Totally 67 mtPPs with 124 phosphorylation sites were identified, which of them included 48 differential mtPPs (mtDPPs). Eighteen mtPPs were reported previously in OCs, and they were consistent in this study compared to previous literature. GO analysis revealed those mtPPs were involved in multiple cellular processes. PPI network indicated that those mtPPs were correlated mutually, and some mtPPs acted as hub molecules, such as EIF2S2, RPLP0, RPLP2, CFL1, MYH10, HSP90, HSPD1, PSMA3, TMX1, VDAC2, VDAC3, TOMM22, and TOMM20. Totally 32 mtPP-pathway systems (p<0.05) were enriched and clustered into 15 groups, including mitophagy, apoptosis, deubiquitination, signaling by VEGF, RHO-GTPase effectors, mitochondrial protein import, translation initiation, RNA transport, cellular responses to stress, and c-MYC transcriptional activation. Totally 29 mtPPs contained a certain protein domains. Upstream regulation analysis showed that TP53, TGFB1, dexamethasone, and thapsigargin might act as inhibitors, and L-dopa and forskolin might act as activators. This study provided novel insights into mitochondrial protein phosphorylations and their potential roles in OC pathogenesis and offered new biomarker resource for OCs.To investigate the existence and their potential biological roles of mitochondrial phosphoproteins (mtPPs) in human ovarian carcinoma (OC), mitochondria purified from OC and control tissues were analyzed with TiO The completion of human genome sequencing has identified about 20,300 human genes . ResearcMitochondria are crucial and multifunctional subcellular organelle, and are associated with multiple cellular activities and diseases, such as metabolic reprogramming of OC cells . Mitocho4) group is added to amino acid residues serine , threonine , and tyrosine , which promotes a conformational alteration via interacting with other hydrophilic and hydrophobic residues [Phosphorylation is a common PTM that phosphate , signaling pathways, protein domains, and upstream regulation, which was used to more accurately determine the roles of phosphorylation in OC mitochondria. The current study deepened understanding of the reported biomarkers in OCs, and indicated the relationship of phosphorylation with those molecules. Simultaneously, those non-reported mitochondrial phosphoproteins (mtPPs) might add novel scientific merits for OCs. One should pay more attention to the hub molecules within PPI network, key molecules in enriched pathways, and important phosphorylations within or adjacent to protein domains, which had important scientific value in cell functions, or drug targets in OCs. The experimental flow-chart was used to identify mtPPs and reported biomarkers .2 enrichment-LC-MS/MS between OC mitochondria relative to controls  derived from CAV1 (Swiss-Prot No.: C9JKI3) . The phosphorylation site was localized to amino acid residue S*6, and the phosphorylation level was significantly decreased in OCs (T) compared to controls (N)   derived from CFL1 (Swiss-Prot No.: E9PS23) . The phosphorylation site was localized to amino acid residue S*3, and the phosphorylation level was significantly decreased in OCs compared to controls   derived from XRN2 (Swiss-Prot No.: B4E0B9) . The phosphorylation site was localized to amino acid residues S*423 and S*425, and the phosphorylation level was not significantly changed in OCs compared to controls  , and PUS1 . Some mtPPs were phosphorylated at residue Thr, such as KIN27 . Some mtPPs were phosphorylated at both residues Ser and Tyr, such as NBAS .A total of 67 mtPPs based on 85 phosphopeptides with 124 phosphorylation sites were identified in the control and OC samples with iTRAQ-TiOcontrols ; and amo C9JKI3) , with a .07E-07) . Another E9PS23) , with a .25E-05) . The thi B4E0B9) , with a .16E-02) . With th.16E-02) . Among 6.16E-02) . In termThose 67 mtPPs between OCs and controls were analyzed with GO enrichment methods, in biological process (BP) , cellulaThe PPI network of 67 mtPPs was constructed with String analysis database . First, CooLGeN (Version GenCLiP 3) was used to map 67 mtPPs to the corresponding molecules  for pathway network analysis. A total of 32 statistically significantly signaling pathway systems (p < 0.05) were identified with 67 mtPPs, and were grouped into 15 clusters . Cluster135 and S138 , TOMM20 at residue S135 , TOMM22 at residue S15 , and VDAC1 at residue S104 , which might be novel therapeutic targets and fundamental improvement of the damaged mitochondria in OC patients. These data showed that mitophagy might play important role in mediating tumorigenesis and tumor progression.When mitochondria were in the status of imbalance, such as stress or damage, defective mitochondria might enter selective degradation process by autophagy, named mitophagy. Mitophagy is a crucial mechanism of quality control in mitochondria and can be regarded as a double-edged sword in cancer cells. The defective mitochondria that responded to oncogenic stresses could either stimulate or block tumorigenesis through mitophagy . Here, tin vivo. At the same time, VEGF can increase vascular permeability and mobility of cancer cells, induce tumor angiogenesis, and maintain the growth of tumors [6 , CAV1 at residue S26 , CTNNA1 at residue S518 , HSP90 at residue S217 , CFL1 at residue S3 , and MYH9 at residue S1943 , which might be novel molecules and regulation mechanism for tumor angiogenesis.VEGF signaling is related to many physiological and pathological processes in human. VEGF is a specific heparin-binding growth factor of vascular endothelial cells, which can induce angiogenesis f tumors . Here, t3 , MYH10 at residue S1975 , MYH10 at residue S1956 , MYH10 at residues S1975 and S1956 , MYH9 at residue S1943 , and CTNNA1 at residue S518 , which might be novel molecules and regulation mechanism for tumor invasion and metastasis.Rho GTPases are the members of small GTPases, and Rho GTPase pathway is closely related to cytoskeleton reorganization, cell morphology, apoptosis, cell migration, and cell cycle. Recent studies showed that Rho GTPases mediated tumor invasion and metastasis by regulating extracellular matrix remodeling, loss of epithelial polarity, lymphatic vasculature, cytoskeleton reorganization, and between-cell junction and adhesion . Here, t2+), so intracellular calcium ions are the necessary condition for occurrence, development, and proliferation of tumors through various pathways [2+ concentrations in cells. This study found that mitochondrial calcium ion transport pathway and calcium signaling pathway were enriched to involve mtPPs, including phosphorylations of VDAC1 at residue S104 , VDAC2 at residue S140 , VDAC3 at residue T6, and ATP2A2 at residue S554 . Imbalance of Ca2+ might be the cause of tumorigenesis, so it is definitely worth further exploring.Multiple signal transduction pathways are initiated by calcium ions , and at residues S88, S92, and S93, HSP90 at residue S217 , and HSPD1 at residue S70 . Enhanced c-Myc-mediated transcriptional activity might induce cancer.C-Myc-mediated transcriptional activity promotes tumorigenesis and development. The major oncogenic mechanisms of c-MYC are related to promotion of cell cycle, drug resistance, reprogramming of cell metabolism, and rapid proliferation . Here, t89. ANKIB1 also contained ankyrin-repeated region circular (sites: 45-197), with phosphorylation at residue S89. EIF2B5 contained W2 domain (sites: 543-720), with phosphorylation at residues S717 and S718. HMGA1 contained HMGI_Y domain (sites: 21-89), with phosphorylation at residue S88. PSMA3 contained proteasome alpha domain (sites: 8-30 and 23-237), with phosphorylation at residue S78. VANGL2 contained TPR regions (sites: 114-578), with phosphorylation at S517. Second, CFL1 and HMGA1 were phosphorylated near protein domains. CFL1 contained actin-depolymerising factor homology domain (ADF-H domain) (sites: 4-153), with phosphorylation at residue S3. HMGA1 contained HMG-Y DNA-binding domain (AT-hooks) (sites: 21-89), with phosphorylation at residues S92 and S93. Third, the phosphorylation sites of the other 20 mtPPs with structural and functional domains were neither presented within domains nor near domains. However, it doesn't mean that those mtPPs were not important. The between-molecule abscopal effects could not been ignored. Additionally, phosphorylation at residue Y was a low abundance event compared to that at residues S and T. Specific tyrosine kinase inhibitors had been discovered to have some pretty amazing therapeutic effects in different types of cancers. NBAS contained ribosomal protein S14 signature (sites: 2207-2229), with phosphorylations at residues S473, S475 and Y477. This protein was associated with transporting proteins from Golgi complex to endoplasmic reticulum.A total of 29 identified mtPPs contained a certain structural and functional domains . Conjoin2+ ATPase. Upstream regulation analysis showed that dexamethasone might be an inhibitor for ATP2A2, and CAV1. L-dopa was also known as L-3,4-dihydroxyphenylalanine. Upstream regulation analysis showed that L-dopa might be an activator for VDAC1, PKP2, CLN6, VANGL2, ATP2A2, and PA2G4. Forskolin was a commonly used chemical toxicant in laboratory to increase cyclic AMP level. Upstream regulation analysis showed that forskolin might be an activator for PGRMC1, OSTF1, BNIP3L, and PRKAR2A.Upstream regulation analysis of 67 mtPPs indicated potential regulatory mechanisms and provided potential anti-cancer drugs . Upstrea397) and PTK2B (pY402) was altered in tumor tissues [Protein phosphorylation regulates multiple cellular processes through complex and highly dynamic signaling pathways. Multiple PTMs are one of the reasons to form proteoforms that are ultimate actors for cellular activity. Distinguishing of different proteoforms benefits for real precision medicine practice . In canc tissues . Taken t3 had a significant effect on glutamine uptake by cells, which facilitated nuclear translocation, and changed actin organization [14 was related to the insulin-resistant state in endometrial tissue of polycystic ovary syndrome patients. Plasma membrane was required for phosphorylation of membrane-associated MARCKS to reduce the amount of F-actin [http://ci.smu.edu.cn/CooLGeN/Home.php). These new identified mtPPs and their phosphorylation sites might be malignancy-associated intracellular events in OCs.Every subcellular component is highly dynamic because of their PTMs, abundance, and the protein expressions depending on cell physiological state. Therefore, it is very important to identify subcellular phosphoproteins for understanding biological functions of proteins in specific compartment. Mitochondria are classically viewed as isolated, spherical, and energy metabolism-related organelles, which is going through a revolutionary change. Literature data showed that mitochondria were associated with energy metabolism, oxidative stress, cell apoptosis, cell cycle, autophagy, and immunity process. In fact, mitochondrial pathways were involved in many diseases, including malignant tumors. For example, high levels of AKAP1 were found in variety of high-grade cancer tissues. AKAP1 scaffolding protein integrated Src and cAMP signaling on mitochondria to regulated oxidative metabolism, organelle biogenesis, and cell survival . These fnization . Blockagnization . Compare F-actin . A totalin vitro, which provided a clue to high amounts of diverse PTMs of HMGA1 protein [A total of 32 statistically significant pathways (p<0.05) was identified to involve mtPPs, which associated closely with the occurrence of cancer to indicate potential molecular mechanisms that mtPPs played in OCs. Additionally, a total of 29 identified mtPPs contained a certain structural and functional domains. This study provided novel insights into phosphorylations of mitochondrial proteins and their potential roles in contribution to molecular mechanisms of an OC. ANK1, ANKIB1, EIF2B5, HMGA1, CS, PSMA3, and VANGL2 were phosphorylated within protein domains. A certain structural and functional domains played important roles in tumor biological behaviors. For example, ANK1 contained ankyrin-repeated region cicular. Ankyrins, which link the membrane proteins to the spectrin-actin cytoskeleton, were encoded by ANK1 gene, and played crucial roles in multiple cellular activities such as proliferation, motility, and membrane domain activation. The 24 tandem ankyrin repeats could recognize a wide range of membrane proteins through non-specific interactions, such as hydrogen bonding, electrostatic interactions, and hydrophobic interactions . EIF2B5  protein .2+-ATPases, which influenced intracellular calcium concentration by blocking cell capacity to pump calcium into the sarco/endoplasmic reticular [in vivo [Furthermore, upstream regulation analysis showed that TP53, TGFB1, dexamethasone, and thapsigargin might act as inhibitors, and that L-dopa, and forskolin might act as activators. TP53 gene was one of the most frequently mutated genes (>50%) in human cancers, it might play a crucial role in cancer formation. TP53 gene also encoded a certain proteins that bond to DNA to prevent mutations of genome. In human, the secreted protein TGF-β1 was encoded by TGFB1 gene. TGF-β1 performed various cellular functions, including cell proliferation, cell growth, apoptosis, and cell differentiation. Upstream regulation analysis showed that dexamethasone might be an inhibitor for ATP2A2 and VCL. Dexamethasone, a kind of corticosteroid medication, was one of essential medicines in the World Health Organization's List, which was usually given to counteract a certain side-effects for cancer patients. Actually, dexamethasone was also used as a chemotherapeutic agent in some malignancies, in which it was given alone or combined with other chemotherapeutic drugs such as lenalidomide, thalidomide, vincristine, bortezomib or doxorubicin . Thapsigeticular . L-dopa,[in vivo . Forskol[in vivo . Therefo2 enrichment-LC-MS/MS method. Many identified mtPPs and their phosphorylation sites have not been reported previously, which benefit for the discovery of novel biomarkers to clarify basic molecular mechanisms of human OC formation, and truly predict OC progression.This study is the first report to investigate globally mitochondrial protein phosphorylations and their potential biological roles in the pathological processes of human OCs with iTRAQ-labeled TiOFrozen OC tissues (n = 8) and control ovaries with benign gynecologic disease (n = 11) were obtained from Department of Gynecology, Xiangya Hospital, Central South University, China. This study was approved by the Medical Ethics Committee of Xiangya Hospital, and the written informed consent was obtained from each patient. Both OC and control tissues were verified with histological analysis. Each tissue sample was immediately placed in liquid nitrogen and then stored at −80°C.Ovarian tissue samples were fully minced in pieces and homogenized in mitochondrial isolation buffer that contained Nagarse, followed by differential-speed centrifugation to obtain crude mitochondria. The crude mitochondria were further purified with Nycodenz-gradient centrifugation. The purified mitochondria samples were verified by western blot and electron microscopy. The detailed experimental procedure was described previously , 5. The An amount (200 μg) of extracted mitochondrial proteins were mixed with 30 μl solution that contained 4% SDS, 100 mM DTT, and 150 mM Tris-HCl pH 8.0. The detergent, DTT, and other low-molecular-weight components were removed by repeated ultrafiltration with the solution of 8 M urea and 150 mM Tris-HCl pH 8.0. The reduced cysteine residues were blocked with adding 100 μl solution that contained 100 mM iodoacetamide, 8 M urea, and 150 mM Tris-HCl pH 8.0, for incubation in darkness for 30 min, followed by washing with 100 μl solution (8 M urea and 150 mM Tris-HCl pH 8.0) for 3 times and then 100 μl dissolution buffer for 2 times. The protein suspensions were digested with 4 μg trypsin (Promega) . The tryptic peptides were desalted on C18 Cartridges, concentrated by vacuum centrifugation. The peptide content was estimated by UV light spectral density at 280 nm. A amount (100 μg) of tryptic peptide mixture of each sample was labeled using iTRAQ reagent. Each sample was labeled by three different iTRAQ reagents. The detailed experimental procedure was described previously [2 beads were added and agitated for 2 h, followed by centrifugation  to keep the beads, washing with 50 μL of washing buffer I (30% acetonitrile and 3% trifluoroacetic acid) (3 x) and then 50 μL of washing buffer II (80% acetonitrile and 0.3% trifuoroacetic acid) (3 x) to remove the remaining non-adsorbed material. Finally, phosphopeptides were eluted with 50 μL of elution buffer (40% acetonitrile and 15% NH3·H2O) (3 x), and then lyophilized.Six iTRAQ-labeled peptides were equally mixed, concentrated by a vacuum concentrator, and resuspended in 500 μl DHB buffer. TiO2-enriched phosphopeptides were analyzed with LC-MS/MS. Briefly, the enriched phosphopeptides were loaded onto a reverse phase trap column  connected to the C18-reversed phase analytical column  in buffer A (0.1% formic acid) and separated with a linear gradient of buffer B (84% acetonitrile and 0.1% formic acid) at a flow rate of 300 nl/min controlled by IntelliFlow technology. The linear gradient was 0–55% buffer B for 220 min, 55-100% buffer B for 8 min, and then 100% buffer B for 12 min. MS/MS analysis was performed on a Q Exactive mass spectrometer (Thermo Scientific), with the following parameters, including positive ion mode, data-dependent top10 method to choose the most abundant precursor ions from the survey scan (300–1800 m/z) for HCD fragmentation, automatic gain control (AGC) target 3e6, and maximum inject time to 10 ms, dynamic exclusion duration 40.0 s, resolution for MS set to 70,000 at m/z 200, resolution for MS/MS set to 17,500 at m/z 200, isolation width set to 2 m/z, normalized collision energy set to 30 eV, the underfill ratio set to 0.1%. MS/MS spectra were used to search protein database with MASCOT engine  embedded into Proteome Discoverer 1.4. The intensities of iTRAQ reporter ions were used to determine differentially phosphorylated proteins between OC and control mitochondrial samples.The TiOhttps://www.ncbi.nlm.nih.gov/pubmed/). Gene ontology, including BP and CC, was analyzed with Cytoscape ClueGO to obtain more insights into the biological information of mtPPs. PPI network was analyzed with String database (http://string-db.org/cgi/input.pl). Pathway enrichment was analyzed by CooLGeN (http://ci.smu.edu.cn/CooLGeN/Home.php) based on Reactome, Wikipathways, NetPath, and KEGG database. Each MS/MS-derived phosphoprotein amino acid sequence was input into the ScanProsite program (http://prosite.expasy.org/scanprosite) to determine its protein domains and relationship of phosphorylation sites and protein domains. The Swiss-Prot accession numbers and corresponding fold-changes between OCs and controls were input to the Ingenuity Pathway Analysis (IPA) data-upload workflow. The upstream regulation analysis was generated to involve those phosphoproteins.The reported biomarkers for OC based on the identified mtPPs were obtained from NCBI (t-test was used to assess mtDPPs between OC and control groups. P-value for GO enrichment analysis was obtained by two-sided hypergeometric test and corrected by Benjamini-Hochberg. P-value for pathway enrichment analysis was obtained by two-sided hypergeometric test and corrected by Q-value. The level of statistical significance was set as p < 0.05.The Student’s All the patients were informed about the purposes of the study and consequently have signed their “consent of the patient”. All investigations conformed to the principles outlined in the Declaration of Helsinki and were performed with permission by the responsible Medical Ethics Committee of Xiangya Hospital, Central South University, China.Supplementary Table 1Supplementary Table 2Supplementary Table 3


Hyperglycemia and hyperlipidemia are the hallmarks of diabetes and obesity. Experimental and epidemiological studies have suggested that dietary management and caloric restriction are beneficial in reducing the complications of diabesity. Studies have suggested that increased availability of energy metabolites like glucose and saturated fatty acids induces metabolic, oxidative, and mitochondrial stress, accompanied by inflammation that may lead to chronic complications in diabetes. In the present study, we used human hepatoma HepG2 cells to investigate the effects of high glucose (25 mM) and high palmitic acid (up to 0.3 mM) on metabolic-, inflammatory-, and redox-stress-associated alterations in these cells. Our results showed increased lipid, protein, and DNA damage, leading to caspase-dependent apoptosis and mitochondrial dysfunction. Glucolipotoxicity increased ROS production and redox stress appeared to alter mitochondrial membrane potential and bioenergetics. Our results also demonstrate the enhanced ability of cytochrome P450s-dependent drug metabolism and antioxidant adaptation in HepG2 cells treated with palmitic acid, which was further augmented with high glucose. Altered NF-kB/AMPK/mTOR-dependent cell signaling and inflammatory (IL6/TNF-α) responses were also observed. Our results suggest that the presence of high-energy metabolites enhances apoptosis while suppressing autophagy by inducing inflammatory and oxidative stress responses that may be responsible for alterations in cell signaling and metabolism. Most of the complications associated with metabolic disorders, including type 2 diabetes, obesity, insulin resistance, non-alcoholic fatty liver diseases (NAFLD), and non-alcoholic associated steatohepatitis (NASH) are associated with hyperglycemia and increased free saturated fatty acid concentration in the blood ,2. GlucoTherapeutic intervention to target the autophagy/apoptosis machinery that ultimately reprograms the cellular metabolism under nutrient overload conditions may therefore provide strategies for better management and treatment of diabesity-associated complications. Augmentation of PA-induced-lipotoxicity by HG, or vice versa, is controversial, as actual in vivo concentrations of glucose and PA under conditions of diabetes, fasting or after meal, in normal and obese subjects have not yet been defined conclusively, and might be highly variable due to various physiological and endocrine factors involved in regulating the bioavailability of nutrients. Therefore, we designed in vitro experiments to determine the molecular mechanisms of action of glucose and PA overload in HepG2 cells. Our aim was to elucidate the mechanism of glucotoxicity alone (25 mM) or in combination with high PA (up to 0.3 mM) under in vitro conditions. We have shown that mitochondrial respiratory function, energy/drug metabolism, redox homeostasis, and cellular oxidative stress are affected in glucolipotoxicity. These changes appear to be associated, at least in part, with altered inflammatory and NF-kB/mTOR/AMPK-dependent cell signaling pathways. Furthermore, in the presence of HG, PA treatment augments glucotoxicity, as shown by the reduction in autophagy with increased apoptosis in HepG2 cells.Palmitic acid, fatty acid-free bovine serum albumin (BSA), 3--2,5-diphenyltetrazolium bromide (MTT), reduced and oxidized glutathione, 1-chloro 2,4-dinitrobenzene (CDNB), cumene hydroperoxide, glutathione reductase, glucose-6-phosphate, NADH, NADPH, cytochrome c, coenzyme Q2, antimycin A, dodecyl maltoside, N-nitrosodimethylamine(NDMA), erythromycin,7-ethoxyresorufin, methoxyresorufin, resorufin, Hoechst 33342, Oil Red O stain, and kits for ATP and hexokinase (HK )were purchased from Sigma . 2′, 7′-Dichlorofluorescein diacetate (DCFDA) was procured from Molecular Probes . Kits for nitric oxide and mitochondrial membrane potential assays were purchased from R & D Systems , while those for lipid peroxidation (LPO) and aconitase were procured from Oxis Int, Inc. . Kits for GSH/GSSG assays were procured from Promega Corp. . Apoptosis detection kits for flow cytometry and IL6 and TNF-α measurement kits were purchased from BD Pharmingen . Kits for catalase and protein carbonylation assays were purchased from Cayman Chemical . Kits for superoxide dismutase (SOD) were purchased from Trevigen , while those for glutamate dehydrogenase . HepG2 cells were purchased from American Type Culture Collection . Polyclonal antibodies against PARP, caspase-3, NFκB, and beta-actin were procured from Santa Cruz Biotechnology Inc.  while those for mTOR, p-mTOR, AMPK, and p-AMPK were purchased from Cell Signaling Technology, Inc. . Polyclonal antibodies against CYP2E1 and CYP3A4 were purchased from Aviva Systems Biology . Reagents for cell culture, SDS-PAGE, and western blot analysis were purchased from Gibco BRL  and Bio-Rad Laboratories .2 at 37 °C. Cells were cultured to 80% confluence and then treated with normal (5.5 mM)  or high glucose (25 mM) (to mimic the in vivo diabetic condition) with/without high fatty acids  for 24 h. A 100 mM stock solution of palmitic acid was prepared in warm ethanol and then conjugated to 1% fatty acid free-BSA in a molar ratio of 6:1. The cells were treated with different concentrations of palmitate by adding appropriate amounts of the palmitate/BSA conjugate to the cultured cells in DMEM media supplemented with 1% FBS. Control cells for normal and high glucose media were treated with vehicle (BSA/ethanol) alone. Concentrations and time points used for high glucose or high fatty acid treatments were based on the MTT-based cytotoxicity studies and literature search. After the desired time of treatment, cells were harvested, washed with PBS (pH 7.4) and homogenized in H-medium buffer  at 4 °C. Cellular fractionation to prepare mitochondria and postmitochondrial (PMS) fractions was performed by centrifugation, as described previously [HepG2 cells were cultured in DMEM supplemented with 2 mM glutamine, 10% fetal calf serum, and non-essential amino acids in the presence of 5% CO glucose  mM  were grown on cover slips and incubated with 5 µM DCFDA for 30 min at 37 °C. Cells were washed twice with PBS, and the fluorescence analyzed microscopically, as described above [Intracellular production of ROS was measured flurometrically using the cell permeable probe, DCFDA, which preferentially measures peroxides. Briefly, treated and control cells  in the cell lysate from HG/HFA-treated and control cells was measured using the LPO-586 kit as per the manufacturer’s protocol and the concentration of MDA calculated from the standard curve as described previously ,28.Protein carbonylation, an oxidative modification of proteins under oxidative stress conditions was measured by dinitrophenylhydrazine (DNPH) coupling method according to the vendor’s protocol as described previously . Briefly6 cells/mL) in binding buffer . A fraction (100 µL/1 × 105 cells) of the cell suspension was incubated with 5 µL Annexin V conjugated to FITC and 5 µL propidium iodide (PI) for 15 min at 25 °C in the dark. A total of 400 µL of binding buffer was added to the suspension and apoptosis was measured immediately using a Becton Dickinson FACSCanto II analyzer. This method was able to distinguish the apoptotic cells from the viable and necrotic cells.Apoptosis measurement for DNA damage was performed by Hoechst dye staining of fragmented nuclei. Cells grown on cover slips were treated with HG/HFA, fixed with 3.7% formaldehyde, and stained with Hoechst33342 (10 µg/mL) for 20 min at room temperature. The cover slips were then washed, mounted on glass slides, and analyzed by fluorescence microscopy. Cells with signs of apoptosis showed fragmented nuclei. DNA damage caused by ROS due to oxidative stress was also measured by DNA fragmentation using standard 2% agarose gel electrophoresis of damaged DNA, as described previously ,29. ApopHepG2 cells were treated with different concentrations of palmitic acid in the presence of normal/high glucose for 24 h, as mentioned above. GSH/GSSG ratios and activities of GSH-metabolizing enzymes, GSH-Px, GSH-reductase, and glutathione S-transferase (GST) were measured in the HG/HFA treated and control cell extracts. GSH/GSSG ratios were measured using the GSH/GSSG-Glo kit as per the manufacturer’s instructions, as described previously ,28. GSH-Antioxidant enzymes, catalase, and SOD were measured using kits according to the vendor’s protocols. The measurement of catalase was based on its ability to catalyze the oxidation of methanol by hydrogen peroxide. The formaldehyde thus formed was measured colorimetrically in the presence of a chromogen at 450 nm.The measurement of SOD was based on the conversion of xanthine to uric acid and hydrogen peroxide, in the presence of xanthine oxidase. The superoxide ions produced, in turn, reduced the NBT (nitroblue tetrazolium) to NBT-diformazan. The rate of the reduction with a superoxide anion is linearly related to the xanthine oxidase activity, and is inhibited by SOD. Thus, the percentage inhibition of SOD activity was measured colorimetrically at 550 nm.Post-mitochondrial supernatants from control and HG/HFA-treated HepG2 cells were analyzed for CYP2E1, CYP3A4, CYP1A1, and CYP1A2 activities using N-nitosodimethylamine, erythromycin, 7-ethoxyresorufin, and methoxyresorufin, respectively, as substrates by standard protocols ,34,35,36Mitochondrial membrane potential was measured in cells after treatment with varying concentrations of glucose/palmitic acid for 24 h using a fluorescent cationic dye, DePsipher TM (R&D System Inc.), as described previously ,26,27. IFreshly isolated mitochondria (5 µg protein) from untreated and HG/HFA-treated cells were suspended in 1.0 mL of 20 mM potassium phosphate buffer, pH 7.4, in the presence of the lauryl maltoside (0.2%). Oxidative phosphorylation enzymes, NADH-ubiquinone oxidoreductase , and cytochrome c oxidase (Complex IV) were measured using the substrates coenzymeQ2 and reduced cytochrome c, respectively, according to the method of Birch-Machin and Turnbull , as descThe ATP content in the HG/HFA-treated and untreated control cell lysates was determined using an ATP Bioluminescent cell assay kit according to the manufacturer’s recommended protocol, and samples were analyzed using the TD-20/20 Luminometer .Activity of hexokinase, a cytosolic enzyme phosphorylating glucose to glucose 6-phosphate, was measured using the HK assay kit  as per the vendor’s instructions. Additionally, activity of an NADPH-producing cytosolic enzyme, glucose 6-phosphate dehydrogenase (G6PDH), was measured in HepG2 cell extracts treated with HG/HPA using glucose-6-phosphate as the substrate by following the increase in absorption due to the reduction of NADP to NADPH at 340 nm.HepG2 cells were treated with HG/HFA, as described above, and inflammatory markers TNF-α and IL6 were measured in the media using ELISA kits from BD Pharmingen  as described in the vendor’s protocols, as described previously .5 cells/well) were cultured in six well plates for 24 h prior to HG/HPA treatments. NO production was determined by measuring the concentration of total nitrite in the culture supernatants using Griess reagent (R &D Systems Inc.).For NO assay, cells . Lipid droplets appear red and nuclei appear blue.To measure triglyceride lipid accumulation, Oil Red O staining was carried out by the method of Pittenger MF et al.  with sigCell extract proteins (50 µg) were separated on 12% SDS-PAGE and electrophoretically transferred on to nitrocellulose paper by western blotting, as described previously ,26,27,28p-values < 0.05 were considered statistically significant.Results are expressed as mean + S.E.M. of three individual experiments. Statistical significance of the data was assessed using SPSS software (version 23) by analysis of variance followed by LSD post-hoc analysis. A significant increase (31%) in ROS production was seen with 0.3 mM PA in the presence of NG, while 0.06 mM PA showed no appreciable effects. On the other hand, PA treatment markedly increased (60–70%) ROS production in the presence of HG A. These Increased ROS production also resulted in the increased oxidative peroxidation of lipids A, proteiIn support of increased ROS production and oxidative-stress-related peroxidation, our results also demonstrated increased nuclear condensation  by PA treatments, particularly at higher concentrations of PA A. On theCYP450s and GSTs play important roles in maintaining the redox homoeostasis and metabolite pools for many physiological substrates, especially lipids, and xenobiotics. Our results shown in A significant loss of mitochondrial membrane potential was observed in HepG2 cells after PA treatment . HG treaNo significant alterations were observed in the activity of NADH-dehydrogenase, Complex I A. HoweveIn contrast, cytosolic metabolism of glucose by hexokinase and G6PDH seemed to increase when HepG2 cells were treated with HG alone A,B. HoweHepG2 cells treated with HG/PA also exhibited increased deposits of TG-enriched lipid droplets in the cytosol  which agHepG2 cells treated with 0.06 mM PA exhibited increased release of inflammatory markers TNF-α and IL6 in the presence of NG and HG A,B. SimiGlucolipotoxicity induces metabolic syndrome, as seen in insulin resistant type 2 diabetes, obesity, nonalcoholic fatty liver disease (NAFLD), and cardiovascular complications ,40,41. BOur results suggest that under nutrient overload conditions, in the presence of HG and high PA, a saturated fatty acid, HepG2 cells undergo severe metabolic and oxidative stress, causing increased ROS production, lipid, protein, and DNA damage, NF-kB-, TNF-α-, IL6-, and NO-dependent inflammatory responses, increased apoptosis, and decreased mitochondrial function leading to altered energy metabolism. The increased glucolipotoxicity also caused alterations in GSH-dependent redox metabolism, as well as in CYP-dependent metabolism of endogenous and exogenous compounds. NF-kB/AMPK/mTOR-dependent metabolic signaling appears, at least in part, to play a key role in the metabolic adaptation of cells under glucolipotoxicity conditions. These results might have implications in understanding and elucidating the molecular mechanisms of hyperglycemia/hyperlipidemia-induced complications in diabesity and cardiovascular diseases, as well as in identifying molecular targets for therapeutic management.


All biguanides inhibited CLIC1-mediated ion current, showing the same potency observed in the antiproliferative effects, with the exception of proguanil which was ineffective. These effects were specific for GSCs, since no (or little) cytotoxicity was observed in normal umbilical cord mesenchymal stem cells, whose viability was not affected by metformin and moroxydine, while cycloguanil and phenformin induced toxicity only at much higher concentrations than required to reduce GSC proliferation or invasiveness. Conversely, proguanil was highly cytotoxic also for normal mesenchymal stem cells. In conclusion, the inhibition of CLIC1 activity represents a biguanide class-effect to impair GSC viability, invasiveness, and self-renewal, although dissimilarities among different drugs were observed as far as potency, efficacy and selectivity as CLIC1 inhibitors. Being CLIC1 constitutively active in GSCs, this feature is relevant to grant the molecules with high specificity toward GSCs while sparing normal cells. These results could represent the basis for the development of novel biguanide-structured molecules, characterized by high antitumor efficacy and safe toxicological profile.The antidiabetic biguanide metformin exerts antiproliferative effects in different solid tumors. However, during preclinical studies, metformin concentrations required to induce cell growth arrest were invariably within the mM range, thus difficult to translate in a clinical setting. Consequently, the search for more potent metformin derivatives is a current goal for new drug development. Although several cell-specific intracellular mechanisms contribute to the anti-tumor activity of metformin, the inhibition of the chloride intracellular channel 1 activity (CLIC1) at G1/S transition is a key events in metformin antiproliferative effect in glioblastoma stem cells (GSCs). Here we tested several known biguanide-related drugs for the ability to affect glioblastoma  stem cell viability, and in particular: phenformin, a withdrawn antidiabetic drug; moroxydine, a former antiviral agent; and proguanil, an antimalarial compound, all of them possessing a linear biguanide structure as metformin; moreover, we evaluated cycloguanil, the active form of proguanil, characterized by a cyclized biguanide moiety. All these drugs caused a significant impairment of GSC proliferation, invasiveness, and self-renewal reaching IC Novel drug development is currently facing significant drawbacks due to the increasing costs of preclinical screening and the more stringent rules on drug safety adopted by government agencies. Thus, a system already intrinsically inefficient  is now becoming economically unsustainable . This isGlioblastoma  is a rapidly growing brain tumor characterized by invasive and pro-angiogenic behavior leading to poor prognosis . GBM is in vitro and in vivo cancer models, including breast  activity , similarly to what observed in liver to inhibit glucose release . In turnactivity . CLIC1 iactivity . Importaactivity . We repoactivity . As expeactivity .in vitro studies, although in vivo metformin intratumoral concentrations were reported to be several fold higher than in plasma , type 1 rat tail collagen from SERVA Electrophoresis GmbH , and methylcellulose from Sigma-Aldrich.Metformin , phenformin [1-(2-phenylethyl)-biguanide hydrochloride], dissolved in water, and IAA94 (indanyloxyacetic acid 94), in ethanol, were from Sigma-Aldrich . Moroxydine, cycloguanil and proguanil, were synthesized according to the procedures previously reported  and stocThe following antibodies were used: anti-CLIC1 cl. F9 and cl. 356.1  for immunofluorescence and Western blot experiments, respectively; anti-GFAP, anti-nestin, anti-Oct4, anti-Sox2, and anti-MAP-2 ; anti-Olig2 and anti-Sox2, ; anti-mouse and anti-rabbit Alexa Fluor 488 and 568 secondary antibodies .Table 1.Cultures were obtained from post-surgical samples of GBM grade IV (WHO classification) derived from 7 patients  and coded as GBM1 to GBM7. Post-surgical samples were used after patients’ informed and written consent and Institutional Ethical Committee (IEC) approval. All patients underwent surgery at Neurosurgery Department  and had not received therapy prior to the intervention. Detailed information on individual patients is reported in Figure 1). For survival and electrophysiology experiments, GSCs were grown as monolayer on growth factor-reduced Matrigel coating, allowing easier evaluation without affecting stem cell features . All the cultures analyzed in this study have been characterized in previous works for tumor-initiating capacity by orthotopic injection of 10,000 sphere-derived cells in 6–8 weeks old non-obese diabetic severe combined immunodeficient (NOD/SCID) mice   . Cell di States) .Genetic alteration analysis in GSCs was performed by multiplex ligation-dependent probe amplification (MLPA) analysis. Briefly, genomic DNA was isolated from GSCs and normal human astrocytes  using QIAamp DNA microkit (Qiagen). The MLPA analysis  was performed using 100ng of genomic DNA, following the manufacturer’s instructions. The resulting DNA fragments were identified and quantified by using capillary electrophoresis on an ABI XL3130 genetic analyzer  and the Genemapper program (version 4.0 – Applied Biosystems). Data were analyzed with the Coffalyser software (MRC-Holland). For each GBM, gains and losses were assigned by comparing the peaks between the patient and the reference samples , as previously described , 2014.CLIC1 gene down-regulation: short hairpins mRNA specific for human CLIC1 (5′-GATGATGAGGAGATCGAGCTC-3′) and firefly luciferase (5′-CGTACGCGGAATACTTCGA-3′) were cloned into the XhoI/HpaI sites of the pLentiLox 3.7 lentiviral vector and stably expressed in GBM2 CSCs, as reported . CLIC1 dTM , underwent flow cytometry phenotypical characterization , showing that more than 95% of the cells, in each independent culture, were negative for hematopoietic antigens  and MHC class-II, but expressed CD73, CD105, CD90, CD29, and MHC class-I. Multipotency was demonstrated showing that after incubation in selective media, ucMSCs differentiate into osteocytic, chondrocytic, and adipocytic lineages  following informed consent and approval by IEC, as reported . Brieflylineages .Glioblastoma stem cells and differentiated GBM cells were fixed with 4% paraformaldehyde, permeabilized with PBS/0.1% Triton X-100, blocked with normal goat serum and immunostained with the appropriate antibody, followed by fluorochrome-conjugated secondary antibody . Nuclei Cells were lysed in RIPA buffer and processed as reported . ProteinMitochondrial function, as index of cell viability, was evaluated by measuring the reduction of 3--2,5-diphenyltetrazolium bromide , as reported . BrieflyGlioblastoma stem cells from different tumors, grown in standard conditions for 3 days in the presence or absence of vehicle or biguanides, were counted with an automated cell counter  every 24 h. Briefly, after treatments, cells were harvested and the suspension was diluted 1:10 in sterile PBS and mixed with an equal volume of 0.4% Trypan Blue solution. Cell viability was calculated as % of viable cells divided by the total number of cells .50, and stained with Annexin V-FITC and propidium iodide (PI) . Samples were analyzed by FACScalibur (BD Biosciences), equipped with BD CellQuest Pro software. The percentage of cell death was obtained by summing-up the percentages of early and late apoptosis , and 5–8 unit fields per filter were counted by a scorer, blind to the experimental conditions, with a Zeiss microscope.Experiments were performed in a 48-well modified Boyden chamber using 8 μm nuclepore polyvinylpyrrolidine-free polycarbonate filters, coated with Matrigel (100 μl/filter). Boyden chamber was assembled with the coated side of the filter facing the cells in the upper compartment. GSCs were suspended at a density of 1 × 10g immediately after plating. Tumor spheroids were harvested within 24 h, suspended in DMEM/F12 containing 10% FBS and 0.9% methylcellulose, and mixed with an equal volume of a collagen stock solution prepared by mixing at 4°C acidic rat tail collagen  with 1 vol of 10× DMEM and 0.1 M NaOH, to adjust pH to 7.4. Drugs, or the corresponding vehicles, were added to suspended spheroids before embedding them into collagen. Spheroid-containing gel was rapidly transferred into pre-warmed 24-well plates, and incubated for 24 h at 37°C in 5% CO2. In-gel invasion was quantified by measuring the length of all of the processes originating from individual spheroids using the ImageJ software . At least 10 randomly selected spheroids per experimental group were measured in each experiment.Multicellular tumor spheroids were generated in the presence of methylcellulose as previously described , with mi50, and monitored for 5 days, to allow sphere generation. The number of spheres/well was quantified using a digital camera mounted on a transmitted light microscope to image each individual well, and visually calculated by three independent operators, as reported  were pulled from hard borosilicate glass on a Brown-Flaming P-87 puller  and fire-polished to a tip diameter of 1–1.5 μm and an electrical resistance of 5–7 MΩ. Patch-clamp electrophysiology was performed in perforated-patch whole cell configurations, as reported .2, 2 CaCl2, 5 D-Glucose, pH 7.4. Pipette solution (mM): 135 KCl, 10 HEPES, 10 NaCl, 1 MgCl2, 2 CaCl2, 5 D-Glucose, pH 7.4.The voltage protocol consisted of 800 ms pulses from −40 mV to +60 mV every 10 s. Current amplitude was measured as trace average between 700 and 750 ms. Patch clamp solutions were the following: bath solution (mM): 140 NaCl, 5 KCl, 10 HEPES, 1 MgClChloride intracellular channel 1 activity was detected using electrophysiological measurements. Cells under continuous membrane current monitoring were constantly perfused with external solution at 1 ml/min. Once current recordings reached a constant value, cells were challenged with the different biguanides, followed by addition of IAA94 (100 μM) to get maximum inhibition . Data rep-value ≤ 0.05. Prism version 5.02  software was used to analyze the results. All experiments were repeated independently at least three times (n) each performed in triplicate or quadruplicate (as indicated). The half maximal inhibitory concentration IC50 was calculated using non-linear regression curve fit analysis selecting the log(drug) vs. response-variable slope (four parameters) equation Statistical significance between two independent groups was assessed by t-test  or one-way ANOVA for multiple groups, followed by Tukey’s, Dunnett’s or Bonferroni’s post hoc tests (post hoc tests were only applied when ANOVA gave p < 0.05). Direct comparison between GSCs and differentiated GBM cells after biguanides treatment were performed using two-way ANOVA with interaction terms between cell type and concentration; this analysis was followed by post hoc Bonferroni test to determine at which concentration the effect of treatment was statistically significant.All data are presented as means ± SEM. Statistical significance was established at Table 1 for patient and tumor characteristics). As expected, GBM analyzed were heterogeneous with 3 classified as neural  and 1 as “mesenchymal” (GBM 3) and only GBM2 showed unmethylated MGMT promoter. Similar to normal stem cells, isolated GSCs, cultured in chemically defined medium (without serum and containing EGF and bFGF), are able to grow in vitro as spheroids or loosely adherent clones expressing stem cell-related markers  . Alternatively, to perform a better analysis of GSC pharmacological responses, cells were cultured on Matrigel in the same medium  and the tumorigenic ability , might TF and MM conceived and designed the experiments. FB, RW, AP, IV, CM, MC, AS, AB, AD, and LV performed the experiments. AD and MT contributed reagents, materials, and analysis tools. TF, FB, AD, LV, and MM wrote the manuscript. All authors analyzed the data.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Phenolic compounds have been considered inhibitors of various cancers.in vitro. We also evaluated ligand interaction and ligand binding with estrogen receptor alpha by molecular docking. To determine half maximal inhibitory concentration, MCF-7 cells were treated with different concentrations of caffeic acid and gallic acid by 3--2, 5-diphenyltetrazolium bromide assay. Furthermore, morphological changes in cells and alterations in P53, Mcl-1 and P21 gene expression were studied by real-time RT-PCR. Also, protein network and different interactions between the desired genes were analyzed using GeneMANIA database. In this study, caffeic acid and gallic acid were appraised for their possible effects on apoptotic genes expression in a breast cancer cell line P53, Mcl-1 and P21 gene expression; this alteration in gene expression probably occurred along with the activation of intrinsic apoptotic signaling pathway. Evaluation of cell survival by MTT assay revealed that the half-maximal inhibitory concentration values for caffeic acid and gallic acid against MCF-7 cells, were 159 and 18 µg/ml, respectively. These compounds were found to affect Via apoptosis induction, caffeic acid and gallic acid have induce toxic effects and morphological changes in breast cancer cells, suggesting their possible future application as antitumor agents. Breast cancer is the most common cause of cancer mortality , with moHuman and animal studies have revealed the roles of estrogen receptor (ER) in female and male sexual development and behavior, reproductive function, regulation of the neuroendocrine and cardiovascular systems and bone metabolism . The losBcl-2 gene family, including myeloid cell leukemia-1(Mcl-1). The oncogene Mcl-1 is involved in hematogenous and solid tumor cell survival   and gallic acid (GA)  found in fruits, coffee and vegetables are potential anticancer, anti-inflammatory, antimicrobial, immunoregulatory, and antioxidant compounds . Mechaniucidated . P53, P21 and Mcl-1 gene expression in an ERα-positive breast cancer cell line in silico and in vitro, simultaneously. For the first time, we focused on the intrinsic apoptotic signaling pathway, as a major apoptosis pathway, and the relationship between these genes and tested compounds. Therefore, to explore mechanisms associated with CA- and GA-induced modulation of breast carcinogenesis, we assessed the ligand interaction and binding mode of these compounds with ERα using molecular docking, analyzed associated protein networks and gene interactions using GeneMANIA database, and investigated their effects on hormone receptor positive breast cancer cells (MCF-7), morphological alteration and proliferation by MTT -2, 5-diphenyltetrazolium bromide) assay. Ultimately, using real-time RT-PCR, we measured the effects of CA and GA on the expression of apoptotic genes, including P53, Mcl-1 and P21 in MCF-7 cell line.The objective of this study was to simultaneously study the anti-breast cancer effects of gallic acid and caffeic acid, as two important phenolic compounds, on the MCF-7 cell line. In addition, the effects of these compounds were investigated, for the first time, on Reagents CA and GA with the chemical structure shown in Molecular docking6 and the number of runs [ga_run] altered to 100. AutoDockTools includes several methods for analyzing the results of docking simulations, including tools for clustering the results by conformational similarity, and visualizing conformations, interactions between ligands and proteins, and the affinity potentials created by AutoGrid. Results were visualized via PyMol (http://www.pymol.org).The molecular docking of all compounds was done into the three-dimensional X-ray structure (PDB code: 3ERT). The AutoDock 4.2 software was used for the docking studies. This automated method is useful for studying the binding mode of ligands binding to biomacromolecules. The three-dimension structures of ligand molecules were built, optimized (PM3), and saved in PDB format using the molecular modeling program SPARTAN (Wavefunction Inc.). All non-protein atoms were deleted, and AutoDockTools was used for creating PDBQT files from primary PDB files. A grid box was constructed around the binding site of protein with number of grid points [npts]: X:60, Y:60, and Z:60 in the three dimensions, coordinates of three dimensions [gridcenter]: X:121.952, Y:90.314, and Z:5.066, and spacing of 0.375. Hydrogen atoms and Gasteiger partial charges were added using AutoDockTools (Ver. 4.2). In the present docking study, the Lamarckian genetic algorithm (LGA) method, implemented in the program AutoDock 4.2, was employed. All default docking parameters were maintained except for the maximum number of energy evaluations [ga_nim_evals] that was changed to 2.5×10Preparation of compounds solutionsTo prepare different concentrations of CA and GA , 2 mg of each compound was dissolved in 100 μl dimethyl sulfoxide and diluted with complete culture medium before the experiments. To better evaluate the various treatments, the viability of each group was compared with its relevant control containing equal amounts of DMSO.Culture of MCF-7 cells2. Cells were routinely sub-cultured using 0.25% trypsin and 1 mM EDTA. The human primers were commercially purchased form DENA ZIST Asia, Mashhad, Iran.The human breast cancer cell line (MCF-7) was purchased from Pasteur Institute . The cells were cultured in RPMI1640 medium (pH 7.2-7.4), supplemented with penicillin G (100 U/ml), streptomycin (100 U/ml) and 10% fetal bovine serum (FBS), and incubated at 37°C in a humidified atmosphere with 5% COMorphological alterations50) dose of each compound and morphological changes were observed under a light inverted microscope  following 48 hr of treatment.MCF-7 cells were treated with half maximal inhibitory concentration (ICMTT assayin vitro tetrazolium-based colourimetric assay (MTT) is a rapid method developed based on the cleavage of a yellow tetrazolium salt to purple formazan crystals by mitochondrial enzymes in metabolically active cells (The ve cells . The log%viability of cells = the mean absorbance of treated cells in each well/the mean absorbance of control cells (DMSO) × 100. Expression of P53, Mcl-1 and P21 in MCF-7 cell line assessed by Real-time PCRP53, Mcl-1, and P21 transcript levels. QRT PCR reaction was carried out in thermocycler detection system  under the following conditions: 10 min at 95°C, 15 sec at 94°C, 30 sec at 55°C, and 30 sec at 72°C; each cycle was repeated 40 times. Primers used in this study are listed in Total RNA was extracted using TRIzol reagent and used as a template for the production of cDNA which was employed in SYBR-based quantitative real-time PCR for the quantification of Analysis of protein network using GeneMANIA databaseP53, Mcl-1 and CDKN1 (P21) genes (supplementary data). In each cluster network, display-colored lines indicate a direct link, blue lines indicate the link provided by the pathway, pink lines represent the physical interactions, and purple lines show interactions with co-expression  genes analyzed by GeneMANIA database. Furthermore, all data were analyzed using Kolmogorov–Smirnov test. Based on the normality test, data distribution in most experiments was normal. Differences were determined by one way ANOVA and Student's t-test (two-tailed), followed by Tukey multiple comparison test , and Mann–Whitney U test , using GraphPad software (Ver. 6). Values were expressed as mean±SD, and a p value less than 0.05 was considered statistically signiﬁcant.The direct networks between Molecular Dockingα was -11.21 kcal/mol. Other properties of reference drug are given in para position of the ring binds Arg-394 and Glu-353, like tamoxifen, blocks ER functions, and prevents the proliferation of breast cells.Interactions and binding mode of caffeic acid, gallic acid, and tamoxifen with ERα, are illustrated in Cytotoxic activity50 values of compounds against MCF-7 cells were further determined . Our data showed that GA also significantly impacted the expression of P53, P21, and Mcl-1 in MCF-7 cells . P53, P21 and Mcl-1 genes.The possible roles of CA and GA in altering the expression of InteractionsP53, Mcl-1, and P21). The network also shows a correlation between intended compound-targeted genes and other genes effective in intrinsic apoptotic signaling pathway, such as BCL2l11, BAK1, TP73, BOK, BAX, TP63, and PMAIP1. By this analysis, we showed probable interactions between certain CA and GA regulated genes. CA and GA targeted genes with a functional connection with intended genes involved in intrinsic apoptotic signaling pathway (Generally, the results of this study are in agreement with previously published researches, which also showed the anticancer effects of these compounds against different types of cancers including breast , prostatP53, Mcl-1 and P21 had a direct association concerning physical interactions, co-expression and signaling pathway; hence, we focused on the effect of CA and GA on the gene expression of P53, P21 and Mcl-1 in MCF-7 cell line. As specified in the results, after treatment with CA and GA, the expression of these genes was altered, reinforcing our hypothesis regarding the possible effects of these compounds on the intrinsic apoptotic signaling pathway.To further clarify the possible mechanism of action of these compounds in the present research, we focused on the expression of a set of master genes associated with apoptosis in breast cancer. Network connectivity, for the first time, showed the functional interactions between investigated CA/GA-regulated genes and other genes associated with intrinsic apoptotic signaling pathway. It was also found that P53, also known as TP53, codes a protein that functions as a tumor suppressor and partakes in the initiation of apoptosis process (P53 gene, yet GA increases its expression. This gene induces apoptosis by target gene activation and transactivation-independent in mitochondria (WAF1/Cip1, mediates P53-dependent G1 growth arrest (P21 helps apoptosis is not clear, but might depend on both P53-independent and p53-dependent upregulation of pro-apoptotic protein BAX, activation of the members of the tumor necrosis factor family of death receptors, or via affecting DNA repair (P21 gene expression following cell treatment with CA and GA, showed that the overexpression of this gene occurred at different times post treatment. Growth arrest by P21 can promote cellular differentiation, thereby inhibiting cell proliferation. Several other studies have shown that GA improves the viability of HL-60 cell line via P21 overexpression, and suppression of apoptosis induced by the overexpression of caspase-3, caspase-8 and caspase-9 ( process . In resp process . Based ochondria . It has chondria . Proteinh arrest . How P21A repair . Resultsaspase-9 . Furthermore, the overexpression of P21, by inhibiting several CDKs activity, such as CDK2, CDK3, and CDK4, ensues cell cycle arrest in G1 or G2 . P21 canMcl-1 mRNA is up-regulated as part of an early rapid cellular response to cytotoxic stimuli such as calcium ionophores, chemotherapeutic agents, pneumococcal infection and UV irradiation (Mcl-1, unlike the two other genes, is slightly overexpressed following treatment with the two phenolic compounds; However, it is noted that MCL-1 is a major cause of resistance to chemo and radiotherapies, hence, inhibiting the expression of this gene is an important issue in cancer-related research. (Moreover, Bcl-2 and its related proteins are among the most significant regulators of apoptosis . Variousadiation . After sesearch.  .P53 and P21WAF-1/CIP-1 are essential players; apoptosis plays an important role in myriad physiologic procedures in cell development; therefore, failure in apoptosis facilitates tumor formation and development (Furthermore, sub-apoptotic concentrations of cytotoxic compounds can induce growth arrest with senescence features, in which, elopment . AccordiP53, P21 and Mcl-1. Accordingly, we suggest that these phenolic compounds are probable candidates for more research in animal models of breast cancer, as therapeutic agents, and adjuvants to standard chemotherapeutic drugs. By further cellular, molecular and in vivo studies on the effects and probable side effects of CA and GA, maybe in the not too distant future, these materials can be applied for possible health benefits as food supplements and medication for patients with breast cancer.Findings of this study showed that MCF-7 cells treated with the intended compounds underwent several changes associated with cell death, such as morphological changes, and their numbers were reduced. Moreover, it was found that these compounds had significant effects on the expression of apoptosis genes such as


Urinary bladder cancer is a common malignancy, being characterized by substantial patient mortality and management cost. Its high somatic-mutation frequency and molecular heterogeneity usually renders tumors refractory to the applied regimens. Hitherto, methotrexate-vinblastine-adriamycin-cisplatin and gemcitabine-cisplatin represent the backbone of systemic chemotherapy. However, despite the initial chemosensitivity, the majority of treated patients will eventually develop chemoresistance, which severely reduces their survival expectancy. Since chromatin regulation genes are more frequently mutated in muscle-invasive bladder cancer, as compared to other epithelial tumors, targeted therapies against chromatin aberrations in chemoresistant clones may prove beneficial for the disease. “Acetyl-chromatin” homeostasis is regulated by the opposing functions of histone acetyltransferases (HATs) and histone deacetylases (HDACs). The HDAC/SIRT (super-)family contains 18 members, which are divided in five classes, with each family member being differentially expressed in normal urinary bladder tissues. Since a strong association between irregular HDAC expression/activity and tumorigenesis has been previously demonstrated, we herein attempt to review the accumulated published evidences that implicate HDACs/SIRTs as critical regulators in urothelial bladder cancer. Moreover, the most extensively investigated HDAC inhibitors (HDACis) are also analyzed, and the respective clinical trials are also described. Interestingly, it seems that HDACis should be preferably used in drug-combination therapeutic schemes, including radiation. Cancer of the urinary bladder is a highly prevalent disease, and is closely associated with substantial morbidity, mortality and cost. It is the 9th most frequent malignancy and the 13th most common cause of death, worldwide. In the year 2012, more than 429,000 cases of bladder cancer were diagnosed and more than 165,000 deaths were recorded, globally ,8,9,10.N-acetyltransferase 2 (NAT2). Moreover, genome-wide association studies (GWAS) have identified sequence variants that can increase susceptibility to the disease, with alterations in the urea transporter SLC14A1 gene being characteristic examples [The most common risk factor for bladder cancer is cigarette smoking, with tobacco being estimated to be responsible for almost half of all cases ,12. Ambiexamples ,21,22,23In North America and Europe, more than 90% of bladder cancers belong to urothelial carcinomas. These tumors are graded according to their cellular properties and staged using the Tumor-Node-Metastasis (TNM) system, which describes their invasion competence (Tis-T4). The low grade, non-muscle-invasive bladder cancer (NMIBC) infrequently acquires invasion features, but can usually recur, following an approximate 5-year survival rate of 90%. In contrast, the high grade, muscle-invasive bladder cancer  often progresses to metastasis, presenting an unfavorable patient prognosis with a five year survival rate of less than 50%. Since therapy protocols have not advanced for many years, new systemic strategies are necessitated to be promptly developed for the clinical management of the disease ,25,26.TP53 and Rb1 genes are frequently mutated, while regulators of their cognate pathways are also altered, with MDM2 amplification and CDKN2A deletion being two representative paradigms, respectively. Other tumor suppressor genes being inactivated by mutation in MIBC, are the ones encoding the ARID1A, TXNIP, ELF3, NFE2L2, FBXW7, APC, CDKN1A, EP300, TSC1, PTEN, ATM, KMT2D, KLF5 and TSC2 proteins. The MIBC-specific molecular signature also contains mutation-driven activation of several oncogenes, including the E2F3, PIK3CA, FGFR3, HRAS, ERBB3, CCND1, RXRA, HER2/ERBB2, EGFR, FGFR1, KRAS and AKT1 ones. In contrast to MIBC, NMIBC is genomically stable, with chromosome 9 deletion representing the most common copy number aberration. This leads to CDKN2A and TSC1 loss, and thus subsequent perturbation of the Rb-/p53- and mTOR-dependent pathways, respectively. Additional tumor suppressor genes are also inactivated in NMIBC, containing, among others, the KDM6A, STAG2, RBM10, CREBBP and ERCC2 ones. The NMIBC-specific genomic landscape is also characterized by activating mutations in several key oncogenes , with TERT and FGFR3 exhibiting the highest frequency among all. Interestingly, a powerful, multiplatform analysis has recently identified 5 MIBC subtypes that may stratify response to different treatments. The new subtypes have been characterized as: (a) luminal-papillary , (b) luminal-infiltrated  markers), (c) luminal , (d) basal-squamous  and (e) neuronal . Additional integrative analyses could further refine this novel MIBC subtyping, and notably support the current research for personalized targeted therapies [Among the studied malignancies, bladder cancer is presented with one of the highest somatic mutation frequencies, bearing a median value of more than five mutations per megabase. MIBC genome carries a number of gene aberrations that resemble critical ones of other solid tumors. Particularly, loss of function of key tumor suppressors compels cancer cells to evade cell cycle control and apoptosis, and to also deregulate major signaling pathways. Accordingly, the herapies ,29,30.ARID1A, CREBBP, EP300, KDM6A and KMT2D genes encode chromatin modifiers, epigenetic alterations may hold a central position in the therapy-resistance map of urothelial carcinoma. In accordance, chromatin regulation genes are more frequently mutated in MIBC than in other epithelial cancers examined so far, thus suggesting the strong potential of targeted therapy for chromatin abnormalities in chemoresistant clones [The mortality rate of patients being affected by urothelial carcinoma has not essentially changed over the past 30 years, thus reflecting the limitations of applied therapeutic protocols. Regimens containing platinum-based agents, such as the MVAC (methotrexate-vinblastine-adriamycin-cisplatin) and GC (gemcitabine-cisplatin) ones, represent the backbone of first-line systemic chemotherapy for patients suffering from advanced-stage urothelial carcinoma. Despite the initial chemosensitivity, chemoresistance will eventually develop in the majority of patients, resulting in a median, overall, survival between 13 and 19 months. In the second-line therapy, cisplatin-resistant urothelial carcinomas can be treated with immune-checkpoint inhibitors , but the clinical benefits remain limited, as more than 60% of the patients die after 12 months. It seems that chemotherapy-resistant urothelial carcinoma has undergone a dynamic and extensive clonal-evolution program, with critical changes in its mutational landscape being accumulated during and after chemotherapy. Hence, capture and quantification of heterogeneity in a urothelial tumor’s molecular architecture, will enable the early identification and targeting of key molecular events occurring during a tumor’s evolutionary trajectory, favoring the emergence of chemotherapy-sensitive and immune-responsive clones. Given that the t clones ,38,39,40Eukaryotic DNA is tightly wrapped around a cluster of eight proteins called core histones, in order to form nucleosomes, the fundamental units of chromatin. Several post-translational modifications of histones, such as ubiquitylation, phosphorylation, methylation and acetylation, are critical components of the epigenome , with “acetyl” marks, besides being classified among the most abundant modifications, also providing a large collection of druggable proteins that control the “acetyl-genome” dynamics and functions. Acetylation neutralizes the positive charge of lysine, thus weakening the charge-dependent interactions between nucleosomal DNA and histones. As a result, chromatin becomes structurally more relaxed, and DNA accessibility to the transcription machinery is increased. In general, hyper-acetylated histones are associated with transcriptionally active genes, whereas hypo-acetylated histones are mechanistically linked to transcriptionally repressed genes . InteresThere have been identified nine mammalian nuclear HATs, with structurally defined acetyltransferase domains and histone lysine-specific acetylation activities, mainly organized in pairs: GCN5/KAT2A and PCAF/KAT2B (pair I); CREBBP (CBP)/KAT3A and EP300/KAT3B (pair II); MOZ (MYST3)/KAT6A and MOZ2 (MYST4)/KAT6B (pair III); TIP60/KAT5 and MOF (MYST1)/KAT8 (pair IV); and HBO1 (MYST2)/KAT7 (KAT: lysine (K) acetyltransferase; alternative nomenclature) . For exa2+) for catalysis, while SIRTs function as NAD+-dependent enzymes, carrying protein deacetylase and ADP-ribosylase activities, as well [Mammalian genomes contain 11 HDAC family members, which are divided into four “phylogenetic” classes: HDAC1, 2, 3 and 8 (class I); HDAC4, 5, 7 and 9 (class IIa); HDAC6 and 10 (class IIb); and HDAC11 (class IV). Another group of deacetylases, the SIRTUINS (SIRTs), compose the class III (SIRT1-7) of the deacetylase super-family. Enzymes that belong to class I, II (a and b) and IV require a divalent metal ion  expression, thus regulating the G1- to S-phase transition of the cell cycle [ll cycle ,70,71,72Class IIa HDACs: Class IIa members are presented with rather restricted and tissue-specific expression profiles. HDAC5 and 9 are enriched in brain, heart and muscles, HDAC4 in skeleton growth plates and brain, and HDAC7 in thymocytes and endothelial cells. In response to their phosphorylation by protein kinases (e.g. CaMK and PKD), they bind to the 14-3-3 anchor protein(s) and shuttle from the nucleus to cytoplasm. Hence, MEF2 transcription factor is dissociated from the HDAC-dependent complex and subsequently interacts with the HAT EP300, converting itself from a transcriptional repressor to a transcriptional activator. Class IIa HDAC deacetylase functions are minimal, and their transcription repressive activities partly derive from the recruitment of class I HDACs (e.g. HDAC3), and also from the interaction with the HP1 and CTBP transcriptional repressors. Interestingly, a substitution of a critical tyrosine to histidine in the catalytic pocket of class IIa HDACs seems to account for their significantly reduced catalytic activities (as compared to the class I HDAC respective ones). Most likely, the intrinsic catalytic domains of class IIa HDACs are not required for the repression process to occur [to occur ,82,83,84Class IIb HDACs: Despite our poor knowledge regarding its function, HDAC10 seems able to promote angiogenesis in endothelial cells. However, it is the other class IIb member, HDAC6, that is considered as the major cytoplasmic deacetylase in mammalian cells. HDAC6 differs from the other HDACs, since it carries 2 deacetylase domains and a C-terminal Zn2+-finger. Among the targets being directly deacetylated by HDAC6, there have been recognized two cytoskeletal proteins, the α-Tubulin and Cortactin, which play important roles in cell mitosis and migration, respectively. HSP90 and IFNαR, also, represent genuine targets of the HDAC6 enzyme for direct deacetylation. In addition, HDAC6, together with HDAC9 and SIRT1, can mediate the FOXP3 deacetylation, thus controlling T(reg) cell development and immunosuppressive activity [activity ,92,93.Class IV HDACs: HDAC11 expression is enriched in the brain, heart, muscles, kidney and testis. It contains a central deacetylase domain flanked by small N- and C-terminal extensions. Although it is predominantly localized in the nucleus, HDAC11 co-precipitates with the cytoplasmic HDAC6. Notwithstanding the development-dependent and region-specific expression profiles having been observed in mouse brain, its function remains poorly understood. Interestingly, HDAC11 has been critically implicated in the regulation (suppression) of IL-10 expression in antigen presenting cells. Moreover, its key role in the control of metabolism and obesity has been recently reported [reported ,95,96,97Class III HDACs: SIRT1 is predominantly nuclear, and is expressed in the majority of mammalian tissues. It is activated upon nutrient deprivation, and, besides histones, is able to deacetylase transcription factors (e.g. p53) and signaling proteins (β-Catenin), thereby altering their activities. SIRT1 is associated with an increase in lifespan and memory, and presents beneficial effects in metabolic syndrome, neurodegeneration and cancer [d cancer ,99,100. d cancer ,103,104.d cancer ,109,110.d cancer ,112,113.d cancer ,117,118.d cancer ,122,123.d cancer ,127,128.HDAC expression among the 18 family members (as evidenced by an RNA sequencing strategy), with HDAC1 and nine being presented with the highest (37.5) and lowest (0.6) median RPKM (reads per kilo base per million mapped reads) values, respectively . APL is characterized by leukemic cell arrest at the promyelocytic stage of myeloid differentiation, mechanistically caused by the production of fusion proteins, with PML-RARα being detected in the majority of examined cases. Normally, RARα acts as a transcription factor, which can heterodimerize with RXR and bind to DNA, while in the absence of retinoic acid (its cognate ligand) it can recruit HDAC-containing complexes to transcriptionally repress genes. Retinoic acid induces a conformational switch and release of HDAC-dependent repressing complexes, promoting the interaction of RARα with transcriptional co-activators, including HATs, to activate genes. In APL disease, RARα fusion proteins retain the ability to bind to DNA of target promoters, but cannot respond to physiological doses of retinoic acid and, thus, HDAC-carrying complexes remain associated with retinoic acid target genes, causing their constitutive transcriptional silencing. Strikingly, the repression effects are further reinforced by fusion protein oligomerization, that leads to an increased stoichiometric interaction with HDAC-containing complexes and the recruitment of additional chromatin modifiers, such as DNA and histone methyl-transferases. Similarly, the AML1-ETO oncogenic fusion protein identified in t AML (acute myeloid leukemia) patients has the capacity to recruit the HDAC1, 2 and/or 3 family members, thereby repressing AML1 target gene expression, which ultimately results in the prevention of myeloid differentiation and induction of cellular transformation ,138,139.A molecular link between mitogenic signaling and altered HDAC activity is provided by the RAS-MAPK/ERK-HDAC4 axis. Expression of oncogenic RAS  causes a significant increase in the number of cells that contain HDAC4 in the nucleus, a compartmentalization process that is associated with kinase activity. In the same direction, an ERK kinase-mediated phosphorylation of HDAC6 is able to promote cell migration via a tubulin deacetylation mechanism. Threonine 1031 and serine 1035 of HDAC6 can be phosphorylated by ERK1 in vitro, while serine 1035 is phosphorylated in response to activation of the EGFR-RAS-ERK signaling pathway in vivo. Therefore, the RAS-ERK-HDAC4/6 axis may critically contribute to cancer initiation, progression and metastasis. Interestingly, phosphorylation of HDAC1 at the critical serine 421 by the NLK serine/threonine kinase  results in reduced β-Catenin/LEF1 promoter activation, thus indicating the proficiency of NLK-HDAC1 axis to downregulate WNT signaling, which is vital for the prevention of aberrant proliferation of non-transformed, primary, fibroblast cells. Likewise, the c-ABL (ABL1/ABL) protein kinase can induce tyrosine phosphorylation of HDAC2, affecting (upregulating) both its stability and repression activity in neurons. Given the major importance of the oncogenic fusion protein BCR-ABL (carries aberrant tyrosine kinase activity) in t CML (chronic myeloid leukemia) patients, a pivotal role of aberrant HDAC2 function in CML pathogenesis is herein suggested to occur. Remarkably, the JNK kinase -dependent phosphorylation of HDAC3 is associated with a five times upregulation of its enzyme activity and three to 16 times increase in HDAC inhibitor selectivity for HDAC3 in triple-negative breast cancer cells, as compared to luminal subtypes. These findings highlight how HDAC phosphorylation affects HDAC inhibitor binding and selectivity, and underscore the significance of JNK-HDAC3 axis in -breast- cancer clinical management and therapy ,145,146.G12V-driven lung adenocarcinomas [HDAC3 gene leads to hepatocellular carcinoma, while reduced HDAC1/2 enzyme activity causes neoplastic transformation of immature T cells concomitant with increased MYC levels [min mice is able to produce significantly lower, as compared to APCmin mice (with unimpaired HDAC2), tumor rates [Besides their aberrant (oncogenic) recruitment and signaling, individual HDACs are also subjected to altered expression in a tumor-specific fashion. For example, HDAC1 is over-expressed in breast, gastric and prostate carcinomas, while elevated HDAC2 levels have been detected in cervical and colorectal cancers ,150,151.rcinomas ,156,157.C levels ,159,160.or rates ,151,161,or rates .HDAC1 mRNA was expressed at notably higher levels in urinary bladder cancer versus normal tissues, while the cognate protein was intensively compartmentalized in the tumorigenic, but not control, nuclei [HDAC1 mRNA levels in the malignant state, as compared to the healthy one [HDAC2 gene in metastatic advanced Egyptian bladder cancer [HDAC2 mRNA, as compared to normal uroepithelial cells obtained from healthy human ureters. Notably, HDAC8 mRNA was also presented with elevated levels in a distinct collection of cell lines, while HDAC8 gene proved to follow an upregulated pattern of activity in tumor versus control specimens [HDAC2 and 8 transcript levels with malignancy grades or stages of the disease could be observed [HDAC8 knockout could not impair the viability of urothelial cancer cell lines in a therapeutically useful manner, thus questioning its capacity to serve as a promising and powerful drug target for bladder cancer [HDAC3 gene transcription cannot be excluded. The bladder cancer cell line-dependent regulation of HDAC1 gene (and its cognate protein product) [Class I HDACs are the most extensively characterized members of the family in urothelial bladder cancers. The ability of, siRNA-mediated, HDAC1 and 2 double knockout to cause significant impairment in cell proliferation and clonogenic growth, and to also induce apoptotic death of urothelial carcinoma cell lines strongly indicate the critical contribution of class I HDAC member activities to disease initiation and progression . In acco, nuclei ,165. A slthy one . Furtherlthy one . Intereslthy one . The utilthy one . In the r cancer . Likewispecimens . Howeverobserved . Intrigur cancer . Regardir cancer ,168,171.product) ,165,171 HDAC9 mRNA was shown with upregulated levels in 5/18 of the examined cell lines, indicating, again, the importance of specific oncogenic components to the control of each HDAC expression and activity. Accordingly, but unlike HDAC2 and 8, protein levels of HDAC4 and 7 were found generally reduced in malignant versus normal uro(epi)thelial cells. However, no statistically significant difference of HDAC9 mRNA contents was observed in 24 bladder cancer specimens when compared to 12 control  tissues. The mechanistic complexity of HDAC9-mediated deacetylation of bladder cancer cell substrates is further underlined by patterns of decreased HDAC9 expression being derived from publicly available microarray data. In contrast, using the same platform, HDAC2, 3 and 8 were presented with a proclivity towards tumor-dependent augmented transcription, thus indicating the differential contribution of each HDAC enzyme to bladder cancer development [All HDAC expression landscapes have been molecularly mapped in a number of urothelial bladder cancer cell lines being typified by different malignancy grades and stages ,171. Deselopment ,171. Witelopment ,173. It HDAC6 mRNA levels, although the cognate protein is presented with a notably varying expression among them [HDAC6 over-expression was observed in urothelial cancer versus control  tissues, while in 1/6 (publicly available) microarray data reports, elevated transcriptional activity of the gene was also detected. However, these findings could not be confirmed in the remaining five microarray studies and in a different cohort of bladder cancer patients, as well [HDAC10, the second gene member of this class, it has been previously presented with varying mRNA expression profiles in a selected collection of bladder cancer cell lines, although a tendency towards its moderate upregulation in carcinoma versus normal urothelial tissues has been also observed [Hitherto, only a few studies investigating the expression patterns of class IIb HDAC enzymes in bladder cancer have been published. It has been shown that a significant number of bladder cancer cell lines are characterized by downregulated ong them ,171,174. as well ,171,174.observed ,171,174.HDAC11 gene activity, with 10/18 lines carrying decreased, 5/18 unchanged and 3/18 elevated mRNA levels [HDAC11 transcriptional expression may be critically controlled by specific oncogenic networks differentially operating in each bladder cancer cell line examined. Since its depletion seems sufficient to cause death in neuroblastoma, breast, ovarian, prostate and colon cancer cell lines [The class IV family member HDAC11 represents a poorly investigated enzyme in urothelial bladder cancer. Again, examination of several bladder cancer cell lines, being typified by distinct malignancy grades and stages, revealed varying patterns of A levels ,171. Simll lines ,176, HDASIRT1 upregulation in bladder cancer (as compared to matched control) tissues was observed, thus indicating its oncogenic role in urothelial malignancies. In accordance, SIRT1 knockout has proved able to suppress bladder cancer cell proliferation, viability and migration, and also reduce the EMT program potency [SIRT2 mRNA, combined with a tubacin-induced inhibition of HDAC6 family member, resulted in significant suppression of bladder cancer cell migration and invasion capacities, strongly supporting their (SIRT2 and HDAC6) cooperative actions and tumor promoting roles in urothelial malignancies [SIRT6 also acts as a tumor suppressor gene, since its over-expression can inhibit proliferation of bladder cancer cells, while its expression  decreases  with progression of urothelial bladder cancer from T2 to T4 stage [SIRT7 has been previously presented with an upregulated transcriptional activity in bladder carcinomas versus matched normal tissues, with the high-grade and stage tumors being characterized by elevated SIRT7 expression levels, as compared to the low-grade and stage ones [SIRT7 downregulation that causes increased apoptosis, and reduced proliferation and motility of bladder cancer cells [To elucidate the role of SIRT1, a key member of class III HDAC enzyme family, in urothelial tumorigenesis, its levels of expression in clinical cancer samples were appropriately measured and quantified. In one report, using publicly available data sets, its expression levels were downregulated in bladder carcinomas versus normal tissues, suggesting a tumor suppressor function for the disease ,156. How potency . It seemgnancies . Similargnancies  points ognancies ,113, theT4 stage . On the age ones , thus iner cells  further HDAC4 is subjected to deep deletion more frequently, when compared to the rest of the 17 genes of the family, while HDAC9 and 11 show the highest percentage of amplification incidents among all examined members   has reve members . It mustThe epigenetic dysregulation of tumor suppressor genes is considered as an alternative to gene mutations mechanism of cancer pathogenesis. Therefore, epigenetic modification-related enzymes, such as HDACs, can and have already been successfully used as cancer therapeutic targets, since epigenetic control seems to be reversible, and the engaged regulatory proteins can be targeted using small molecules . HDACs aHDACis are typically characterized by a molecular structure being comprised of a surface recognition motif (CAP), a zinc-binding group (ZBG) and a linker domain connecting the CAP and ZBG motifs. HDACis’ common mechanism of action is to bind to the catalytic pocket of HDAC and chelate the critical zinc ion required for the catalytic function of the enzyme  186,187,189,190.WAF1 levels via a p53 independent pathway and apoptosis induction [RAS genes. These observations strongly suggest a potential value of romidepsin in developing anti-cancer therapeutic regimens against RAS-related bladder cancers [Romidepsin  is a Class I-specific HDACi, which belongs to the chemical class of cyclic peptides B. Under nduction . A time-nduction . Similarnduction . Interesnduction . These anduction . In the nduction . A quantnduction . Oncogen cancers ,207,208.Efficient adenoviral (Ad) infection, which is commonly used as a delivery system in gene-therapy protocols, requires the presence of coxsackievirus and adenovirus type-5 receptor (CAR). CAR, typically, is a cell-adhesion molecule linked to cell motility and tumor invasiveness , and itsWAF1 expression, and a decrease in invasion rate (except RT4), but had no effect in migration capacity. Moreover, valproic acid significantly inhibited tumor growth of T24 xenograft models [WAF1 protein expression [KIP1 and reduced the cell-cycle regulators Cdk1, 2 and 4, and Cyclin B, D1 and E [Valproic acid (VPA) is a pan-HDACi of the short-chain fatty acids chemical class C and an t models . Exposurpression . Time- aD1 and E . ValproiD1 and E .XPC gene, while prior treatment of these cells with valproic acid enhanced cisplatin-induced activation of caspase-3 [XPC is a DNA-damage recognition protein required for the initiation of the nucleotide-excision repair (NER) process and reduced levels of XPC protein have been reported in tumors derived from bladder cancer patients. Valproic acid administration in T24 and 5637 bladder cancer cells induced transcription of aspase-3 . In anotaspase-3 . The effaspase-3 . Similaraspase-3 . Notablyaspase-3 . Interesaspase-3 . Finallyaspase-3 .WAF1 mRNA and protein, an induction that may be regulated, at least in part, by histone acetylation in chromatin associated with the promoter and coding regions of WAF1p21 gene [WAF1p21, Hep27, Histone H2B, TRPM2, Gelsolin, α-Tubulin, Glutaredoxin and Metallothionein 1L, and a repression in Thymidylate Synthase, CTP synthase, APRIL and TRP gene expression [WAF1 upregulation, a Thymidylate Synthase downregulation and an induction of Caspase 3/7 activities [Notch3 contents in bladder tumor tissues, as compared to control (non-cancerous) samples. This Notch3 over-expression was associated with poor prognosis and shorter overall patient survival. Treatment of T24 bladder cancer cells with vorinostat upregulated Notch3 protein-acetylation levels, decreased Notch3 expression, reduced proliferation and induced cell-cycle arrest, thus indicating the integration of vorinostat in bladder cancer therapeutic schemes [Vorinostat (SAHA: suberanilohydroxamic acid) is a pan-HDACi that belongs to the class of hydroxamic acids A and undAF1 gene . Microarpression . In anottivities . In bladtivities . Finally schemes .WAF1p21 transcript contents, Plakoglobin upregulation, and cell death [WAF1p21 mRNA expression, G1 cell-cycle arrest and apoptotic cell death were obtained in BIU-87 cells, in response to treatment with TSA [TRPM2 promoter [E-cadherin and RARβ2, thus suggesting a reversible cross-talk between histone acetylation and DNA demethylation processes [473) expression in bladder cancer cells, and to inhibit TGIF (TG-Interacting Factor), whose over-expression is correlated with poor prognosis and chemoresistance to gemcitabine [L anti-apoptotic proteins [Trichostatin A (TSA) is a pan-HDACi that belongs to the chemical class of hydroxamic acids D. TSA adll death . Similarwith TSA . Cao et promoter . In anotpromoter . In vivopromoter . Using Trocesses . As it hrocesses . TSA courocesses . Furthercitabine . These dcitabine . As mentcitabine ,211. TSAcitabine . Notablycitabine . Most imcitabine ,237. Int0/G1-phase and activating apoptosis. They could significantly increase the acetylation contents of Histone H3, induce WAF1p21 mRNA expression and downregulate Cyclin A mRNA levels [Entinostat (MS275) is a Class I HDACi, which belongs to the chemical group of benzamides E. In a sA levels . In anotA levels . ExposurA levels . Lee et A levels .WAF1p21 expression [Belinostat (PXD101) is a pan-HDACi, which belongs to the class of hydroxamic acids F and undpression . A new mpression . Most impression .Panobinostat (LBH589) is a pan-HDACi, which belongs to the class of hydroxamic acids G and undTubacin (Tubulin acetylation inducer) is a highly potent and selective HDAC6 inhibitor that belongs to the class of hydroxamic acids H and is Conclusively, although HDAC inhibition seems to serve as a promising strategy for bladder cancer therapy, accumulated evidences suggest that for maximizing the anti-tumor effects of HDACis, these may not be used as monotherapy agents, but in combination with other already established drugs and therapeutic schemes, including radiation.Hitherto, four different HDACis have been approved by the FDA for cancer therapy. In 2006, vorinostat was the first HDACi to be approved for the treatment of progressive, persistent or recurrent CTCL, following two systemic therapies . SimilarThe principle in using HDACis as, small-molecule, anti-cancer drugs is their capacity to increase core histones’ acetylation, hence strongly triggering an open-chromatin state, and consequently the high accessibility of DNA-targeting agents. In this regard, the mode-of-action of HDACis mainly includes cell-cycle arrest, terminal differentiation and cell death in transformed cancer cells, but not in normal cells, which are presented as either insensitive or capable to effectively overcome the drug-induced stress. Additionally, HDACis can also exert their tumor-suppressing activities by inhibiting angiogenesis and/or modulating immune responses ,253,254.Combination schemes of HDACis with agents targeting DNA damage-response (DDR) pathways or topoisomerase inhibitors have been reported to effectively increase DNA damage, cell-cycle arrest and death of cancer cells, as compared to monotherapy. Interestingly, HDACis have been documented to induce DNA double-strand breaks (DSBs), as well as to attenuate DDR, through reduction of ATM and p53 activities, specifically in cancer cells ,258. SimThe ability of HDACis to attenuate DDR leads to an enhanced radio-sensitivity of cancer cells, with favorable results having been reported by the combination of HDACis with radiotherapy . In the Focusing on platinum-based chemotherapy, phase-I trials of belinostat or vorinostat, in combination with carboplatin and paclitaxel, in patients with solid tumors, underscored the well-tolerance of the scheme, along with notable benefits in patients’ response rates ,267. In Additionally, numerous phase-I trials have also focused on the combination of HDACis with Receptor Tyrosine Kinase (RTK)-targeted agents, including sorafenib (a multi-kinase inhibitor of MAPK pathway) and gefitinib (an EGFR inhibitor). Interestingly, an ongoing phase-I/II single group trial (NCT00918333) on the combination of everolimus (an mTOR inhibitor) with panobinostat for the treatment of recurrent MM, non-Hodgkin’s and Hodgkin’s lymphoma has already shown the absence of severe adverse effects. However, a phase-Ib/IIa trial on the combination of trastuzumab  with panobinostat in HER2-positive metastatic breast cancer did not prove a clinical benefit for the patients (NCT00567879).Immune-checkpoint inhibitors represent one of the most widely examined categories of anti-cancer agents nowadays and are already approved by the FDA for cancer therapy. Several studies have pointed out the ability of HDACis to regulate immune responses, and their combination with immune-checkpoint inhibitors has been presented to improve clinical outcomes ,270. In CREBBP and/or EP300 acetyltransferase genes, previously treated for locally advanced or metastatic urothelial bladder carcinoma, has been studied in a completed phase-II trial (NCT02236195). This trial represents the first genomic-based approach for the identification of patients that are suitable (or more likely to benefit) for treatment with HDACis; however, severe toxicities were observed, compromising the overall clinical therapeutic success [Focusing on urothelial bladder carcinoma , vorinos success . On the HAT/HDAC gene combinations with variable oncogenic activities. To further complicate things, there could be more than one family member able to modulate the “acetyl-load” of a given target, while many substrates might be specifically targeted by only one (HAT/HDAC) member. Moreover, presumable operation of redundant responses, during which changes in one protein’s acetylation levels are compensated by critical changes in a different protein, most likely stiffen and worsen our comprehension, and view of the system’s functionality. These are most likely some of the reasons justifying the “failure” of HDACi-mediated monotherapy to meet its promise, since only combination schemes are presented beneficial for the patients. A new generation of HAT/HDAC-specific inhibitors and novel cocktail-drug/radiation protocols are expected to overcome the existing limitations, and offer the desired therapeutic success for several human malignancies.The cellular “acetyl-proteome” and especially “acetyl-chromatin” have emerged as major druggable landscapes for human malignancies. Targeting the plethora of molecular “writers” (HATs) and “erasers” (HDACs), along with their cognate substrates, opens a new therapeutic window for the disease. However, the presumable tissue-specific expression and/or activity of HAT/HDAC family members necessitate their cancer cell-dependent molecular profiling before treatment. In the same direction, given the heterogeneity of a solid tumor, different sub-populations may express distinct In recent years, HDACs have emerged as key players to cellular homeostasis. Deregulation of these enzymes ultimately leads to several malignancies, including bladder cancer, by various mechanisms. This review brings together important information about HDACs and their specific inhibitors, with some of them already being tested in clinical trials, demonstrating potential for treating urothelial cancers. The knowledge gathered in this study regarding HDACis, provides insights necessary for directing future research in this field.