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Autophagy, the process of lysosomal degradation of biological materials within cells, is often halted abnormally in proteopathies, such as tauopathy and amyloidopathy. Thus, autophagy regulators that rescue dysregulated autophagy have great potential to treat proteopathies. We previously reported that the natural small molecule kaempferide (Kaem) induces autophagy without perturbing mTOR signaling. Here, we report that Kaem promotes lysosomal degradation of microtubule-associated protein tau (MAPT) in inducible MAPT cells. Kaem enhanced autophagy flux by mitigating microtubule-associated protein 1 light chain 3 (LC3) accumulation when MAPT expression was induced. Kaem also promoted activation of transcription factor EB (TFEB) without inhibiting mTOR and without mTOR inhibition-mediated cytotoxicity. In addition, Kaem-induced MAPT degradation was abolished in the absence of mitochondrial elongation factor Tu (TUFM), which was previously shown to be a direct binding partner of Kaem. Collectively, these results demonstrate that Kaem could be a potential therapeutic for tauopathy and reveal that TUFM can be a drug target for autophagy-driven disorders.

Small molecules targeting autophagy have been highly implicated as new therapeutic agents to treat diseases of interest. With the increasing demand for autophagy-targeting drugs, this review attempts to provide an efficient strategy to explore major autophagy-based human disease interventions with newly explored mechanisms using small molecules and promising therapeutic approaches.Introduced in this review are direct links and applications among autophagy pathways, their modulators, and phenotypic diseases, along with recent approaches. Autophagy-related diseases, machinery, and compounds are introduced to guide the appropriate investigation of autophagy in the pharmaceutical industry. The authors then provide their expert perspectives on the subject.The self-catabolic intracellular process autophagy occurs in organisms throughout their lifetime, supporting its critical role in organismal health across life stages. Because of the detrimental influence of dysfunctional cells to an organism and their etiology in numerous diseases, maintaining cellular quality control by recycling components through autophagy is essential to prevent health decline.

In the advanced stages, malignant melanoma (MM) has a very poor prognosis. Due to tremendous efforts in cancer research over the last 10 years, and the introduction of novel therapies such as targeted therapies and immunomodulators, the rather dark horizon of the median survival has dramatically changed from under 1 year to several years. With the advent of proteomics, deep-mining studies can reach low-abundant expression levels. The complexity of the proteome, however, still surpasses the dynamic range capabilities of current analytical techniques. Consequently, many predicted protein products with potential biological functions have not yet been verified in experimental proteomic data. This category of ‘missing proteins’ (MP) is comprised of all proteins that have been predicted but are currently unverified. As part of the initiative launched in 2016 in the USA, the European Cancer Moonshot Center has performed numerous deep proteomics analyses on samples from MM patients. In this study, nine MPs were clearly identified by mass spectrometry in MM metastases. Some MPs significantly correlated with proteins that possess identical PFAM structural domains; and other MPs were significantly associated with cancer-related proteins. This is the first study to our knowledge, where unknown and novel proteins have been annotated in metastatic melanoma tumour tissue.

The MM500 meta‐study aims to establish a knowledge basis of the tumor proteome to serve as a complement to genome and transcriptome studies. Somatic mutations and their effect on the transcriptome have been extensively characterized in melanoma. However, the effects of these genetic changes on the proteomic landscape and the impact on cellular processes in melanoma remain poorly understood. In this study, the quantitative mass‐spectrometry‐based proteomic analysis is interfaced with pathological tumor characterization, and associated with clinical data. The melanoma proteome landscape, obtained by the analysis of 505 well‐annotated melanoma tumor samples, is defined based on almost 16 000 proteins, including mutated proteoforms of driver genes. More than 50 million MS/MS spectra were analyzed, resulting in approximately 13,6 million peptide spectrum matches (PSMs). Altogether 13 176 protein‐coding genes, represented by 366 172 peptides, in addition to 52 000 phosphorylation sites, and 4 400 acetylation sites were successfully annotated. This data covers 65% and 74% of the predicted and identified human proteome, respectively. A high degree of correlation (Pearson, up to 0.54) with the melanoma transcriptome of the TCGA repository, with an overlap of 12 751 gene products, was found. Mapping of the expressed proteins with quantitation, spatiotemporal localization, mutations, splice isoforms, and PTM variants was proven not to be predicted by genome sequencing alone. The melanoma tumor molecular map was complemented by analysis of blood protein expression, including data on proteins regulated after immunotherapy. By adding these key proteomic pillars, the MM500 study expands the knowledge on melanoma disease., The MM500 meta‐study aims to establish a knowledge basis of the tumor proteome to serve as a complement to genome and transcriptome studies. The melanoma proteome landscape, obtained by the analysis of 505 well‐annotated melanoma tumor samples, is defined based on almost 16 000 proteins, including mutated proteoforms of driver genes. This data covers 65% and 74% of the predicted and identified human proteome, respectively.

Genomic analysis of the clavulanic acid (CA)-high-producing Streptomyces clavuligerus strains, OL13 and OR, developed through random mutagenesis revealed a frameshift mutation in the cas1 gene-encoding clavaminate synthase 1. Overexpression of the intact cas1 in S. clavuligerus OR enhanced the CA titer by approximately 25%, producing ~ 4.95 g/L of CA, over the OR strain in the flask culture. Moreover, overexpression of the pathway-specific positive regulatory genes, ccaR and claR, in the OR strain improved CA yield by approximately 43% (~ 5.66 g/L) in the flask. However, co-expression of the intact cas1 with ccaR-claR did not further improve CA production. In the 7 L fermenter culture, maximum CA production by the OR strain expressing the wild-type cas1 and ccaR-claR reached approximately 5.52 g/L and 6.01 g/L, respectively, demonstrating that reverse engineering or simple rational metabolic engineering is an efficient method for further improvement of industrial strains.

Melanoma of the skin is the sixth most common type of cancer in Europe and accounts for 3.4% of all diagnosed cancers. More alarming is the degree of recurrence that occurs with approximately 20% of patients lethally relapsing following treatment. Malignant melanoma is a highly aggressive skin cancer and metastases rapidly extend to the regional lymph nodes (stage 3) and to distal organs (stage 4). Targeted oncotherapy is one of the standard treatment for progressive stage 4 melanoma, and BRAF inhibitors (e.g. vemurafenib, dabrafenib) combined with MEK inhibitor (e.g. trametinib) can effectively counter BRAFV600E-mutated melanomas. Compared to conventional chemotherapy, targeted BRAFV600E inhibition achieves a significantly higher response rate. After a period of cancer control, however, most responsive patients develop resistance to the therapy and lethal progression. The many underlying factors potentially causing resistance to BRAF inhibitors have been extensively studied. Nevertheless, the remaining unsolved clinical questions necessitate alternative research approaches to address the molecular mechanisms underlying metastatic and treatment-resistant melanoma. In broader terms, proteomics can address clinical questions far beyond the reach of genomics, by measuring, i.e. the relative abundance of protein products, post-translational modifications (PTMs), protein localisation, turnover, protein interactions and protein function. More specifically, proteomic analysis of body fluids and tissues in a given medical and clinical setting can aid in the identification of cancer biomarkers and novel therapeutic targets. Achieving this goal requires the development of a robust and reproducible clinical proteomic platform that encompasses automated biobanking of patient samples, tissue sectioning and histological examination, efficient protein extraction, enzymatic digestion, mass spectrometry-based quantitative protein analysis by label-free or labelling technologies and/or enrichment of peptides with specific PTMs. By combining data from, e.g. phosphoproteomics and acetylomics, the protein expression profiles of different melanoma stages can provide a solid framework for understanding the biology and progression of the disease. When complemented by proteogenomics, customised protein sequence databases generated from patient-specific genomic and transcriptomic data aid in interpreting clinical proteomic biomarker data to provide a deeper and more comprehensive molecular characterisation of cellular functions underlying disease progression. In parallel to a streamlined, patient-centric, clinical proteomic pipeline, mass spectrometry-based imaging can aid in interrogating the spatial distribution of drugs and drug metabolites within tissues at single-cell resolution. These developments are an important advancement in studying drug action and efficacy in vivo and will aid in the development of more effective and safer strategies for the treatment of melanoma. A collaborative effort of gargantuan proportions between academia and healthcare professionals has led to the initiation, establishment and development of a cutting-edge cancer research centre with a specialisation in melanoma and lung cancer. The primary research focus of the European Cancer Moonshot Lund Center is to understand the impact that drugs have on cancer at an individualised and personalised level. Simultaneously, the centre increases awareness of the relentless battle against cancer and attracts global interest in the exceptional research performed at the centre.

Clavulanic acid (CA) produced by Streptomyces clavuligerus is a clinically important β-lactamase inhibitor. It is known that glycerol utilization can significantly improve cell growth and CA production of S. clavuligerus. We found that the industrial CA-producing S. clavuligerus strain OR generated by random mutagenesis consumes less glycerol than the wild-type strain; we then developed a mutant strain in which the glycerol utilization operon is overexpressed, as compared to the parent OR strain, through iterative random mutagenesis and reporter-guided selection. The CA production of the resulting S. clavuligerus ORUN strain was increased by approximately 31.3% (5.21 ± 0.26 g/l) in a flask culture and 17.4% (6.11 ± 0.36 g/l) in a fermenter culture, as compared to that of the starting OR strain. These results confirmed the important role of glycerol utilization in CA production and demonstrated that reporter-guided mutant selection is an efficient method for further improvement of randomly mutagenized industrial strains.

The MM500 study is an initiative to map the protein levels in malignant melanoma tumor samples, focused on in‐depth histopathology coupled to proteome characterization. The protein levels and localization were determined for a broad spectrum of diverse, surgically isolated melanoma tumors originating from multiple body locations. More than 15,500 proteoforms were identified by mass spectrometry, from which chromosomal and subcellular localization was annotated within both primary and metastatic melanoma. The data generated by global proteomic experiments covered 72% of the proteins identified in the recently reported high stringency blueprint of the human proteome. This study contributes to the NIH Cancer Moonshot initiative combining detailed histopathological presentation with the molecular characterization for 505 melanoma tumor samples, localized in 26 organs from 232 patients., The MM500 study is an initiative to map the protein levels in malignant melanoma tumor samples, focused on in‐depth histopathology coupled to proteome characterization. The protein levels and localization were determined for a broad spectrum of diverse melanoma tumors originating from multiple body locations. More than 15,500 proteoforms were identified by mass spectrometry, from which chromosomal and sub‐cellular localization was annotated within both primary and metastatic melanoma.

Ciliogenesis is often impaired in some cancer cells, leading to acceleration of cancer phenotypes such as cell migration and proliferation. From the investigation of primary cilia of 16 gastric cancer cells (GCs), we found that GCs could be grouped into four primary cilia (PC)–positive GCs and 12 PC-negative GCs. The proliferation of the PC-positive GCs was lower than that of PC-negative GCs. To explore the role of fatty acid binding protein 4 (FABP4), which is a known oncogenic factor, in ciliogenesis, FABP4 expression and function were inhibited by transfection of cells with short interfering RNA targeting FABP4 (siFABP4) or FABP4 inhibitor treatment. Notably, the proliferation and migration of the cilia-forming GCs was effectively suppressed by inhibition of FABP4. In addition, the primary cilia in GCs were restored by a factor greater than two, suggesting a negative role of FABP4 in ciliogenesis in these GCs and FABP4 as a potential anticancer target.

Simple Summary Non-small-cell lung carcinoma (NSCLC) covers for almost 85% of all lung cancers and a major contributor to the overall cancer death rate. Erlotinib is used to treat NSCLC via inhibition of epithelial growth factor receptor (EGFR) kinase activity. Despite its high efficacy, recurrence can occur in patients who become resistant to the drug. We performed DARTS LC-MS/MS with SWATH of DIA analysis and identified a novel binding protein of Erlotinib that may underlie NSCLC resistance. Our study indicated that Erlotinib binds POLA2 in addition to EGFR. This was confirmed by DARTS and CETSA results. Importantly, POLA2 expression levels in four NSCLC cell lines were positively correlated with anti-proliferative Erlotinib efficacy (Pearson correlation coefficient, R = 0.9886). These results suggest that POLA2 is a novel complementary target protein of Erlotinib, and could clinically provide validity as a surrogate marker for drug resistance in patients with NSCLC. Abstract Erlotinib inhibits epithelial growth factor receptor (EGFR) kinase activity and is used to treat non-small cell lung cancer (NSCLC). Despite its high efficacy, recurrence can occur in patients who become resistant to the drug. To address the underlying mechanism of Erlotinib resistance, we investigated additional mechanisms related to mode-of-drug-action, by multiple protein-binding interactions, besides EGFR by using drug affinity responsive target stability (DARTS) and liquid chromatography-mass spectrometry (LC-MS/MS) methods with non-labeled Erlotinib. DNA polymerase alpha subunit B (POLA2) was identified as a new Erlotinib binding protein that was validated by the DARTS platform, complemented with cellular thermal shift assays. Genetic knock-down of POLA2 promoted the anti-proliferative effect of the drug in the Erlotinib-resistant cell line H1299 with high POLA2 expression, whereas the overexpression of POLA2 restored anti-proliferative effects in the Erlotinib-sensitive cell line HCC827 with low POLA2 expression. Importantly, POLA2 expression levels in four NSCLC cell lines were positively correlated with anti-proliferative Erlotinib efficacy (Pearson correlation coefficient, R = 0.9886). These results suggest that POLA2 is a novel complementary target protein of Erlotinib, and could clinically provide validity as a surrogate marker for drug resistance in patients with NSCLC.

Erlotinib inhibits epithelial growth factor receptor (EGFR) kinase activity and is used to treat non-small cell lung cancer (NSCLC). Despite its high efficacy, recurrence can occur in patients who become resistant to the drug. To address the underlying mechanism of Erlotinib resistance, we investigated additional mechanisms related to mode-of-drug-action, by multiple protein-binding interactions, besides EGFR by using drug affinity responsive target stability (DARTS) and liquid chromatography-mass spectrometry (LC-MS/MS) methods with non-labeled Erlotinib. DNA polymerase alpha subunit B (POLA2) was identified as a new Erlotinib binding protein that was validated by the DARTS platform, complemented with cellular thermal shift assays. Genetic knock-down of POLA2 promoted the anti-proliferative effect of the drug in the Erlotinib-resistant cell line H1299 with high POLA2 expression, whereas overexpression of POLA2 restored anti-proliferative effects in the Erlotinib-sensitive cell line HCC827 with low POLA2 expression. Importantly, POLA2 expression levels in four NSCLC cell lines were positively correlated with anti-proliferative Erlotinib efficacy, verified by bio-statistical analysis (Pearson correlation coefficient, R=0.9886). These results suggest that POLA2 is a novel complementary target protein of Erlotinib, and could clinically provide validity as a surrogate marker for drug resistance in patients with NSCLC.

Background A gel‐free proteomic approach was utilized to perform in‐depth tissue protein profiling of lung adenocarcinoma (ADC) and normal lung tissues from early and advanced stages of the disease. The long‐term goal of this study is to generate a large‐scale, label‐free proteomics dataset from histologically well‐classified lung ADC that can be used to increase further our understanding of disease progression and aid in identifying novel biomarkers. Methods and results Cases of early‐stage (I‐II) and advanced‐stage (III‐IV) lung ADCs were selected and paired with normal lung tissues from 22 patients. The histologically and clinically stratified human primary lung ADCs were analyzed by liquid chromatography‐tandem mass spectrometry. From the analysis of ADC and normal specimens, 4863 protein groups were identified. To examine the protein expression profile of ADC, a peak area‐based quantitation method was used. In early‐ and advanced‐stage ADC, 365 and 366 proteins were differentially expressed, respectively, between normal and tumor tissues (adjusted P‐value

Manipulating autophagy is a promising strategy for treating cancer as several autophagy inhibitors are shown to induce autophagic cell death. One of these, autophagonizer (APZ), induces apoptosis-independent cell death by binding an unknown target via an unknown mechanism. To identify APZ targets, we used a label-free drug affinity responsive target stability (DARTS) approach with a liquid chromatography/tandem mass spectrometry (LC–MS/MS) readout. Of 35 protein interactors, we identified Hsp70 as a key target protein of unmodified APZ in autophagy. Either APZ treatment or Hsp70 inhibition attenuates integrity of lysosomes, which leads to autophagic cell death exhibiting an excellent synergism with a clinical drug, temozolomide, in vitro, in vivo, and orthotropic glioma xenograft model. These findings demonstrate the potential of APZ to induce autophagic cell death and its development to combinational chemotherapeutic agent for glioma treatment. Collectively, our study demonstrated that APZ, a new autophagy inhibitor, can be used as a potent antitumor drug candidate to get over unassailable glioma and revealed a novel function of Hsp70 in lysosomal integrity regulation of autophagy.

Background: The cellular senescence of primary cultured cells is an irreversible process characterized by growth arrest. Restoration of senescence by ginsenosides has not been explored so far. Rg3(S) treatment markedly decreased senescence-associated β-galactosidase activity and intracellular reactive oxygen species levels in senescent human dermal fibroblasts (HDFs). However, the underlying mechanism of this effect of Rg3(S) on the senescent HDFs remains unknown. Methods: We performed a label-free quantitative proteomics to identify the altered proteins in Rg3(S)-treated senescent HDFs. Upregulated proteins induced by Rg3(S) were validated by real-time polymerase chain reaction and immunoblot analyses. Results: Finally, 157 human proteins were identified, and variable peroxiredoxin (PRDX) isotypes were highly implicated by network analyses. Among them, the mitochondrial PRDX3 was transcriptionally and translationally increased in response to Rg3(S) treatment in senescent HDFs in a time-dependent manner. Conclusion: Our proteomic approach provides insights into the partial reversing effect of Rg3 on senescent HDFs through induction of antioxidant enzymes, particularly PRDX3. Keywords: Ginsenoside Rg3(S), Human dermal fibroblast, Label-free quantitative proteomics, Restoration, Senescence

We present the Cancer Moonshot clinical project located at the European center in Lund. Here, tissue and blood samples have been collected and stored in a large-scale biobank. Multiple clinical centers around the world are participating and tissue and blood samples are sent to the European Cancer Moonshot Lund Center that acts as the clinical hub. Our center has been developed to generate and build large-scale biostorage archives of patient melanoma samples, which is then combined with a histopathological capability to characterize the patient tumours. Such a large-scale clinical sample processing initiative has begun with the aim of creating high-end histopathology indexing with database computational power and including proteogenomic analysis. The biobank at Lund has become an important resource in clinical research worldwide. Following suite, several national health programs are being initiated with the aim of also building large-scale biobank storages with a wealth of high-quality patient samples. In our Cancer Moonshot R&D activities, samples in the biobanks and the data derived from these samples are being used to build an understanding of disease presentation and using this information to move towards ‘Big Data’ proteogenomic and mass spectrometry imaging studies. Additionally, we report here a sample processing workflow that has been adapted to a fully-automated biobank processing strategy for large-scale studies.

Cholesterol plays a key role in membrane protein function and signaling in endothelial cells. Thus, disturbing cholesterol trafficking is an effective approach for inhibiting angiogenesis. We recently identified astemizole (AST), an antihistamine drug, as a cholesterol trafficking inhibitor from a phenotypic screen. In this study, we found that AST induced cholesterol accumulation in the lysosome by binding to the sterol-sensing domain of Niemann-Pick disease, type C1 (NPC1), a lysosomal surface protein responsible for cholesterol transport. Inhibition of cholesterol trafficking by AST led to the depletion of membrane cholesterol, causing SREBP1 nuclear localization. The depletion of membrane cholesterol resulted in dissociation of mammalian target of rapamycin (mTOR) from the lysosomal surface and inactivation of mTOR signaling. These effects were effectively rescued by addition of exogenous cholesterol. AST inhibited endothelial cell proliferation, migration and tube formation in a cholesterol-dependent manner. Furthermore, AST inhibited zebrafish angiogenesis in a cholesterol-dependent manner. Together, our data suggest that AST is a new class of NPC1 antagonist that inhibits cholesterol trafficking in endothelial cells and angiogenesis.

Background Cyclosporine (CsA) usually reduces glomerular filtration rate (GFR) but also can induce tubular injury without resulting in GFR reduction. Apelin is an endogenous ligand for the apelin receptor and has diverse physiologic roles related to hemodynamic or metabolic processes. We investigated the renoprotective role of apelin against CsA-induced tubular toxicity in rats. Methods Rats were given CsA (15 mg/kg/day) and/or apelin-13 (15 μg/kg/day) for 7 days via subcutaneous injection. We performed serum and urinary assays of creatinine and neutrophil gelatinase-associated lipocalin (NGAL) to estimate renal injury and performed Western blotting for endothelial nitric oxide synthase and nuclear factor of activated T-cell cytoplasmic 1 (NFATc1) to document the underlying mechanism. Results The CsA-treated group showed increased urinary creatinine excretion, polyuria, and renal glycosuria without GFR reduction, suggesting adequate CsA-induced renal tubular injury. Urinary NGAL excretion also increased significantly in the CsA group. Conversely, apelin attenuated CsA-induced tubular injury and had no effect on urinary NGAL excretion. In histopathologic examination, the apelin-treated group had lower tubulo-interstitial injury scores compared with those in the CsA group. Regarding the effects of apelin, our results indicate that apelin provides protection against CsA-induced tubular injury by activating nitric oxide and/or the NFATc1 pathway. Notably, we also found that CsA inhibits renal glucose reabsorption by reducing Na+-K+ ATPase expression and that apelin reverses reduced renal glucose reabsorption by CsA in tubular cells. Conclusions Our study demonstrates the renoprotective effect of apelin against CsA-induced renal tubular toxicity and provides novel insights into the effects of CsA and apelin on renal tubular cells.

Background Daptomycin is a recently introduced, last-resort antibiotic that displays a unique mode of action against Gram-positive bacteria that is not fully understood. Several bacterial targets have been proposed but no human binding partner is known. Methods In the present study we tested daptomycin in cell viability and proliferation assays against six human cell lines, describe the synthesis of biotinylated and fluorescently labeled analogues of daptomycin. Biotinylated daptomycin was used as bait to isolate the human binding partner by the application of reverse chemical proteomics using T7 phage display of five human tumor cDNA libraries. The interaction between the rescued protein and daptomycin was validated via siRNA knockdown, DARTS assay and immunocytochemistry. Results We have found that daptomycin possesses selective growth inhibition of some cancer cell lines, especially MCF7. The unbiased interrogation of human cDNA libraries, displayed on bacteriophage T7, revealed a single human target of daptomycin; ribosomal protein S19. Using a drug affinity responsive target stability (DARTS) assay in vitro, we show that daptomycin stabilizes RPS19 toward pronase. Fluorescently labeled daptomycin stained specific structures in HeLa cells and co-localized with a RPS19 antibody. Conclusion This study provides, for the first time, a human protein target of daptomycin and identifies RPS19 as a possible anticancer drug target for the development of new pharmacological applications and research. Electronic supplementary material The online version of this article (doi:10.1186/s12953-017-0124-2) contains supplementary material, which is available to authorized users.

The present research reports a novel biological activity of indatraline, a compound clinically used as an antidepressant. We previously identified indatraline as an autophagy inducer. Autophagy is an intracellular catabolic pathway for degrading or recycling unnecessary organelles in response to cellular stress. Indatraline-mediated autophagy induction results from mTOR inhibition. The mTOR is a negative regulator of autophagy as well as a master regulator of angiogenesis. Angiogenesis defines the process by which new vessels are formed from pre-existing vascular tissues, providing nutrients to cancer cells, allowing rapid tumor progression. Accordingly, targeting angiogenesis to prevent cancer is an attractive therapeutic strategy. Here, we demonstrate that indatraline possibly acts to suppress tumor-mediated angiogenesis via downregulation of HIF-1α-mediated VEGF expression. The effects of indatraline on autophagy and angiogenesis could make it a potential drug candidate toward cancer treatment.

Autophagy is a critical regulator of cellular homeostasis, dysregulation of which is associated with diverse diseases. Here we show therapeutic effects of a novel autophagy enhancer identified by high-throughput screening of a chemical library against metabolic syndrome. An autophagy enhancer increases LC3-I to LC3-II conversion without mTOR inhibition. MSL, an autophagy enhancer, activates calcineurin, and induces dephosphorylation/nuclear translocation of transcription factor EB (TFEB), a master regulator of lysosomal biogenesis and autophagy gene expression. MSL accelerates intracellular lipid clearance, which is reversed by lalistat 2 or Tfeb knockout. Its administration improves the metabolic profile of ob/ob mice and ameliorates inflammasome activation. A chemically modified MSL with increased microsomal stability improves the glucose profile not only of ob/ob mice but also of mice with diet-induced obesity. Our data indicate that our novel autophagy enhancer could be a new drug candidate for diabetes or metabolic syndrome with lipid overload., Autophagy plays an important role in metabolic functions and increased autophagic activity may be beneficial for metabolic disorders. Here the authors screen a chemical library for enhancer of autophagic flux and identify small molecules that improve the metabolic profile by increasing lysosomial functions.

In comparison to other human cancer types, malignant melanoma exhibits the greatest amount of heterogeneity. After DNA-based detection of the BRAF V600E mutation in melanoma patients, targeted inhibitor treatment is the current recommendation. This approach, however, does not take the abundance of the therapeutic target, i.e., the B-raf V600E protein, into consideration. As shown by immunohistochemistry, the protein expression profiles of metastatic melanomas clearly reveal the existence of inter- and intra-tumor variability. Nevertheless, the technique is only semi-quantitative. To quantitate the mutant protein there is a fundamental need for more precise techniques that are aimed at defining the currently non-existent link between the levels of the target protein and subsequent drug efficacy. Using cutting-edge mass spectrometry combined with DNA and mRNA sequencing, the mutated B-raf protein within metastatic tumors was quantitated for the first time. B-raf V600E protein analysis revealed a subjacent layer of heterogeneity for mutation-positive metastatic melanomas. These were characterized into two distinct groups with different tumor morphologies, protein profiles and patient clinical outcomes. This study provides evidence that a higher level of expression in the mutated protein is associated with a more aggressive tumor progression. Our study design, comprised of surgical isolation of tumors, histopathological characterization, tissue biobanking, and protein analysis, may enable the eventual delineation of patient responders/non-responders and subsequent therapy for malignant melanoma.

In comparison to other human cancer types, malignant melanoma exhibits the greatest amount of heterogeneity. After DNA-based detection of the BRAF V600E mutation in melanoma patients, targeted inhibitor treatment is the current recommendation. This approach, however, does not take the abundance of the therapeutic target, i.e., the B-raf V600E protein, into consideration. As shown by immunohistochemistry, the protein expression profiles of metastatic melanomas do clearly reveal the existence of inter- and intra-tumor variability. Nevertheless, the technique is only semi-quantitative. To quantitate the mutant protein there is a fundamental need for more precise techniques that are aimed at defining the currently non-existent link between the levels of the target protein and subsequent drug efficacy. Using cutting-edge mass spectrometry combined with DNA and mRNA sequencing, the mutated B-raf protein within metastatic tumors was quantitated for the first time. B-raf V600E protein analysis revealed a subjacent layer of heterogeneity for mutation-positive metastatic melanomas. These were characterized into two distinct groups with different tumor morphologies, protein profiles and patient clinical outcomes. This study provides evidence that a higher level of expression for the mutated protein is associated with a more aggressive tumor progression. Our study design that is comprised of surgical isolation of tumors, histopathological characterization, tissue biobanking, and protein analysis may enable the eventual delineation of patient responders/non-responders and the subsequent therapy of malignant melanoma.

When the production of Reactive Oxygen Species exceeds the capacity for anti-oxidation, the result is cell damage or cell death in melanoma caused by toxicity. This paper conducts a study on the chemo-preventive agents and the resulting chemoprevention. For example, plant polyphenols are examined for their potential to inhibit the process of reactive chain reactions in the UV-exposed skin. In addition, the paper analyzes the chemical and physical properties of the polyphenols that may be useful for skin diseases associated with solar UV radiation-induced inflammation and oxidative stress. Using computational programs, various information about molecules of polyphenols with functional groups is extracted. The optimization energies (kJ/mol) of the molecules are computed and used to determine the characteristics and stability of the molecules. Dipole moments (D) are used to determine the reactivity of the molecules. Measurement of the molecules' stability and reactivity allows evaluation of polyphenols and their ability to inhibit reactive chain reactions caused by Reactive Oxygen Species.

Ubiquinol-cytochrome c reductase (UQCRB), a subunit of the mitochondrial complex III, is highly expressed in tissues from colorectal cancer patients. Since UQCRB is highly expressed in colorectal cancer, we investigated miRNAs from mutant UQCRB-expressing cell lines to identify new miRNA biomarkers. After sequencing miRNAs in the mutant UQCRB-expressing cell lines, miR-4435 was selected as a potential biomarker candidate from the six up-regulated miRNAs. The expression level of miR-4435 in the mutant UQCRB-expressing cell lines and colon cancer was increased. Notably, the expression level of miR-4435 was increased in exosomes isolated from cell culture medium, suggesting that miR-4435 is closely related to colon cancer and that large amounts of miR-4435 may be secreted outside of the cells through exosomes. Additionally, exosomes extracted from the serum samples of colorectal cancer patients showed increased miR-4435 levels depending on the cancer progression stage. Moreover, analyses of a miRNA database and mRNA-sequencing data of the mutant UQCRB-expressing cell lines revealed that TIMP3, a tumor suppressor, could be a target of miR-4435. Additionally, the expression of miR-4435 was suppressed by UQCRB inhibitor treatment whereas TIMP3 was up-regulated. Upregulation of TIMP3 decreased proliferation of the mutant UQCRB-expressing cell lines and a colorectal cancer cell line. TIMP3 was also upregulated in response to miR-4435 inhibitor and UQCRB inhibitor treatments. Furthermore, these findings suggest that miR-4435 is related to an oncogenic function in UQCRB related disease, CRC, and that effects migration and invasion on mutant UQCRB-expressing cell lines and colorectal cancer cell. In conclusion, our results identified miR-4435 as a potential circulating miRNA biomarker of colorectal cancer associated with UQCRB.

Background and purpose Although non-alcoholic fatty liver disease (NAFLD) shares common cardiovascular risk factors with cerebral white matter hyperintensity (WMH), few studies have reported the association between NAFLD and WMH. The association between the presence of NAFLD with its severity and the volume of WMH was investigated. Methods This cross-sectional study was conducted for 2460 subjects who voluntarily participated in health screening check-ups including brain magnetic resonance imaging and liver ultrasonography at the Health Promotion Center at Seoul National University Hospital from 2009 to 2013. Ultrasonography was used to detect the presence and severity of NAFLD combined with the NAFLD fibrosis score and the FIB-4 index. The volume of WMH was measured using a semi-automated quantification method by a trained neurologist. Results The prevalence of NAFLD was 36.5%, and the median volume of WMH in all the subjects was 1.1 ml (interquartile range 0.2-2.7 ml). The presence of NAFLD was associated with a smaller volume of WMH [β (standard error, SE) -0.051 (0.046); P = 0.012]. Moderate to severe NAFLD was associated with a smaller volume of WMH than was non-NAFLD [β (SE) -0.067 (0.061); P = 0.002]. The negative correlation observed between NAFLD severity and WMH volume was persistent only in those with low FIB-4 index and low NAFLD fibrosis scores, whereas there was a positive association in those with high FIB-4 index and NAFLD fibrosis scores. Conclusions Non-alcoholic fatty liver disease, and its severity, showed a favorable association with WMH volume. However, its causality and mechanism should be evaluated in further relevantly designed studies.

Angiogenesis generates new blood vessels from pre-existing vessels. Tumors induce the formation of new blood vessels to ensure sufficient oxygen and nutrients for their growth. Normally, angiogenesis is induced by various pro-angiogenesis factors, including vascular endothelial growth factor (VEGF). Inhibition of VEGF is a promising approach to cancer treatment. A guanidine-based synthetic compound, E2, was identified as a potent hit from 68 guanidine-based derivatives by screening for angiogenesis inhibitors showing antiproliferative activity in human umbilical vein endothelial cells (HUVECs). To explore the mode of action of E2, target proteins were investigated using phage display biopanning, and acid ceramidase 1 (ASAH1) was identified as an E2-binding protein. Drug affinity responsive target stability (DARTS) and ASAH1 activity assays revealed the direct binding of E2 to ASAH1. Moreover, siRNA knockdown of ASAH1 demonstrated its role as an angiogenesis factor. Consequently, E2 inhibited chemoinvasion and tube formation of HUVECs in a dose-dependent manner. E2 also potently suppressed neo-vascularization of chorioallantoic membranes in vivo. Collectively, these data suggest that E2 is a novel angiogenesis inhibitor and ASAH1 is proposed to be a new antiangiogenesis target.

[Background]: Currently, only a limited number of molecular biomarkers for malignant melanoma exist. This is the case for both diagnosing the disease, staging, and efficiently measuring the response to therapy by tracing the progression of disease development and drug impact. There is a great need to identify novel landmarks of disease progression and alterations., [Methods]: Matrix-assisted laser desorption ionisation mass spectrometry imaging (MALDI-MSI) has been developed within our group to study drug localisation within micro environmental tissue compartments. Here, we expand further on this technology development and introduce for the frst time melanoma tumour tissues to map metabolite localisation utilising high resolution mass spectrometry. MALDI-MSI can measure and localise the distribution pattern of a number of small molecule metabolites within tissue compartments of tumours isolated from melanoma patients. Data on direct measurements of metabolite identities attained at the local sites in tissue compartments has not been readily available as a measure of a clinical index for most cancer diseases. The current development on the mapping of endogenous molecular expression melanoma tumours by mass spectrometry imaging focuses on the establish‑ment of a cancer tissue preparation process whereby a matrix crystal formation is homogenously built on the tissue surface, providing uniform molecular mapping. We apply this micro-preparation technology to disease presentation by mapping the molecular signatures from patient tumour sections., [Results]: We have automated the process with a micro-technological dispensing platform. This provides the basis for thin film generation of the cancer patient tissues prior to imaging screening. Compartmentalisation of the tumour regions are displayed within the image analysis interfaced with histopathological grading and characterisation., [Conclusions]: This enables site localisation within the tumour with image mapping to disease target areas such as melanoma cells, macrophages, and lymphocytes., This study was supported by a joint grant from the Berta Kamprad Founda‑tion (Melanoma Cancer Moonshot) and by grants from the National Research Foundation of Korea (NRF-2015K1A1A2028365, 2016K2A9A1A03904900), the Brain Korea 21 Plus Project, Republic of Korea, and KNN121510 by the National Research, Development and Innovation Ofce of Hungary.

Ciliopathies are clinically overlapping genetic disorders involving structural and functional abnormalities of cilia. Currently, there are no small-molecule drugs available to treat ciliary defects in ciliopathies. Our phenotype-based screen identified the flavonoid eupatilin and its analogs as lead compounds for developing ciliopathy medication. CEP290, a gene mutated in several ciliopathies, encodes a protein that forms a complex with NPHP5 to support the function of the ciliary transition zone. Eupatilin relieved ciliogenesis and ciliary receptor delivery defects resulting from deletion of CEP290. In rd16 mice harboring a blinding Cep290 in-frame deletion, eupatilin treatment improved both opsin transport to the photoreceptor outer segment and electrophysiological responses of the retina to light stimulation. The rescue effect was due to eupatilin-mediated inhibition of calmodulin binding to NPHP5, which promoted NPHP5 recruitment to the ciliary base. Our results suggest that deficiency of a ciliopathy protein could be mitigated by small-molecule compounds that target other ciliary components that interact with the ciliopathy protein.

Ubiquinol–cytochrome c reductase binding protein (UQCRB), a component of the mitochondrial complex III, has been recently implicated in angiogenesis. Targeting mitochondria to balance vascular homeostasis has been widely recognized. However, the effect of UQCRB replenishment by direct delivery remains unknown. To explore the biological function of UQCRB in angiogenesis, a novel protein transduction domain (PTD)-conjugated UQCRB fusion protein was generated. PTD-UQCRB localized to mitochondria as does endogenous UQCRB. Treatment with PTD-UQCRB generated mitochondrial reactive oxygen species (mROS) without cytotoxicity, following hypoxia inducible factor-1α (HIF-1α) stabilization and downstream vascular endothelial growth factor (VEGF) expression. Accordingly, PTD-UQCRB induced angiogenesis in vitro and PTD-UQCRB pro-angiogenic activity was further validated in matrigel plug assay and in cutaneous wound-healing mouse models in vivo. Together, these results demonstrate that UQCRB plays a role in angiogenesis and the developed cell-permeable PTD-UQCRB can be utilized as a pro-angiogenic agent.

Glioblastoma stem cell targeted therapies have become a powerful strategy for the treatment of this deadliest brain tumor. We demonstrate for the first time that downregulation of mitochondrial ubiquinol-cytochrome c reductase binding protein (UQCRB) inhibits the cancer stem cell-like properties in human glioblastoma cells. The synthetic small molecules targeting UQCRB significantly suppressed not only the self-renewal capacity such as growth and neurosphere formation, but also the metastatic potential such as migration and invasion of glioblastoma stem‑like cells (GSCs) derived from U87MG and U373MG at subtoxic concentrations. Notably, the UQCRB inhibitors repressed c‑Met-mediated downstream signal transduction and hypoxia‑inducible factor‑1α (HIF‑1α) activation, thereby reducing the expression levels of GSC markers including CD133, Nanog, Oct4 and Sox2 in the GSCs. Furthermore, the UQCRB inhibitors decreased mitochondrial ROS generation and mitochondrial membrane potential in the GSCs, indicating that they regulate the mitochondrial function in GSCs. Indeed, the knockdown of UQCRB gene by UQCRB siRNA significantly inhibited the cancer stem cell-like phenotypes as well as the expression of stemness markers by blocking mitochondrial ROS/HIF‑1α/c‑Met pathway in U87MG GSCs. These findings suggest that UQCRB and its inhibitors could be a new therapeutic target and lead compounds for eliminating cancer stem cells in glioblastoma.

Cholesterol is an important modulator of membrane protein function and signaling in endothelial cells, thus making it an emerging target for anti-angiogenic agents. In this study, we employed a phenotypic screen that detects intracellular cholesterol distribution in endothelial cells (HUVEC) and identified 13 existing drugs as cholesterol trafficking inhibitors. Cepharanthine, an approved drug for anti-inflammatory and cancer management use, was amongst the candidates, which was selected for in-depth mechanistic studies to link cholesterol trafficking and angiogenesis. Cepharanthine inhibited the endolysosomal trafficking of free-cholesterol and low-density lipoprotein in HUVEC by binding to Niemann-Pick disease, type C1 (NPC1) protein and increasing the lysosomal pH. The blockade of cholesterol trafficking led to a cholesterol-dependent dissociation of mTOR from the lysosomes and inhibition of its downstream signaling. Cepharanthine inhibited angiogenesis in HUVEC and in zebrafish in a cholesterol-dependent manner. Furthermore, cepharanthine suppressed tumor growth in vivo by inhibiting angiogenesis and it enhanced the antitumor activity of the standard chemotherapy cisplatin in lung and breast cancer xenografts in mice. Altogether, these results strongly support the idea that cholesterol trafficking is a viable drug target for anti-angiogenesis and that the inhibitors identified among existing drugs, such as cepharanthine, could be potential anti-angiogenic and antitumor agents.

Background and Purpose Activation of hepatic stellate cells (HSCs) is a crucial step in the pathogenesis of hepatic fibrosis. Histone deacetylase (HDAC) is an attractive target in liver fibrosis because it plays a key role in gene expression and cell differentiation. We have developed a HDAC inhibitor, N-hydroxy-7-(2-naphthylthio)heptanomide (HNHA), and investigated the anti-fibrotic activity of HNHA in vitro and in vivo. Experimental Approach We investigated the anti-fibrotic effect of HNHA on mouse and human HSC activation in vitro and in the liver of bile duct-ligated (BDL) rats in vivo using cell proliferation assays, cell cycle analysis, biochemical assay, immunohistochemistry and Western blots. Liver pathology was assessed with histochemical techniques. Key Results HNHA inhibited proliferation and arrested the cell cycle via p21 induction in HSCs. In addition, HNHA induced apoptosis of HSCs, which was correlated with reduced COX-2 expression, NF-κB activation and cell death signals. HNHA restored liver function and decreased the accumulation of extracellular matrix in the liver via suppression of HSC activation in BDL rats in vivo. HNHA administration also increased survival in BDL rats. Conclusions and Implications HNHA improved liver function, suppressed liver fibrosis and increased survival of BDL rats, accompanied by reduction of cell growth, activation and survival of HSCs. These findings show that HNHA may be a potent anti-fibrosis agent against hepatic fibrosis because of its multi-targeted inhibition of HSC activity in vivo and in vitro.

For this study, we examined the effects of curcumin against acute and chronic stress, paying specific attention to ROS. We also aimed to clarify the differences between acute and chronic stress conditions. We investigated the effects of curcumin against acute stress (once/1 day CCl4 treatment) and chronic-stress (every other day/4week CCl4 treatment). Compared with acute stress, in which the antioxidant system functioned properly and aspartate transaminase (AST) and ROS production increased, chronic stress increased AST, alanine aminotransferase (ALT), hepatic enzymes, and ROS more significantly, and the antioxidant system became impaired. We also found that ER-originated ROS accumulated in the chronic model, another difference between the two conditions. ER stress was induced consistently, and oxidative intra-ER protein folding status, representatively PDI, was impaired, especially in chronic stress. The PDI-associated client protein hepatic apoB accumulated with the PDI-binding status in chronic stress, and curcumin recovered the altered ER folding status, regulating ER stress and the resultant hepatic dyslipidemia. Throughout this study, curcumin and curcumin-rich Curcuma longa L. extract promoted recovery from CCl4-induced hepatic toxicity in both stress conditions. For both stress-associated hepatic dyslipidemia, curcumin and Curcuma longa L. extract might be recommendable to recover liver activity.

Background Curcumin, a major active component of turmeric, has previously been reported to alleviate liver damage. Here, we investigated the mechanism by which turmeric and curcumin protect the liver against carbon tetrachloride (CCl4)-induced injury in rats. We hypothesized that turmeric extract and curcumin protect the liver from CCl4-induced liver injury by reducing oxidative stress, inhibiting lipid peroxidation, and increasing glutathione peroxidase activation. Methods Chronic hepatic stress was induced by a single intraperitoneal injection of CCl4 (0.1 ml/kg body weight) into rats. Turmeric extracts and curcumin were administered once a day for 4 weeks at three dose levels (100, 200, and 300 mg/kg/day). We performed ALT and AST also measured of total lipid, triglyceride, cholesterol levels, and lipid peroxidation. Result We found that turmeric extract and curcumin significantly protect against liver injury by decreasing the activities of serum aspartate aminotransferase and alanine aminotransferase and by improving the hepatic glutathione content, leading to a reduced level of lipid peroxidase. Conclusions Our data suggest that turmeric extract and curcumin protect the liver from chronic CCl4-induced injury in rats by suppressing hepatic oxidative stress. Therefore, turmeric extract and curcumin are potential therapeutic antioxidant agents for the treatment of hepatic disease. Electronic supplementary material The online version of this article (doi:10.1186/s12906-016-1307-6) contains supplementary material, which is available to authorized users.

Tumor growth and metastasis depend on angiogenesis triggered by chemical signals, such as vascular endothelial growth factor (VEGF), released from tumor cells. Therefore, the specific perturbation of angiogenesis has been considered a powerful strategy for the treatment of cancer. Herein, we report that tricin, 4',5,7-trihydroxy-3',5'-dimethoxyflavone, exhibits potent antiangiogenic activity in vitro. Tricin effectively suppressed the proliferation as well as VEGF-induced invasion and tube formation of human umbilical vein endothelial cells (HUVECs) at subtoxic doses. Furthermore, tricin significantly inhibited the angiogenesis of the chorioallantoic membrane from growing chick embryos without showing cytotoxicity. We also found that tricin blocked tumor cell-induced angiogenesis. Notably, tricin downregulated not only the VEGFR2 signal transduction by reducing reactive oxygen species (ROS) generation in endothelial cells, but also the expression of VEGF by inhibiting hypoxia inducible factor-1α (HIF-1α) accumulation in tumor cells. Moreover, combined treatment with tricin and bevacizumab, an anti-VEGF drug, ameliorated the antiangiogenic effect of bevacizumab. Taken together, our findings demonstrate for the first time that tricin possesses promising antiangiogenic potential and thus may be applied to anticancer therapy by targeting tumor angiogenesis.

Indatraline is an antidepressive agent and a non-selective monoamine transporter inhibitor that blocks the reuptake of neurotransmitters (dopamine, serotonin, and norepinephrine). In this study, we report that indatraline induces autophagy via the suppression of mTOR/S6 kinase signaling. Autophagy induction was examined by a cell-based high content screening system using LysoTracker, which was followed by monodansylcadaverine staining and transmission electron microscope observation. Indatraline increased the number of EGFP-LC3 cells expressing autophagosomes in the cytoplasm. Conversion of LC3 was further validated by immunoblotting. Indatraline induced autophagy by affecting the AMPK/mTOR/S6K signaling axis and had no influence on the PI3K/AKT/ERK signaling. Moreover, indatraline induced autophagy in smooth muscle cells (SMCs); further, it exhibited therapeutic potential for restenosis by inhibiting SMC accumulation in a rat restenosis model. These results provide new insights into the role of monoamine transporters in autophagy regulation and identify indatraline as a novel agent for inducing autophagy.

Angiogenesis, the formation of new blood vessels from pre-existing ones, plays a critical role in normal and pathological phenotypes, including solid tumor growth and metastasis. Accordingly, the development of new anti-angiogenic agents is considered an efficient strategy for the treatment of cancer and other human diseases linked with angiogenesis. We have identified voacangine, isolated from Voacanga africana, as a novel anti-angiogenic agent. Voacangine inhibits the proliferation of HUVECs at an IC(50) of 18 μM with no cytotoxic effects. Voacangine significantly suppressed in vitro angiogenesis, such as VEGF-induced tube formation and chemoinvasion. Moreover, the compound inhibits in vivo angiogenesis in the chorioallantoic membrane at non-toxic doses. In addition, voacangine decreased the expression levels of hypoxia inducible factor-1α and its target gene, VEGF, in a dose-dependent manner. Taken together, these results suggest that the naturally occurring compound, voacangine, is a novel anti-angiogenic compound.

Sphingosine-1-phosphate (S1P) plays an important role in angiogenesis by stimulating DNA synthesis, chemotactic motility, and early blood vessel formation. Accordingly, the S1P signaling pathway is an attractive target for novel anti-angiogenic therapeutics. Here, we describe a small synthetic derivative of S1P that acts as an anti-angiogenic agent. We found that the S1P derivative NHOBTD [N-((2S,3R)-3-hydroxy-1-morpholino-4-(3-octylphenyl)butan-2-yl)tetradecanamide] suppressed S1P-induced invasion and tube formation by human umbilical vein endothelial cells. NHOBTD also suppressed S1P signaling, as seen by destabilization of hypoxia inducible factor-1 alpha (HIF-1α) and secretion of VEGF, a transcriptional target of HIF-1α. Moreover, NHOBTD profoundly blocked endogenous neovascularization of the chick embryo chorioallantoic membrane, without rupturing any existing vessels. Together, these results demonstrate that NHOBTD is a new anti-angiogenic molecule that is capable of perturbing S1P signaling, and provides the basis for developing new anti-angiogenic drugs.

We evaluated the cytotoxic effects of combining suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor, with taxanes in human gastric cancer cell lines and assessed the pre-treatment difference of gene expression to identify genes that could potentially mediate the cytotoxic response. Gastric cancer cell lines were treated with SAHA and paclitaxel or docetaxel, and the synergistic interaction between the drugs was evaluated in vitro using the combination index (CI) method. We performed significance analysis of microarray (SAM) to identify chemosensitivity-related genes in gastric cancer cell lines that were concomitantly treated with SAHA and taxane. We generated a correlation matrix between gene expression and CI values to identify genes whose expression correlated with a combined effect of taxanes and SAHA. Combination treatment with taxane and SAHA had a synergistic cytotoxic effect against taxane-resistant gastric cancer cells. We identified 49 chemosensitivity-related genes via SAM analysis. Among them, nine common genes (SLIT2, REEP2, EFEMP2, CDC42SE1, FSD1, POU1F1, ZNF79, ETNK1, and DOCK5) were extracted from the subsequent correlation matrix analysis. The combination of taxane and SAHA could be efficacious for the treatment of gastric cancer. The genes that were related to the synergistic response to taxane and SAHA could serve as surrogate biomarkers to predict the therapeutic response in gastric cancer patients.

Autophagy is an apoptosis-independent mechanism of cell death that protects the cell from environmental imbalances and infection by pathogens. We identified a novel small molecule, 2-(3-Benzyl-4-oxo-3,4,5,6,7,8-hexahydro-benzo[4,5]thieno[2,3-d]pyrimidin-2-ylsulfanylmethyl)-oxazole-4-carboxylic acid (2-pyrrolidin-1-yl-ethyl)-amide (referred as autophagonizer), using high-content cell-based screening and the autophagosome marker EGFP-LC3. Autophagonizer inhibited growth and induced cell death in the human tumor cell lines MCF7, HeLa, HCT116, A549, AGS, and HT1080 via a caspase-independent pathway. Conversion of cytosolic LC3-I to autophagosome-associated LC3-II was greatly enhanced by autophagonizer treatment. Transmission electron microscopy and acridine orange staining revealed increased autophagy in the cytoplasm of autophagonizer-treated cells. In conclusion, autophagonizer is a novel autophagy inducer with unique structure, which induces autophagic cell death in the human tumor cell lines.

Matrix metalloproteinases (MMPs) are key regulators of the skin photoaging process that is set in motion by exposure to ultraviolet (UV) irradiation. This skin damage results from UV-induced generation of reactive oxygen species, which are associated with upregulation of MMPs and decreased collagen synthesis. We investigated the effects of xanthorrhizol, isolated from Curcuma xanthorrhiza, on the expression of MMP-1 and type-I procollagen in UV-irradiated human skin fibroblasts. Fibroblasts cultured in the presence or absence of purified xanthorrhizol or C. xanthorrhiza extract were irradiated with UV (20 mJ/cm(2)), and MMP-1 and type-I procollagen levels were measured using Western blot analysis. Xanthorrhizol (0.001-0.1 microM) and C. xanthorrhiza extract (0.01-0.5 microg/mL) induced a significant, dose-dependent decrease in the expression of MMP-1 protein, and increased the expression of type-1 procollagen. At a concentration of 0.1 microM, xanthorrhizol nearly completely abrogated MMP-1 expression. The MMP-1-suppressing and type-1 procollagen-inducing effects of xanthorrhizol treatment were greater than those of epigallocatechin 3-O-gallate (EGCG), which is known to be a natural anti-aging agent. These results suggest that xanthorrhizol is a potential candidate for the prevention and treatment of skin aging.

Background and Aim: Metastasis is a multistep event in which neoplastic cells detach from the tumor, migrate, disseminate, extravasate, and eventually proliferate at the secondary distant sites. Hepatocellular carcinoma (HCC) is characterized by hypervascularity and frequent metastasis. Recently, metastasis-associated proteins were identified and named metastatic tumor antigens (MTA) 1, 2, and 3. They have been found to be contained in the nucleosome remodeling and histone deacetylase complex. MTA2 has been reported to interact with p53 and inhibit p53-mediated cell growth arrest and apoptosis by deacetylation. Although it has been reported that the expression of MTA1 is related to tumor progression and metastasis, it is still unclear how MTA2 is involved in HCC. In this study, we found that the overexpression of MTA2 is associated with HCC size and differentiation after hepatectomy. Methods: The expression of MTA2 was examined in 506 human HCC samples that underwent hepatic resection using tissue microarray. The expression of MTA2 was classified into 0, 1, 2, and 3, based on immunoreactivity. Results: The expression of MTA2 was predominantly localized to the nucleus. MTA2 was detected in 487 (96.2%) of the 506 human HCC samples. Notably, the MTA2 expression level strongly increased depending on the size and differentiation of HCC. Conclusions: These findings indicate a tight correlation between the MTA2 expression level and HCC size and differentiation. Therefore, MTA2 might be a predictor of aggressive phenotypes and a possible target molecule for anticancer drug design in human HCC.

Histone deacetylase (HDAC) is a key enzyme regulating gene expression, including angiogenic cytokine expression. We have previously identified a novel synthetic HDAC inhibitor, known as N-hydroxy-7-(2-naphthylthio) heptanomide (HNHA), with antitumor properties. Here, we investigated the antiangiogenic properties of this small synthetic molecule both in vitro and in vivo. HNHA inhibited nuclear HDAC enzyme activity in human umbilical endothelial cells (HUVECs), an effect accompanied by histone hyperacetylation, p21 upregulation, and cell cycle arrest. HNHA also inhibited vascular endothelial growth factor-induced tube formation and migration of HUVECs, in the absence of any detectable cellular toxicity. Intravitreous injection of HNHA into mice inhibited retinal neovascularization associated with oxygen-induced retinopathy (OIR) and laser-induced choroidal neovascularization (CNV), as determined through fluorescence angiography and vessel counting. Retinas from HNHA-treated animals had a normal histological appearance without any detectable increase in terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive cells, showing that this compound did not induce retinal toxicity. These findings indicate that HNHA has direct antiangiogenic effects and may be an effective strategy for inhibiting the pathological retinal and choroidal neovascularization underlying blinding eye diseases.

Mycophenolic acid (MPA, 1), an inhibitor of IMP-dehydrogenase (IMPDH) and a latent PPARgamma agonist, is used as an effective immunosuppressant for clinical transplantation and recently entered clinical trials in advanced multiple myeloma patients. On the other hand, suberoylanilide hydroxamic acid (SAHA), a non-specific histone deacetylase (HDAC) inhibitor, has been approved for treating cutaneous T-cell lymphoma. MPA seemed to bear a cap, a linker, and a weak metal-binding site as a latent inhibitor of HDAC. Therefore, the hydroxamic acid derivatives of mycophenolic acid having an effective metal-binding site, mycophenolic hydroxamic acid (MPHA, 2), 7-O-acetyl mycophenolic acid (7-O-Ac MPHA, 3), and 7-O-lauroyl mycophenolic hydroxamic acid (7-O-L MPHA, 4) were designed and synthesized. All these compounds inhibited histone deacetylase with IC50 values of 1, 0.9 and 0.5 microM, and cell proliferation at concentrations of 2, 1.5 and 1 microM, respectively.

We recently developed a specific small molecule inhibitor of aminopeptidase N (APN), named as HNSA, through a high throughput screening. In the present study, we investigated the major cellular phenotypes of HNSA in comparison with those of APN knock-down in human fibrosarcoma cells and the mechanism of angiogenesis inhibition by the compound using DNA microarray analyses. Global gene expression analyses showed that HNSA signatures are significantly correlated with those of APN knock-down in HT1080 cells, suggesting that APN is a primary target of HNSA in the cells. Using the angiogenesis-focused DNA microarrays, nine of angiogenesis-related genes were identified as crucial mediators of angiogenesis inhibition by HNSA. These data demonstrate that HNSA can be used as a valuable tool to decipher the APN function in angiogenesis.

Autophagy is a process where cytoplasmic materials are degraded by lysosomal machinery. Histone deacetylase (HDAC) inhibitors induce autophagy, and HDAC6, one of class II HDAC isotypes, is directly involved in autophagic degradation in the cell. However, it is unclear if class I HDAC isotype such as HDCA1 is involved in this process. To investigate if class I HDAC isotype is involved in autophagy, a specific class I HDAC inhibitor and an siRNA of HDAC1 were used to treat HeLa cells. Autophagic markers were then investigated. Both inhibition and genetic knock-down of HDAC1 in the cells significantly induced autophagic vacuole formation and lysosome function. Moreover, disruption of HDAC1 leads to the conversion of LC3-I to LC3-II. Together, these results demonstrate that HDAC1 could play a role in autophagy and specific inhibition of HDAC1 can induce autophagy.