The neural ganglioside GD2 has recently been reported to be a novel surface marker that is only expressed on human bone marrow mesenchymal stem cells within normal marrow. In this study, an MRI-visible, targeted, non-viral vector for effective gene delivery to human bone marrow mesenchymal stem cells was first synthesized by attaching a targeting ligand, the GD2 single chain antibody (scAbGD2), to the distal ends of PEG-g-PEI-SPION. The targeted vector was then used to condense plasmid DNA to form nanoparticles showing stable small size, low cytotoxicity, and good biocompatibility. Based on a reporter gene assay, the transfection efficiency of targeting complex reached the highest value at 59.6% ± 4.5% in human bone marrow mesenchymal stem cells, which was higher than those obtained using nontargeting complex and lipofectamine/pDNA (17.7% ± 2.9% and 34.9% ± 3.6%, respectively) (P<0.01). Consequently, compared with the nontargeting group, more in vivo gene expression was observed in the fibrotic rat livers of the targeting group. Furthermore, the targeting capacity of scAbGD2-PEG-g-PEI-SPION was successfully verified in vitro by confocal laser scanning microscopy, Prussian blue staining, and magnetic resonance imaging. Our results indicate that scAbGD2-PEG-g-PEI-SPION is a promising MRI-visible non-viral vector for targeted gene delivery to human bone marrow mesenchymal stem cells.
Chronic lymphocytic leukemia (CLL) is an adult lymphoid malignancy with a variable clinical course. There is considerable interest in the identification of new treatments, as most current approaches are not curative. While most patients respond to initial chemotherapy, relapsed disease is often resistant to the drugs commonly used in CLL and patients are left with limited therapeutic options. In this study, we used a luminescent cell viability assay based on ATP levels to find compounds that were potent and efficacious in killing CLL cells. We employed an in-house process of quantitative high throughput screening (qHTS) to assess 8 concentrations of each member of a 2,816 compound library (including FDA-approved drugs and those known to be bio-active from commercial suppliers). Using qHTS we generated potency values on each compound in lymphocytes donated from each of six individuals with CLL and five unaffected individuals. We found 102 compounds efficacious against cells from all six individuals with CLL (“consensus” drugs) with five of these showing low or no activity on lymphocytes from a majority of normal donors, suggesting some degree of specificity for the leukemic cells. To our knowledge, this is the first study to screen a drug library against primary CLL cells to identify candidate agents for anti-cancer therapy. The results presented here offer possibilities for the development of novel drug candidates for therapeutic uses to treat CLL and other diseases.
The ubiquitin–proteasome system (UPS) degrades 80 – 90% of intracellular proteins. Cancer cells take advantage of the UPS for their increased growth and decreased apoptotic cell death. Thus, the components that make up the UPS represent a diverse group of potential anti-cancer targets. The success of the first-in-class proteasome inhibitor bortezomib not only proved that the proteasome is a feasible and valuable anti-cancer target, but also inspired researchers to extensively explore other potential targets of this pathway.
This review provides a broad overview of the UPS and its role in supporting cancer development and progression, especially in aspects of p53 inactivation, p27 turnover and NF-κB activation. Also, efforts toward the development of small molecule inhibitors (SMIs) targeting different steps in this pathway for cancer treatment are reviewed and discussed.
Whereas some of the targets in the UPS, such as the 20S pro-teasome, Nedd8 activating enzyme and HDM2, have been well-established and validated, there remains a large pool of candidates waiting to be investigated. Development of SMIs targeting the UPS has been largely facilitated by state-of-the-art technologies such as high-throughput screening and computer-assisted drug design, both of which require a better understanding of the targets of interest.
cancer therapy; deubiquitinases; E3 ligases; proteasome; small molecule inhibitors; ubiquitin
Firefly luciferase (FLuc) is frequently used as a reporter in high-throughput screening assays owing to the exceptional sensitivity, dynamic range, and rapid measurement that bioluminescence affords. However, interaction of small molecules with FLuc has, to some extent, confounded its use in chemical biology and drug discovery. To identify and characterize chemotypes interacting with FLuc, we determined potency values for 360,864 compounds, found in the NIH Molecular Libraries Small Molecule Repository, available in PubChem. FLuc inhibitory activity was observed for 12% of this library with discernible SAR. Characterization of 151 inhibitors demonstrated a variety of inhibition modes including FLuc-catalyzed formation of multisubstrate-adduct enzyme inhibitor complexes. As in some cell-based FLuc reporter assays compounds acting as FLuc inhibitors yield paradoxical luminescence increases, data on compounds acquired from FLuc-dependent assays requires careful analysis as described in this report.
profiling; PubChem; luciferase; quantitative high-throughput screening; qHTS; firefly luciferase; reporter-gene assays; adenylate forming enzymes
Prostate cancer; metal; arsenic; cadmium; copper; lead; zinc; proteasome activity; erythrocyte
Epidemiological studies suggest that trichloroethylene (TCE) exposure may be associated with renal cancer. The biological mechanisms involved are not exactly known although nephrotoxicity is believed to play a role. Studies on TCE nephrotoxicity among humans, however, have been largely inconsistent. We studied kidney toxicity in Chinese factory workers exposed to TCE using novel sensitive nephrotoxicity markers. Eighty healthy workers exposed to TCE and 45 comparable unexposed controls were included in the present analyses. Personal TCE exposure measurements were taken over a 2-week period before urine collection. Ninety-six percent of workers were exposed to TCE below the current US Occupational Safety and Health Administration permissible exposure limit (100 ppm 8h TWA), with a mean (SD) of 22.2 (35.9) ppm. Kidney injury molecule-1 (KIM-1) and Pi-glutathione S transferase (GST) alpha were elevated among the exposed subjects as compared with the unexposed controls with a strong exposure-response association between individual estimates of TCE exposure and KIM-1 (P < 0.0001). This is the first report to use a set of sensitive nephrotoxicity markers to study the possible effects of TCE on the kidneys. The findings suggest that at relatively low occupational exposure levels a toxic effect on the kidneys can be observed. This finding supports the biological plausibility of linking TCE exposure and renal cancer.
Abbreviations:GSTglutathione-S-transferaseKIM-1kidney injury molecule-1NAGN-acetyl-beta-(d)-glucosaminidaseOVMorganic vapour monitoringTCEtrichloroethyleneVEGFvascular endothelial growth factor.
Cancer cells engage in a metabolic program to enhance biosynthesis and support cell proliferation. The regulatory properties of pyruvate kinase M2 (PKM2) influence altered glucose metabolism in cancer. PKM2 interaction with phosphotyrosine-containing proteins inhibits enzyme activity and increases availability of glycolytic metabolites to support cell proliferation. This suggests that high pyruvate kinase activity may suppress tumor growth. We show that expression of PKM1, the pyruvate kinase isoform with high constitutive activity, or exposure to published small molecule PKM2 activators inhibit growth of xenograft tumors. Structural studies reveal that small molecule activators bind PKM2 at the subunit interaction interface, a site distinct from that of the endogenous activator fructose-1,6-bisphosphate (FBP). However, unlike FBP, binding of activators to PKM2 promotes a constitutively active enzyme state that is resistant to inhibition by tyrosine-phosphorylated proteins. These data support the notion that small molecule activation of PKM2 can interfere with anabolic metabolism.
RNA interference (RNAi) has significant therapeutic promise for the genetic treatment of hepatocellular carcinoma (HCC). Targeted vectors are able to deliver small interfering RNA (siRNA) into HCC cells with high transfection efficiency and stability. The tripeptide arginine glycine aspartic acid (RGD)-modified non-viral vector, polyethylene glycol-grafted polyethylenimine functionalized with superparamagnetic iron oxide nanoparticles (RGD-PEG-g-PEI-SPION), was constructed as a magnetic resonance imaging (MRI)-visible nanocarrier for the delivery of Survivin siRNA targeting the human HCC cell line Bel-7402. The biophysical characterization of the RGD-PEG-g-PEI-SPION was performed. The RGD-modified complexes exhibited a higher transfection efficiency in transferring Survivin siRNA into Bel-7402 cells compared with a non-targeted delivery system, which resulted in more significant gene suppression at both the Survivin mRNA and protein expression levels. Then, the level of caspase-3 activation was significantly elevated, and a remarkable level of tumor cell apoptosis was induced. As a result, the tumor growth in the nude mice Bel-7402 hepatoma model was significantly inhibited. The targeting ability of the RGD-PEG-g-PEI-SPION was successfully imaged by MRI scans performed in vitro and in vivo. Our results strongly indicated that the RGD-PEG-g-PEI-SPION can potentially be used as a targeted non-viral vector for altering gene expression in the treatment of hepatocellular carcinoma and for detecting the tumor in vivo as an effective MRI probe.
In this work we characterize an alcohol dehydrogenase (ADH) from the hyperthermophilic archaeon Pyrobaculum aerophilum (PyAeADHII). We have previously found that PyAeADHII has no activity when standard ADH substrates are used but is active when α-tetralone is used as substrate. Here, to gain insights into enzyme function, we screened several chemical libraries for enzymatic modulators using an assay employing α-tetralone. The results indicate that PyAeADHII activity in the presence of α-tetralone was inhibited by compounds such as flunarizine. We also examined metal coordination of the enzyme in solution by performing metal substitution of the enzyme-bound zinc (Zn2+) with cobalt. The solution-based absorption spectra for cobalt substituted PyAeADHII supports substitution at the structural Zn2+ site. To gain structural insight, we obtained the crystal structure of both wild-type and cobalt-substituted PyAeADHII at 1.75 Å and 2.20 Å resolution, respectively. The X-ray data confirmed one metal ion per monomer present only at the structural site with otherwise close conservation to other ADH enzymes. We next determined the co-crystal structure of the NADPH-bound form of the enzyme at 2.35 Å resolution to help define the active site region of the enzyme and this data shows close structural conservation with horse ADH, despite the lack of a catalytic Zn2+ ion in PyAeADHII. Modeling of α-tetralone into the NADPH bound structure suggests an arginine as a possible catalytic residue. The data presented here can yield a better understanding of alcohol dehydrogenases lacking the catalytic zinc as well as the structural features inherent to thermostable enzymes.
Benzene exposure causes acute myeloid leukemia, and hematotoxicity, shown as suppression of mature blood and myeloid progenitor cell numbers. As the leukemia-related aneuploidies monosomy 7 and trisomy 8 previously had been detected in the mature peripheral blood cells of exposed workers, we hypothesized that benzene could cause leukemia through the induction of these aneuploidies in hematopoietic stem and progenitor cells. We measured loss and gain of chromosomes 7 and 8 by fluorescence in situ hybridization in interphase colony-forming unit-granulocyte-macrophage (CFU-GM) cells cultured from otherwise healthy benzene-exposed (n=28) and unexposed (n=14) workers. CFU-GM monosomy 7 and 8 levels (but not trisomy) were significantly increased in subjects exposed to benzene overall, compared to levels in the control subjects (p=0.0055 and p=0.0034, respectively). Levels of monosomy 7 and 8 were significantly increased in subjects exposed to <10 ppm (20%, p=0.0419 and 28%, p=0.0056, respectively) and ≥10 ppm (48%, p=0.0045 and 32%, p=0.0354) benzene, compared with controls, and significant exposure-response trends were detected (ptrend=0.0033 and 0.0057). These data show that monosomies 7 and 8 are produced in a dose-dependent fashion in the blood progenitor cells of workers exposed to benzene and may be mechanistically relevant biomarkers of early effect for benzene and other leukemogens.
Benzene; leukemia; monosomy; hematopoietic progenitor
The cancer drug discovery field has placed much emphasis on the identification of novel and cancer-specific molecular targets. A rich source of such targets for the design of novel anti-tumor agents is the ubiqutin-proteasome system (UP-S), a tightly regulated, highly specific pathway responsible for the vast majority of protein turnover within the cell. Because of its critical role in almost all cell processes that ensure normal cellular function, its inhibition at one point in time was deemed non-specific and therefore not worth further investigation as a molecular drug target. However, today the proteasome is one of the most promising anti-cancer drug targets of the century. The discovery that tumor cells are in fact more sensitive to proteasome inhibitors than normal cells indeed paved the way for the design of its inhibitors. Such efforts have led to bortezomib, the first FDA approved proteasome inhibitor now used as a frontline treatment for newly diagnosed multiple myeloma (MM), relapsed/refractory MM and mantle cell lymphoma. Though successful in improving clinical outcomes for patients with hematological malignancies, relapse often occurs in those who initially responded to bortezomib. Therefore, the acquisition of bortezomib resistance is a major issue with its therapy. Furthermore, some neuro-toxicities have been associated with bortezomib treatment and its efficacy in solid tumors is lacking. These observations have encouraged researchers to pursue the next generation of proteasome inhibitors, which would ideally overcome bortezomib resistance, have reduced toxicities and a broader range of anti-cancer activity. This review summarizes the success and limitations of bortezomib, and describes recent advances in the field, including, and most notably, the most recent FDA approval of carfilzomib in July, 2012, a second generation proteasome inhibitor. Other proteasome inhibitors currently in clinical trials and those that are currently experimental grade will also be discussed.
Ubiquitin-proteasome system (UP-S); inhibitors of the 20S proteasome; bortezomib; drug discovery; cancer; natural compounds; clinical studies
An approach to selectively and efficiently detect single strand DNA is developed by using streptavidin coated gold nanoparticles (StAuNPs) as efficient quenchers. The central concept for the successful detection is the combination the of streptavidin-biotin interaction with specific probe-target DNA hybridization. Biotin labeled probe DNAs act as “bridges” to bring Cy5 labeled targets to the particle surface and the fluorophore dye can be rapidly and efficiently quenched by StAuPNs. By measuring the changes of photoluminescence intensity of Cy5, an efficient, selective, and reversed detection of DNA hybridization is realized. The methodology may pave a new way for simple and rapid detections of biomolecules.
nanoparticles; fluorophore dye; quenching; streptavidin/biotin; DNA hybridization; sensors
There is an increasing need to understand cell-cell interactions for cell and tissue engineering purposes, such as optimizing cell sheet constructs, as well as for examining adhesion defect diseases. For cell-sheet engineering, one major obstacle to sheet function is that cell sheets in suspension are fragile and, over time, will contract. While the role of the cytoskeleton in maintaining the structure and adhesion of cells cultured on a rigid substrate is well-characterized, a systematic examination of the role played by different components of the cytoskeleton in regulating cell sheet contraction and cohesion in the absence of a substrate has been lacking.
In this study, keratinocytes were cultured until confluent and cell sheets were generated using dispase to remove the influence of the substrate. The effects of disrupting actin, microtubules or intermediate filaments on cell-cell interactions were assessed by measuring cell sheet cohesion and contraction. Keratin intermediate filament disruption caused comparable effects on cell sheet cohesion and contraction, when compared to actin or microtubule disruption. Interfering with actomyosin contraction demonstrated that interfering with cell contraction can also diminish cell cohesion.
All components of the cytoskeleton are involved in maintaining cell sheet cohesion and contraction, although not to the same extent. These findings demonstrate that substrate-free cell sheet biomechanical properties are dependent on the integrity of the cytoskeleton network.
Cell sheet; Cytoskeleton; Adhesion; Contraction
AIM: To examine the clinical features and analyze prognostic factors in a prospective study of primary biliary cirrhosis (PBC) patients.
METHODS: From 1995 to 2010, PBC patients without hepatic decompensation seen at the Peking Union Medical College Hospital were enrolled. Clinical signs and manifestations (pruritus, persistent fatigue, jaundice and pain in the right hypochondrium), laboratory parameters (auto-antibodies for autoimmune hepatic disease, biliary and hepatic enzymes, immunoglobulin, bilirubin, and albumin) and imaging findings were recorded at entry and at specific time points during follow-up. Cox regression and Kaplan-Meier analyses, respectively, assessed the risk factors for hepatic decompensation and survival.
RESULTS: Two hundred and sixty-two PBC patients were enrolled with a median follow-up of 75.2 mo (range, 21-201 mo). The 240 patients were aged 51.5 ± 10.2 years at diagnosis and 91.6% were female. Two hundred and forty-five (93.5%) were seropositive for anti-mitochondrial antibodies. At presentation, 170 patients (64.9%) were symptomatic, while 96 patients (36.6%) had extra-hepatic autoimmune disease. During the follow-up period, 62 (23.7%) patients developed hepatic decompensation of whom four underwent liver transplantation and 17 died. The cumulative survival rate and median survival time were 83.9% and 181.7 mo, respectively. Cox regression analysis revealed that an incomplete ursodeoxycholic acid (UDCA) response or inconsistent treatment [P < 0.001; hazard risk (HR) 95%CI = 2.423-7.541], anti-centromere antibodies (ACA) positivity (P < 0.001; HR 95%CI = 2.516-7.137), alanine aminotransferase ratio (AAR) elevations (P < 0.001; HR 95%CI = 1.357-2.678), and histological advanced liver disease (P = 0.006; HR 95%CI = 1.481-10.847) were predictors of hepatic decompensation. The clinical features and survival of PBC in China are consistent with those described in Western countries.
CONCLUSION: Incomplete UDCA response or inconsistent treatment, ACA positivity, AAR elevations, and advanced histological stage are predictors of decompensation.
Primary biliary cirrhosis; Risk factor; Hepatic decompensation; Survival; Ursodeoxycholic acid response; Anti-centromere antibodies; Histological stage
Phosphoinositide kinases regulate diverse cellular functions and are important targets for therapeutic development for diseases, such as diabetes and cancer. Preparation of the lipid substrate is crucial for the development of a robust and miniaturizable lipid kinase assay. Enzymatic assays for phosphoinositide kinases often use lipid substrates prepared from lyophilized lipid preparations by sonication, which result in variability in the liposome size from preparation to preparation. Herein, we report a homogeneous 1536-well luciferase-coupled bioluminescence assay for PI5P4Kα. The substrate preparation is novel and allows the rapid production of a DMSO-containing substrate solution without the need for lengthy liposome preparation protocols, thus enabling the scale-up of this traditionally difficult type of assay. The Z’-factor value was greater than 0.7 for the PI5P4Kα assay, indicating its suitability for high-throughput screening applications. Tyrphostin AG-82 had been identified as an inhibitor of PI5P4Kα by assessing the degree of phospho transfer of γ-32P-ATP to PI5P; its inhibitory activity against PI5P4Kα was confirmed in the present miniaturized assay. From a pilot screen of a library of bioactive compounds, another tyrphostin, I-OMe tyrphostin AG-538 (I-OMe-AG-538), was identified as an ATP-competitive inhibitor of PI5P4Kα with an IC50 of 1 µM, affirming the suitability of the assay for inhibitor discovery campaigns. This homogeneous assay may apply to other lipid kinases and should help in the identification of leads for this class of enzymes by enabling high-throughput screening efforts.
Extracorporeal Shock Wave Lithotriptors are very popular for the treatment of urinary stones all over the world. They depend basically upon either X-ray fluoroscopy or ultrasound scans to detect the stones before therapy begins. To increase the effectiveness of treatment this study took advantage of both X-ray and ultrasound to develop a dual stone locating system with image processing modules. Its functions include the initial stone locating mode with stone detection by fluorescent images and the follow-up automatic stone tracking mode made by constant ultrasound scanning. The authors have integrated both apparatus and present the operating principles for both modes. The system used two in vitro experiments to justify its abilities of stone location in all procedures.
X-ray; ultrasound; image processing; Extracorporeal Shock Wave Lithotriptor; stone location; stone tracking
This study was designed to investigate whether Resveratrol (Res) could be a prophylactic factor in the prevention of I/R injury and to shed light on its underlying mechanism. Primary culture of neonatal rat cardiomyocytes were randomly distributed into three groups: the normal group (cultured cardiomyocytes were in normal conditions), the I/R group (cultured cardiomyocytes were subjected to 2 h simulated ischemia followed by 4 h reperfusion), and the Res+I/R group (100 µmol/L Res was administered before cardiomyocytes were subjected to 2 h simulated ischemia followed by 4 h reperfusion). To test the extent of cardiomyocyte injury, several indices were detected including cell viability, LDH activity, Na+-K+-ATPase and Ca2+-ATPase activity. To test apoptotic cell death, caspase-3 activity and the expression of Bcl-2/Bax were detected. To explore the underlying mechanism, several inhibitors, intracellular calcium, SOD activity and MDA content were used to identify some key molecules involved. Res increased cell viability, Na+-K+-ATPase and Ca2+-ATPase activity, Bcl-2 expression, and SOD level. While LDH activity, capase-3 activity, Bax expression, intracellular calcium and MDA content were decreased by Res. And the effect of Res was blocked completely by either L-NAME (an eNOS inhibitor) or MB (a cGMP inhibitor), and partly by either DS (a PKC inhibitor) or Glybenclamide (a KATP inhibitor). Our results suggest that Res attenuates I/R injury in cardiomyocytes by preventing cell apoptosis, decreasing LDH release and increasing ATPase activity. NO, cGMP, PKC and KATP may play an important role in the protective role of Res. Moreover, Res enhances the capacity of anti-oxygen free radical and alleviates intracellular calcium overload in cardiomyocytes.
Telomere length plays an important role in chromosomal stability and tumorigenesis, and its measurement in peripheral white blood cell DNA may be a predictor of the development of lung cancer.
Using a new method - monochrome multiplex quantitative PCR -which reduces measurement variability, we compared telomere length relative to standard DNA in white blood cell DNA in 229 incident male lung cancer cases and 229 matched controls within the prospective Alpha-Tocopherol, Beta-Carotene Cancer Prevention (ATBC) Study of male smokers.
Median (10th, 90th percentile) telomere length was 1.13 (0.86, 1.45) in cases and 1.08 (0.85, 1.38) in controls (P = 0.038). Telomere length was inversely associated with pack-years of smoking (Spearman correlation r = −0.16, P = 0.02) among controls. Compared to subjects with shorter telomere length (≤ median), subjects with greater telomere length (> median) had a 1.6-fold (95% CI, 1.06–2.36) increased risk of lung cancer. There was a significant linear relationship between quartiles of telomere length and risk of lung cancer (odds ratios (95% confidence intervals) by quartile: 1.00, 0.98 (0.55–1.73), 1.62 (0.95–2.77), and 1.50 (0.84–2.68); Ptrend = 0.05). In addition, subgroup analysis showed that greater telomere length was associated with increased risk of lung cancer among heavy smokers (> 38 years) (OR, 1.90; 95% CI, 1.00–3.59) but not among light smokers (≤ 38 years) (OR, 1.08; 95% CI, 0.56–2.11) (Pinteraction = 0.01).
Our results suggest that greater telomere length may be associated with higher risk of lung cancer among male smokers.
Telomere length; lung cancer; cohort study
The chemical diversity of nature has tremendous potential for discovery of new molecular probes and medicinal agents. However, sensitivity of HTS assays to interfering components of crude extracts derived from plants, macro- and microorganisms has curtailed their use in lead discovery efforts. Here we describe a process for leveraging the concentration-response curves (CRCs) obtained from quantitative HTS to improve the initial selection of “actives” from a library of partially fractionated natural product extracts derived from marine actinomycetes and fungi. By using pharmacological activity, the first-pass CRC paradigm aims to improve the probability that labor-intensive subsequent steps of re-culturing, extraction and bioassay-guided isolation of active component(s) target the most promising strains and growth conditions. We illustrate how this process identified a family of fungal metabolites as potent inhibitors of firefly luciferase, subsequently resolved in molecular detail by x-ray crystallography.
Pyruvate kinase (PYK) plays a central role in the metabolism of many organisms and cell types, but the elucidation of the details of its function in a systems biology context has been hampered by the lack of specific high-affinity small molecule inhibitors. High-throughput screening has been used to identify a family of saccharin derivatives which inhibit Leishmania mexicana PYK (LmPYK) activity in a time- (and dose-) dependent manner; a characteristic of irreversible inhibition. The crystal structure of 4-[(1,1-dioxo-1,2-benzothiazol-3-yl)sulfanyl]benzoic acid (DBS) complexed with LmPYK shows that the saccharin moiety reacts with an active-site lysine residue (Lys335), forming a covalent bond and sterically hindering the binding of ADP/ATP. Mutation of the lysine residue to an arginine residue eliminated the effect of the inhibitor molecule, providing confirmation of the proposed inhibitor mechanism. This lysine residue is conserved in the active sites of the four human PYK isoenzymes, which were also found to be irreversibly inhibited by DBS. X-ray structures of PYK isoforms show structural differences at the DBS binding pocket, and this covalent inhibitor of PYK provides a chemical scaffold for the design of new families of potentially isoform-specific irreversible inhibitors.
Leishmania mexicana; lysine covalent modification; nucleotide binding; pyruvate kinase; saccharin analogues; covalent inhibitor
The development of tumor drug resistance is one of the biggest obstacles on the way to achieve a favorable outcome of chemotherapy. Among various strategies that have been explored to overcome drug resistance, the combination of current chemotherapy with plant polyphenols as a chemosensitizer has emerged as a promising one. Plant polyphenols are a group of phytochemicals characterized by the presence of more than one phenolic group. Mechanistic studies suggest that polyphenols have multiple intracellular targets, one of which is the proteasome complex. The proteasome is a proteolytic enzyme complex responsible for intracellular protein degradation and has been shown to play an important role in tumor growth and the development of drug resistance. Therefore, proteasome inhibition by plant polyphenols could be one of the mechanisms contributing to their chemosensitizing effect. Plant polyphenols that have been identified to possess proteasome-inhibitory activity include (−)-epigallocatechins-3-gallate (EGCG), genistein, luteolin, apigenin, chrysin, quercetin, curcumin and tannic acid. These polyphenols have exhibited an appreciable effect on overcoming resistance to various chemotherapeutic drugs as well as multidrug resistance in a broad spectrum of tumors ranging from carcinoma and sarcoma to hematological malignances. The in vitro and in vivo studies on polyphenols with proteasome-inhibitory activity have built a solid foundation to support the idea that they could serve as a chemosensitizer for the treatment of cancer. In-depth mechanistic studies and identification of optimal regimen are needed in order to eventually translate this laboratory concept into clinical trials to actually benefit current chemotherapy.
Polyphenols; flavonoids; EGCG; genistein; curcumin; structure-activity relationship; ubiquitin-proteasome pathway; proteasome inhibitors; bortezomib; chemotherapy; drug resistance; NF-κB; Bcl-2; chemosensitization; clinical trials
Control of intracellular reactive oxygen species (ROS) concentrations is critical for cancer cell survival. We show that, in human lung cancer cells, acute increases in intracellular concentrations of ROS caused inhibition of the glycolytic enzyme pyruvate kinase M2 (PKM2) through oxidation of Cys358. This inhibition of PKM2 is required to divert glucose flux into the pentose phosphate pathway and thereby generate sufficient reducing potential for detoxification of ROS. Lung cancer cells in which endogenous PKM2 was replaced with the Cys358 to Ser358 oxidation-resistant mutant exhibited increased sensitivity to oxidative stress and impaired tumor formation in a xenograft model. Besides promoting metabolic changes required for proliferation, the regulatory properties of PKM2 may confer an additional advantage to cancer cells by allowing them to withstand oxidative stress.
Drug repurposing for cancer treatment is an emerging approach to discover clinically approved drugs that demonstrate antineoplastic effect. The effective therapeutics for patients with advanced adrenocortical carcinoma(ACC) are greatly needed. The objective of this study was to identify and validate drugs with antineoplastic effect in ACC cells using a novel quantitative high-throughput drug screening (qHTS) technique.
A quantitative high-throughput proliferation assay of 2,816 clinically approved drugs was performed in the NCI-H295R ACC cell line. We validated the antiproliferative effect of candidate compounds in NCI-H295R cells. Further validation was performed in 3-dimensional multicellular aggregates (MCA) of NCI-H295R and SW-13 cell lines.
We identified 79 active compounds against ACC cells; 21 had an efficacy ≥60% and IC50 <1 μM. The top drug categories enriched were cardiotonic, antiseptic, and antineoplastic. We selected Bortezomib, ouabain, Methotrexate, pyrimethamine for validation. All had an antiproliferative effect in monolayer culture of NCI-H295R cells at clinical achievable serum level. Bortezomib and ouabain inhibited growth of MCA in both cell lines at a low concentration (10 fold below IC50). Methotrexate inhibited growth and caused disintegration of MCA in both cell lines at concentrations well below the maximum serum level (10 to 100 fold of IC50). Pyrimethamine caused growth inhibition in both cell lines at 10 fold of IC50 concentration.
qHTS of previously approved compounds is an effective and efficient method to identify anticancer drugs for a rare cancer such as ACC. We have validated the antineoplastic effect of Bortezomib, ouabain, Methotrexate and pyrimethamine, which could be translated into clinical trials in patients with locally advanced and/or metastatic ACC.
Adrenocortical cancer; High throughput drug screening; Chemotherapy; Drug repurposing; Indication switching
An integrin found on platelets, αIIbβ3 mediates platelet aggregation, and αIIbβ3 antagonists are effective antithrombotic agents in the clinic. Ligands bind to integrins in part by coordinating a magnesium ion (Mg2+) located in the β subunit metal ion–dependent adhesion site (MIDAS). Drugs patterned on the integrin ligand sequence Arg-Gly-Asp have a basic moiety that binds the αIIb subunit and a carboxyl group that coordinates the MIDAS Mg2+ in the β3 subunits. They induce conformational changes in the β3 subunit that may have negative consequences such as exposing previously hidden epitopes and inducing the active conformation of the receptor. We recently reported an inhibitor of αIIbβ3 (RUC-1) that binds exclusively to the αIIb subunit; here, we report the structure-based design and synthesis of RUC-2, a RUC-1 derivative with a ~100-fold higher affinity. RUC-2 does not induce major conformational changes in β3 as judged by monoclonal antibody binding, light scattering, gel chromatography, electron microscopy, and a receptor priming assay. X-ray crystallography of the RUC-2–IIbβ3 headpiece complex in 1 mM calcium ion (Ca2+)/5 mM Mg2+ at 2.6 Å revealed that RUC-2 binds to αIIb the way RUC-1 does, but in addition, it binds to the β3 MIDAS residue glutamic acid 220, thus displacing Mg2+ from the MIDAS. When the Mg2+ concentration was increased to 20 mM, however, Mg2+ was identified in the MIDAS and RUC-2 was absent. RUC-2′s ability to inhibit ligand binding and platelet aggregation was diminished by increasing the Mg2+ concentration. Thus, RUC-2 inhibits ligand binding by a mechanism different from that of all other αIIbβ3 antagonists and may offer advantages as a therapeutic agent.
Polyethylene glycol-grafted polyethylenimine (PEG-g-PEI) which was functionalized with a neuroblastoma cell-specific ligand, the GD2 single chain antibody (scAbGD2), was synthesized in order to effectively deliver Bcl-2 siRNA into neuroblastoma cells. This polymer was complexed first with superparamagnetic iron oxide nanoparticle (SPION) to get a MRI-visible targeted non-viral vector (scAbGD2-PEG-g-PEI-SPION) and then with Bcl-2 siRNA to form nanoparticles showing low cytotoxicity. The targeting capacity of scAbGD2-PEG-g-PEI-SPION was successfully verified in vivo and in vitro by magnetic resonance imaging. The single chain antibody encoded targeted polyplex was more effective in transferring Bcl-2 siRNA than the nontargeting one in SK-N-SH cells, a human neuroblastoma cell line, resulting in a 46.34% inhibition in the expression of Bcl-2 mRNA. Consequently, a high level of cell apoptosis up to 50.76% and a significant suppression of tumor growth were achieved, which indicates that scAbGD2-PEG-g-PEI-SPION is a promising magnetic resonance imaging-visible non-viral vector for targeted neuroblastoma siRNA therapy and diagnosis.
tumor targeting; GD2; non-viral vector; Bcl-2 small interfering RNA; magnetic resonance imaging