gene in tumor cells, impedes cancer cell's migration ability. Overall, a metabolic cooperation between lung cancer cells and fibroblasts has been confirmed in the context of direct Warburg effect, thus the fibroblasts reinforce aerobic metabolism to support the intensified anaerobic glycolytic pathways exploited by cancer cells.