Adiponectin as a growth inhibitor in prostate cancer cells

Prostate cancer is associated with obesity. However, the molecular basis of this association is not well known. Adiponectin is a major adipose cytokine that decreases in circulation in obesity and ameliorates obesity. Here, we identify adiponectin as a novel inhibitor in prostate cancer cell growth. Adiponectin occurs in non-proteolytic (full-length adiponectin: f-adiponectin) and proteolytic (globular adiponectin) forms in various oligomeric states (trimer, hexamer, and high molecular weight complex). The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay demonstrates that f-adiponectin inhibits prostate cancer cell growth drastically at subphysiological concentrations. Furthermore, velocity sedimentation analysis shows that the high molecular weight complex of f-adiponectin is the inhibitory form. Moreover, f-adiponectin suppresses leptin- and/or insulin-like growth factor-I (IGF-I)-stimulated, androgen-independent DU145 cell growth, and dihydrotestosterone-stimulated, androgen-dependent LNCaP-FGC cell growth. In addition, f-adiponectin enhances doxorubicin inhibition of prostate cancer cell growth. Therefore, f-adiponectin is a molecular mediator between prostate cancer and obesity, and may be therapeutic to prostate cancer.     

'Click' synthesis of dextran macrostructures for combinatorial-designed self-assembled nanoparticles encapsulating diverse anticancer therapeutics

With the non-specific toxicity of anticancer drugs to healthy tissues upon systemic administration, formulations capable of enhanced selectivity in delivery to the tumor mass and cells are highly desirable. Based on the diversity of the drug payloads, we have investigated a combinatorial-designed strategy where the nano-sized formulations are tailored based on the physicochemical properties of the drug and the delivery needs. Individually functionalized C(2) to C(12) lipid-, thiol-, and poly(ethylene glycol) (PEG)-modified dextran derivatives were synthesized via 'click' chemistry from O-pentynyl dextran and relevant azides. These functionalized dextrans in combination with anticancer drugs form nanoparticles by self-assembling in aqueous medium having PEG surface functionalization and intermolecular disulfide bonds. Using anticancer drugs with logP values ranging from -0.5 to 3.0, the optimized nanoparticles formulations were evaluated for preliminary cellular delivery and cytotoxic effects in SKOV3 human ovarian adenocarcinoma cells. The results show that with the appropriate selection of lipid-modified dextran, one can effectively tailor the self-assembled nano-formulation for intended therapeutic payload.     

Azidocytidine is a specific inhibitor of deoxyribonucleotide synthesis in 3T6 cells

2'-Azidocytidine is a specific inhibitor of DNA synthesis in cultured 3T6 mouse fibroblasts. Earlier work (Akerblom, L., Pontis, E., and Reichard, P. (1982) 257, 6776-6782) indicated that the nucleoside, after phosphorylation, acted by inhibiting both ribonucleotide reduction and DNA strand elongation. We now demonstrate that the effect on strand elongation was due to a contamination of azidocytidine with less than 0.3% of arabinosyl cytosine. Pure azidocytidine inhibits specifically ribonucleotide reductase and its effects on DNA synthesis are secondary to this inhibition. The results with azidocytidine concerning the size and turnover of deoxyribonucleoside triphosphate pools parallel those of hydroxyurea and are distinct from those of arabinosyl cytosine. Together with hydroxyurea, azidocytidine is a useful compound in studies aiming at a specific block of the production of deoxynucleoside triphosphates in intact cells. Comparisons of the effects of azidocytidine and arabinosyl cytosine complement earlier studies with hydroxyurea and aphidicolin concerning inter-relations between dNTP synthesis and DNA replication.     

Mitochondrial serine acetyltransferase functions as a pacemaker of cysteine synthesis in plant cells

Cysteine (Cys) synthesis in plants is carried out by two sequential reactions catalyzed by the rate-limiting enzyme serine acetyltransferase (SAT) and excess amounts of O-acetylserine(thiol)lyase. Why these reactions occur in plastids, mitochondria, and cytosol of plants remained unclear. Expression of artificial microRNA (amiRNA) against Sat3 encoding mitochondrial SAT3 in transgenic Arabidopsis (Arabidopsis thaliana) plants demonstrates that mitochondria are the most important compartment for the synthesis of O-acetylserine (OAS), the precursor of Cys. Reduction of RNA levels, protein contents, SAT enzymatic activity, and phenotype strongly correlate in independent amiSAT3 lines and cause significantly retarded growth. The expression of the other four Sat genes in the Arabidopsis genome are not affected by amiRNA-SAT3 according to quantitative real-time polymerase chain reaction and microarray analyses. Application of radiolabeled serine to leaf pieces revealed severely reduced incorporation rates into Cys and even more so into glutathione. Accordingly, steady-state levels of OAS are 4-fold reduced. Decrease of sulfate reduction-related genes is accompanied by an accumulation of sulfate in amiSAT3 lines. These results unequivocally show that mitochondria provide the bulk of OAS in the plant cell and are the likely site of flux regulation. Together with recent data, the cytosol appears to be a major site of Cys synthesis, while plastids contribute reduced sulfur as sulfide. Thus, Cys synthesis in plants is significantly different from that in nonphotosynthetic eukaryotes at the cellular level.     

Abl interconnects oncogenic Met and p53 core pathways in cancer cells

The simplicity of BCR-ABL 'oncogene addiction' characterizing leukemia contrasts with the complexity of solid tumors where multiple 'core pathways', including receptor tyrosine kinases (RTKs) and p53, are often altered. This discrepancy illustrates the limited success of RTK antagonists in solid tumor treatment compared with the impact of Imatinib in BCR-ABL-dependent leukemia. Here, we identified c-Abl as a signaling node interconnecting Met-RTK and p53 core pathways, and showed that its inhibition impairs Met-dependent tumorigenesis. Met ensures cell survival through a new path in which c-Abl and p38-MAPK are employed to elicit p53 phosphorylation on Ser(392) and Mdm2 upregulation. We found a clinical correlation between activated Met, phospho-p53, and Mdm2 levels in human tumors, supporting the role of this path in tumorigenesis. Our findings introduce the concept that RTK-driven tumors may be therapeutically treated by hitting signaling nodes interconnecting core pathways. Moreover, they underline the importance of evaluating the relevance of c-Abl antagonists for combined therapies, based on the tumor signaling signature.     

Non-mitotic functions of polo-like kinases in cancer cells

Inhibitors of mitotic protein kinases are currently being developed as non-neurotoxic alternatives of microtubule-targeting agents (taxanes, vinca alkaloids) which provide a substantial survival benefit for patients afflicted with different types of solid tumors. Among the mitotic kinases, the cyclin-dependent kinases, the Aurora kinases, the kinesin spindle protein and Polo-like kinases (PLKs) have emerged as attractive targets of cancer therapeutics.The functions of mammalian PLK1-5 are traditionally linked to the regulation of the cell cycle and to the stress response. Especially the key role of PLK1 and PLK4 in cellular growth and proliferation, their overexpression in multiple types of human cancer and their druggability, make them appealing targets for cancer therapy. Inhibitors for PLK1 and PLK4 are currently being tested in multiple cancer trials. The clinical success of microtubule-targeting agents is attributed not solely to the induction of a mitotic arrest in cancer cells, but also to non-mitotic effects like targeting intracellular trafficking on microtubules. This raises the question whether new cancer targets like PLK1 and PLK4 regulate critical non-mitotic functions in tumor cells. In this article we summarize the important roles of PLK1-5 for the regulation of non-mitotic signaling. Due to these functions it is conceivable that inhibitors for PLK1 or PLK4 can target interphase cells, which underscores their attractive potential as cancer drug targets. Moreover, we also describe the contribution of the tumor-suppressors PLK2, PLK3 and PLK5 to cancer cell signaling outside of mitosis. These observations highlight the urgent need to develop highly specific ATP-competitive inhibitors for PLK4 and for PLK1 like the 3^rd generation PLK-inhibitor Onvansertib to prevent the inhibition of tumor-suppressor PLKs in- and outside of mitosis. The remarkable feature of PLKs to encompass a unique druggable domain, the polo-box-domain (PBD) that can be found only in PLKs offers the opportunity for the development of inhibitors that target PLKs exclusively. Beyond the development of mono-specific ATP-competitive PLK inhibitors, the PBD as drug target will support the design of new drugs that eradicate cancer cells based on the mitotic and non-mitotic function of PLK1 and PLK4.     

Discovery of a novel proteasome inhibitor selective for cancer cells over non-transformed cells

Numerous proteins controlling cell cycle progression, apoptosis, and angiogenesis are degraded by the ubiquitin/proteasome system, which has become the subject for intense investigations for cancer therapeutics. Therefore, we used in silico and experimental approaches to screen compounds from the NCI chemical libraries for inhibitors against the chymotrypsin-like (CT-L) activity of the proteasome and discovered PI-083. Molecular docking indicates that PI-083 interacts with the Thr21, Gly47 and Ala49 residues of the β5 subunit and Asp114 of the β6 subunit of the proteasome. PI-083 inhibits CT-L activity and cell proliferation and induces apoptosis selectively in cancer cells (ovarian T80-Hras, pancreatic C7-Kras and breast MCF-7) as compared to their normal/immortalized counterparts (T80, C7 and MCF-10A, respectively). In contrast, Bortezomib, the only proteasome inhibitor approved by the Food and Drug Administration (FDA), did not exhibit this selectivity for cancer over non-transformed cells. In addition, in all cancer cells tested, including Multiple Myeloma (MM), breast, pancreatic, ovarian, lung, prostate cancer cell lines as well as fresh MM cells from patients, PI-083 required less time than Bortezomib to induce its antitumor effects. Furthermore, in nude mouse xenografts in vivo, PI-083, but not Bortezomib, suppressed the growth of human breast and lung tumors. Finally, following in vivo treatment of mice, PI-083 inhibited tumor, but not hepatic liver CT-L activity, whereas Bortezomib inhibited both tumor and liver CT-L activities. These results suggest that PI-083 is more selective for cancer cells and may have broader antitumor activity and therefore warrants further advanced preclinical studies.     

Aflatoxin B1, a selective inhibitor of DNA synthesis in mammalian cells.

Aflatoxin B1 in concentrations between 0.01 and 0.1 microgram/ml inhibits DNA synthesis in African green monkey cells in culture, but has little effect on RNA synthesis and no effect on protein synthesis. The drug even at concentrations up to 1.0 microgram/ml does not interfere with DNA repair promoted by ultraviolet irradiation nor does it induce DNA repair. The inhibition of DNA synthesis attains maximum values 3 h after addition of aflatoxin B1 and is irreversible upon removal of the drug. Profiles of pulselabeled DNA sedimented in alkaline sucrose gradients indicate that aflatoxin B1 blocks initiation of replication rather than elongation.     

Identification of a selective small molecule inhibitor of breast cancer stem cells

A high-throughput screen (HTS) with the National Institute of Health-Molecular Libraries Small Molecule Repository (NIH-MLSMR) compound collection identified a class of acyl hydrazones to be selectively lethal to breast cancer stem cell (CSC) enriched populations. Medicinal chemistry efforts were undertaken to optimize potency and selectivity of this class of compounds. The optimized compound was declared as a probe (ML239) with the NIH Molecular Libraries Program and displayed greater than 20-fold selective inhibition of the breast CSC-like cell line (HMLE_sh_Ecad) over the isogenic control line (HMLE_sh_GFP).     

Met degradation by SAIT301, a Met monoclonal antibody,reduces the invasion and migration of nasopharyngeal cancer cells via inhibition of EGR-1 expression

Nasopharyngeal carcinoma (NPC) is a common malignant tumor with high invasive and metastatic potential. The hepatocyte growth factor (HGF)-Met signaling pathway has a critical role in mediating the invasive growth of many different types of cancer, including head and neck squamous cell carcinoma. HGF also stimulates NPC cell growth and invasion in the cell line model. In this study, we determined the inhibitory effect of Met, using a Met-targeting monoclonal antibody (SAIT301), on the invasive and growth potential of NPC cell lines. Met inhibition by SAIT301 resulted in highly significant inhibition of cell migration and invasion in both the HONE1 and HNE1 cell lines. In addition, we also found that co-treatment of SAIT301 and HGF decreased the anchorage-independent growth induced by HGF in HNE1 cell lines. After SAIT301 treatment, Met, together with its downstream signaling proteins, showed downregulation of p-Met and p-ERK, but not p-AKT, in both HONE1 and HNE1 cell lines. Interestingly, we found that HGF treatment of NPC cell lines induced early growth response protein (EGR-1) expression, which is involved in cell migration and invasion. In addition, co-treatment with SAIT301 and HGF inhibited the HGF-induced expression of EGR-1. Next, knockdown of EGR-1 using small-interfering RNA inhibited HGF-induced cell invasion in NPC cell lines, suggesting that the expression level of EGR-1 is important in HGF-induced cell invasion of NPC cells. Therefore, the results support that SAIT301 inhibited Met activation as well as the downstream EGR-1 expression and could have therapeutic potential in NPC. Taken together, we suggest that Met is an anticancer therapeutic target for NPC that warrants further investigation and clinical trials and SAIT301 may be a promising tool for NPC therapy.     

A dialyzable protein synthesis inhibitor released by mammalian cells in vitro

Macrophages, lymphocytes, and HeLa cells when incubated $\text{in vitro}$ at 37° in Krebs-Ringer phosphate buffer release a dialyzable, heat-stable, ninhydrin-reacting factor which inhibits protein synthesis by intact cells and isolated ribosomes. Release of the inhibitor appears to be dependent on metabolism. Partial purification of the inhibitor by Sephadex G-10 chromatography suggests it has a molecular weight of 400–600.     

An inhibitor of protein synthesis in cytoplasmic extracts of density inhibited cells

Cytoplasmic extracts of green monkey kidney-Vero M3 cells that have been grown to high cell density and have entered the stationary phase of growth lose the capacity to synthesize proteins after they have been frozen and thawed. The same loss of protein synthetic capacity is not observed when cytoplasmic extracts of low cell density Vero M3 or HeLa S-3 cells are frozen and thawed before assay, nor when high cell density Vero M3 cell extract is assayed without having been frozen. The loss of the protein synthesis capacity of the frozen and thawed extracts of stationary phase Vero M3 cells is accounted for by the appearance of an inhibitor. The inhibitor is precipitated in the 30 to 70% ammonium sulfate fraction of the 100,000 g supernatant. It is inactivated by heat and is non-dialyzable. It blocks the transfer of amino acids from tRNA to growing peptide chains. The amount of this inhibitor in the extract (measured by relative inhibition of in vitro protein synthesis) increases as a function of the density of the cells from which the extract was made, and the increase can be correlated with the progressive turnoff of protein synthesis in whole cells.     

Selective cytotoxicity of a bicyclic Ras inhibitor in cancer cells expressing K-Ras

Mutation of RAS genes is a critical event in the pathogenesis of different human tumors and in some developmental disorders. Here we present an arabinose-derived bicyclic compound displaying selective cytotoxicity in human colorectal cancer cells expressing K-Ras^G13D, that shows high intrinsic nucleotide exchange rate. We characterize binding of bicyclic compounds by docking and NMR experiments and their inhibitory activity on GEF-mediated nucleotide exchange on wild-type and mutant Ras proteins. We demonstrate that the in vitro inhibition of Ras nucleotide exchange depends on the molar ratio between Ras and its GEF activator, suggesting that the observed in vivo selective effect may depend on biochemical parameters and actual intracellular concentration of the Ras protein and its regulators.     

Characteristics of apoptosis induction in human breast cancer cells treated with a ceramidase inhibitor

Cancer is a complex disease with high mortality rates. Breast cancer is one of the most fatal diseases both for men and woman. Despite the positive developments on cancer treatment, a successful treatment agent/method has not been developed, yet. Recently, cancer research has been involved in sphingolipid metabolism. The key molecule here is ceramide. Ceramides mediate growth suppress, apoptosis and aging regulation. Ceramidases metabolize ceramide and decrease its level in cells and cause escape the death. Inhibition of ceramidases as new targets for cancer treatment is shown in the literature. Herein, we found that d-erythro-MAPP and its nanoparticle formulation, reduce the viability of MCF-7 cells in a dose-dependent manner with IC₅₀ value of 4.4 µM, and 15.6 µM, respectively. Confocal and transmission electron microscopy results revealed apoptotic morphological and ultrastructural changes for both agents. Apoptosis and cell cycle arrest were supported by annexin-V, mitochondrial membrane potential changings and cell cycle analysis, respectively.     

Characterization of a novel cellulose synthesis inhibitor

The physiological effects of an experimental herbicide and cellulose synthesis inhibitor, N2-(1-ethyl-3-phenylpropyl)-6-(1-fluoro-1-methylethyl)-1,3,5-triazine-2,4-diamine, called AE F150944, are described. In the aminotriazine molecular class, AE F150944 is structurally distinct from other known cellulose synthesis inhibitors. It specifically inhibits crystalline cellulose synthesis in plants without affecting other processes that were tested. The effects of AE F150944 on dicotyledonous plants were tested on cultured mesophyll cells of Zinnia elegans L. cv. Envy, which can be selectively induced to expand via primary wall synthesis or to differentiate into tracheary elements via secondary wall synthesis. The IC₅₀ values during primary and secondary wall synthesis in Z. elegans were 3.91×10^–8 M and 3.67×10^–9 M, respectively. The IC₅₀ in suspension cultures of the monocot Sorghum halapense (L.) Pers., which were dividing and synthesizing primary walls, was 1.67×10^–10 M. At maximally inhibitory concentrations, 18–33% residual crystalline cellulose synthesis activity remained, with the most residual activity observed during primary wall synthesis in Z. elegans. Addition to Z. elegans cells of two other cellulose synthesis inhibitors, 1 M 2,6-dichlorobenzonitrile and isoxaben, along with AE F150944 did not eliminate the residual cellulose synthesis, indicating little synergy between the three inhibitors. In differentiating tracheary elements, AE F150944 inhibited the deposition of detectable cellulose into patterned secondary wall thickenings, which was correlated with delocalization of lignin as described previously for 2, 6-dichlorobenzonitrile. Freeze-fracture electron microscopy showed that the plasma membrane below the patterned thickenings of AE F150944-treated tracheary elements was depleted of cellulose-synthase-containing rosettes, which appeared to be inserted intact into the plasma membrane followed by their rapid disaggregation. AE F150944 also inhibited cellulose-dependent growth in the rosette-containing alga, Spirogyra pratensis, but it did not inhibit cellulose synthesis in Acetobacter xylinum or Dictyostelium discoideum, both of which synthesize cellulose via linear terminal complexes. Therefore, AE F150944 may inhibit crystalline cellulose synthesis by destabilizing plasma membrane rosettes.Abbreviations AE F150944 N2-(1-ethyl-3-phenylpropyl)-6-(1-fluoro-1-methylethyl)-1,3,5-triazine-2,4-diamine - CBI cellulose biosynthesis inhibiting - CGA CGA 325615, 1-cyclohexyl-5-(2,3,4,5,6-pentafluorophenoxy)-14,2,4,6-thiatriazin-3-amine - DCB 2,6-dichlorobenzonitrile - TE tracheary element