ABSTRACT
Here, we report a non-invasive strategy for isolating cancer cells by autonomously propelled carbon nanotube (CNT) microrockets. H2O2-driven oxygen (O2) bubble-propelled microrockets were synthesized using CNT and Fe3O4 nanoparticles in the inner surface and covalently conjugating transferrin on the outer surface. Results show that self-propellant microrockets can specifically capture cancer cells.
Subject(s)
Nanotubes, Carbon/chemistry , Neoplastic Cells, Circulating/chemistry , Ferrosoferric Oxide/chemistry , HCT116 Cells , Humans , Hydrogen Peroxide/chemistry , Ligands , Magnetite Nanoparticles/chemistry , Microscopy, Electron, Transmission , Oxygen/chemistry , Surface Properties , Time-Lapse ImagingABSTRACT
The mTOR pathway is often upregulated in cancer and thus intensively pursued as a target to design novel anticancer therapies. Approved and emerging drugs targeting the mTOR pathway have positively affected the clinical landscape. Recently, activin receptor-like kinase 1 (ALK1), belonging to the TGFß receptor family, has been reported as an emerging target for antiangiogenic cancer therapy. Here, we describe a novel orally efficacious compound, P7170, that inhibits mTORC1/mTORC2/ALK1 activity with a potent cell growth inhibition. In cell-based assays, P7170 strongly inhibited (IC50 < 10 nmol/L) the phosphorylation of p70S6K (T389) and pAKT (S473). In many cancer cell lines, such as prostate, ovarian, colon, and renal, P7170 treatment resulted in marked cell growth inhibition. Furthermore, it induced G1-S cell-cycle arrest and autophagy. In vitro HUVEC tube formation, in vivo Matrigel plug, and rat aorta ring assays demonstrated that P7170 exhibited significant antiangiogenic activity. In addition, ALK1 knockdown studies in HUVEC confirmed that the antiangiogenic activity of P7170 was primarily due to ALK1 inhibition. Strong inhibition of ALK1 in addition to mTORC1/mTORC2 differentiates P7170 in its mechanism of action in comparison with existing inhibitors. In vivo mouse xenograft studies revealed P7170 to exhibit a significant dose-dependent tumor growth inhibition in a broad range of human tumor types when administered orally at 10 to 20 mg/kg doses. The distinctive pharmacological profile with favorable pharmacokinetic parameters and in vivo efficacy makes P7170 an attractive candidate for clinical development. It is currently being tested in phase I clinical studies.
Subject(s)
Angiogenesis Inhibitors/administration & dosage , Antineoplastic Agents/administration & dosage , Imidazoles/administration & dosage , Prostatic Neoplasms/drug therapy , Quinolines/administration & dosage , Activin Receptors, Type II/antagonists & inhibitors , Administration, Oral , Angiogenesis Inhibitors/pharmacology , Animals , Antineoplastic Agents/pharmacology , Aorta, Thoracic/cytology , Aorta, Thoracic/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Dose-Response Relationship, Drug , Hep G2 Cells , Human Umbilical Vein Endothelial Cells , Humans , Imidazoles/pharmacology , Male , Mice , Prostatic Neoplasms/metabolism , Quinolines/pharmacology , Rats , TOR Serine-Threonine Kinases/antagonists & inhibitors , Xenograft Model Antitumor AssaysABSTRACT
BACKGROUND & OBJECTIVES: Studies involving animal models of experimental tuberculosis have elucidated the predominant role of cytokines secreted by T cells and macrophages to be an essential component of the immune response against Mycobacterium tuberculosis infection. The immune activities of CD4+ T cells are mediated in part by Th1 cytokine interferon gamma (IFN-γ) which is produced primarily by T cells and natural killer (NK) cells and critical for initiating the immune response against intracellular pathogen such as M. tuberculosis. Nuclear matrix protein SMAR1 plays an important role in V(D)J recombination, T helper cell differentiation and inflammatory diseases. In this study a transgenic mouse model was used to study the role of SMAR1 in M. tuberculosis infection. METHODS: Wild type BALB/c, C57BL/6, BALB/c-EGFP-SMAR1 and C57BL/6-SMAR1 transgenic mice were infected with M. tuberculosis (H37Rv). A dose of 100 bacilli was used for infection via respiratory route. Bacterial load in lung and spleen of infected mice was determined at 2, 4, 6 and 8 wk post-infection. Gene expression analysis for Th1 cytokines and inducible nitric oxide synthase (iNOS) was performed in infected lung tissues by quantitative reverse transcription (RT)-PCR. RESULTS: SMAR1 transgenic mice from both BALB/c and C57BL/6 genetic background displayed higher bacillary load and susceptibility to M. tuberculosis infection compared to wild type mice. This susceptibility was attributed due to compromised of Th1 response exhibited by transgenic mice. INTERPRETATION & CONCLUSIONS: SMAR1 transgenic mice exhibited susceptibility to M. tuberculosis infection in vivo irrespective of genetic background. This susceptibility was attributed to downregulation of Th1 response and its hallmark cytokine IFN-γ. Hence, SMAR1 plays an important role in modulating host immune response after M. tuberculosis infection.
Subject(s)
Cell Cycle Proteins/physiology , DNA-Binding Proteins/physiology , Disease Models, Animal , Mycobacterium tuberculosis/genetics , Mycobacterium tuberculosis/pathogenicity , Nuclear Proteins/physiology , Tuberculosis/immunology , Animals , Cell Cycle Proteins/genetics , DNA-Binding Proteins/genetics , Interferon-gamma/metabolism , Mice , Mice, Transgenic , Nuclear Proteins/genetics , Th1 Cells/immunology , Tuberculosis/microbiologyABSTRACT
Carbon nanostructures such as multiwalled carbon nanotubes (CNT) and graphene (G) are potential candidates in a large number of biomedical applications. However, there is limited understanding and connection between the physicochemical properties of diverse carbon nanostructures and biological systems, particularly with regard to cellular responses. It is also crucial to understand how the structure and surface composition of carbon nanostructures affect the cellular internalization process. Here, through in vitro cellular entry kinetics and cytotoxicity studies using MCF-7 breast cancer cells and H460 human lung cancer cells, we show that the structure and surface composition of CNT and G conjugates with various molecules such as PAMAM dendrimers (G4) and G4-poly(ethylene glycol) (PEG) are directly related to their cellular internalization ability and toxicity. Interestingly, the cellular association of CNT and G nanoconjugates was observed to be structure and surface composition dependent. We found that CNT conjugates internalized more compared to G conjugates. Furthermore, G4 conjugated CNT internalized more compared to G4-PEG conjugated CNT, whereas, higher internalization was found for G4-PEG conjugated G than G4 conjugated G. We have also correlated the cytotoxicity and cellular uptake mechanisms of CNT, G, and their conjugates through zeta potential measurements, fluorescence quenching studies and by fluorescence-activated cell sorting. Altogether these studies suggest different biological activities of the carbon nanostructures, with the shape and surface composition playing a primary role.
ABSTRACT
Many polymer based drug delivery nanosystems are currently being explored for delivering cytotoxic agents to the tumors. However, very few strategies delineate the comparative carrier ability of nanosystems, in similar experimental settings. As a result, it remains unclear how to optimally design polymer based multicomponent prodrug systems for delivery applications. The present study is aimed to design polymeric prodrug conjugate carriers for the comparative cellular delivery ability of anticancer drug doxorubicin hydrochloride (DOX) using linear poly(ethylene glycol) (PEG), hyperbranched poly(amido amine) (PAMAM) G4 dendrimer, and PAMAM G4 dendrimer-PEG conjugate using MCF-7 cells. Furthermore, the cellular targetability and in vitro anticancer activity of DOX conjugates is evaluated using transferrin (Tf) as a targeting ligand. Interestingly, conjugation of DOX to PAMAM G4-OH dendrimer significantly influences the cytotoxicity of DOX leading to -4 fold decrease in the IC50 dose when compared to pegylated DOX. This study establishes the rational and comparative structural activity relationship of polymeric prodrug carriers for delivery of anticancer drugs. The schematic representation of design of prodrug conjugates with varied polymeric architecures is as shown below (Fig. 1).
Subject(s)
Cells/metabolism , Dendrimers/chemistry , Doxorubicin , Polyethylene Glycols/chemistry , Prodrugs , Transferrin/pharmacology , Antineoplastic Agents/administration & dosage , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacokinetics , Dendrimers/pharmacokinetics , Doxorubicin/administration & dosage , Doxorubicin/chemistry , Doxorubicin/pharmacokinetics , Drug Delivery Systems , Drug Evaluation, Preclinical , Humans , Inhibitory Concentration 50 , Models, Biological , Polyethylene Glycols/pharmacokinetics , Polymers/chemical synthesis , Polymers/chemistry , Polymers/pharmacokinetics , Prodrugs/administration & dosage , Prodrugs/chemistry , Prodrugs/pharmacokinetics , Structure-Activity Relationship , Transferrin/administration & dosage , Transferrin/chemistry , Tumor Cells, CulturedABSTRACT
A multicomponent magneto-dendritic nanosystem (MDNS) is designed for rapid tumor cell targeting, isolation, and high-resolution imaging by a facile bioconjugation approach. The highly efficient and rapid-acting MDNS provides a convenient platform for simultaneous isolation and high-resolution imaging of tumor cells, potentially leading towards an early diagnosis of cancer.
Subject(s)
Cell Separation/methods , Immunomagnetic Separation/methods , Molecular Diagnostic Techniques/methods , Nanoparticles , Neoplastic Cells, Circulating/metabolism , Neoplastic Cells, Circulating/pathology , Transferrin/pharmacokinetics , Hep G2 Cells , Humans , Immunomagnetic Separation/instrumentation , Molecular Diagnostic Techniques/instrumentation , Nanoparticles/chemistryABSTRACT
We describe a novel multicomponent graphene nanostructured system that is biocompatible, and has strong NIR optical absorbance and superparamagnetic properties. The fabrication of the multicomponent nanostructure system involves the covalent attachment of 3 components; Fe(3)O(4)(Fe) nanoparticles, PAMAM-G4-NH(2) (G4) dendrimer and Cy5 (Cy) on a graphene oxide (GO) surface to synthesize a biologically relevant multifunctional system. The resultant GO-G4-Fe-Cy nanosystem exhibits high dispersion in an aqueous medium, and is magnetically responsive and fluorescent. In vitro experiments provide a clear indication of successful uptake of the GO-G4-Fe-Cy nanosystem by MCF-7 breast cancer cells, and it is seen to behave as a bright and stable fluorescent marker. The study also reveals varied cellular distribution kinetics profile for the GO nanostructured system compared to free Cy. Furthermore, the newly developed GO nanostructured system is observed to be non-toxic to MDA-MB-231 cell growth, in striking contrast to free G4 dendrimer and GO-G4 conjugate. The GO-G4-Fe-Cy nanostructured system characterized by multifunctionality suggests the merits of graphene for cellular bioimaging and the delivery of bioactives.
Subject(s)
Breast Neoplasms/diagnosis , Carbocyanines , Dendrimers , Fluorescent Dyes , Graphite , Magnetite Nanoparticles , Nylons , Carbocyanines/chemistry , Carbocyanines/pharmacokinetics , Cell Line, Tumor , Cell Survival , Dendrimers/chemistry , Dendrimers/pharmacokinetics , Female , Fluorescent Dyes/chemistry , Fluorescent Dyes/pharmacokinetics , Graphite/chemistry , Graphite/pharmacokinetics , Humans , Magnetite Nanoparticles/chemistry , Microscopy, Confocal , Models, Molecular , Nylons/chemistry , Nylons/pharmacokinetics , Optical ImagingABSTRACT
Efficacy of anticancer drug is limited by the severe adverse effects induced by drug; therefore the crux is in designing delivery systems targeted only to cancer cells. Toward this objectives, we propose, synthesis of poly(ethylene glycol) (PEG)-doxorubicin (DOX) prodrug conjugates consisting N-acetyl glucosamine (NAG) as a targeting moiety. Multicomponent system proposed here is characterized by (1)H NMR, UV spectroscopy, and HPLC. The multicomponent system is evaluated for in vitro cellular kinetics and anticancer activity using MCF-7 and MDA-MB-231 cells. Molecular modeling study demonstrated sterically stabilized conformations of polymeric conjugates. Interestingly, PEG-DOX conjugate with NAG ligand showed significantly higher cytotoxicity compared to drug conjugate with DOX. In addition, the polymer drug conjugate with NAG and DOX showed enhanced internalization and retention effect in cancer cells, compared to free DOX. Thus, with enhanced internalization and targeting ability of PEG conjugate of NAG-DOX has implication in targeted anticancer therapy.
Subject(s)
Acetylglucosamine/chemistry , Antibiotics, Antineoplastic/chemistry , Doxorubicin/chemistry , Drug Carriers/chemistry , Polyethylene Glycols/chemistry , Prodrugs/chemical synthesis , Acetylglucosamine/administration & dosage , Antibiotics, Antineoplastic/administration & dosage , Cell Line, Tumor , Cell Survival/drug effects , Doxorubicin/administration & dosage , Drug Carriers/administration & dosage , Humans , Maleimides/chemistry , Models, Molecular , Polyethylene Glycols/administration & dosage , Prodrugs/administration & dosageABSTRACT
A transferrin-conjugated PEG-Fe(3) O(4) nanostructured matrix is developed to explore cellular responses in terms of enhanced cell adhesion, specific interactions between ligands in the matrix and molecular receptors on the cell membrane, comparison of cell shapes on 2D and 3D surfaces, and effect of polymer architecture on cell adhesion. Integration of such advanced synthetic nanomaterials into a functionalized 3D matrix to control cell behavior on surfaces will have implications in nanomedicine.
Subject(s)
Cell Adhesion/physiology , Colonic Neoplasms , Ferric Compounds/chemistry , Nanostructures/chemistry , Transferrin/chemistry , Cell Line, Tumor , Humans , Surface PropertiesABSTRACT
We report synthesis of a highly versatile multicomponent nanosystem by covalently decorating the surface of multiwalled carbon nanotubes (CNTs) by magnetite nanoparticles (Fe(3)O(4)), poly(ethylene glycol) (PEG), and fluorophore fluorescein isothiocyanate (FITC). The resulting Fe(3)O(4)-PEG-FITC-CNT nanosystem demonstrates high dispersion ability in an aqueous medium, magnetic responsiveness, and fluorescent capacity. Transmission electron microscopy images revealed that Fe(3)O(4) nanoparticles were well anchored onto the surfaces of the CNT. In vitro time kinetic experiments using confocal microscopy demonstrated a higher uptake of the Fe(3)O(4)-PEG-FITC-CNT nanosystem localized at the perinuclear region of MCF7 cells compared to the free FITC. In addition, the CNT nanosystem demonstrated no evidence of toxicity on cell growth. Surface conjugation of multicomponents, combined with in vitro non-toxicity, enhanced cellular uptake for FITC and site specific targeting ability makes this fluorescent Fe(3)O(4)-PEG-FITC-CNT nanosystem an ideal candidate for bioimaging, both in vitro and in vivo.
Subject(s)
Magnetite Nanoparticles/chemistry , Nanotubes, Carbon/chemistry , Breast Neoplasms/pathology , Cell Line, Tumor , Cell Survival , Diagnostic Imaging/methods , Female , Fluorescein-5-isothiocyanate/chemistry , Fluorescent Dyes/chemistry , Humans , Magnetite Nanoparticles/ultrastructure , Microscopy, Electron, Transmission , Nanoconjugates/chemistry , Nanoconjugates/ultrastructure , Nanotechnology , Nanotubes, Carbon/ultrastructure , Optical Phenomena , Polyethylene GlycolsABSTRACT
BACKGROUND: Hypoxia-inducible factor-1 (HIF-1) is a master regulator of the transcriptional response to hypoxia. It is essential for angiogenesis and is associated with tumor progression and overexpression of HIF-1α has been demonstrated in many common human cancers. Therefore, HIF-1α is one of the most compelling anticancer targets. METHODS: To identify HIF-1α inhibitors, luciferase reporter gene assay under hypoxia and normoxia was used. Detailed studies such as western blotting, RT-PCR, immunofluorescence were carried out to elucidate its mechanism of action. Antiangiogenic activity of P3155 was demonstrated by migration assay and tube formation assay. Efficacy study of P3155 was performed on PC-3 xenograft model. RESULTS: P3155 showed specific HIF-1α inhibition with IC50 of 1.4 µM under hypoxia. It suppressed HIF-1α expression as well as PI3K/Akt pathway and abrogated expression of HIF-1-inducible gene viz. vascular endothelial growth factor (VEGF). P3155 in combination with HIF-1α siRNA showed significant synergistic effect. In addition, it demonstrated significant in vivo efficacy and antiangiogenic potential in prostate cancer cell lines. CONCLUSION: We have identified a novel HIF-1α inhibitor P3155 that also modulates PI3K/Akt pathway, which may contribute to its significant in vitro and in vivo antitumor activity.
Subject(s)
Angiogenesis Inhibitors/pharmacology , Hypoxia-Inducible Factor 1, alpha Subunit/antagonists & inhibitors , Phosphatidylinositol 3-Kinases/metabolism , Prostatic Neoplasms/drug therapy , Pyridines/pharmacology , Pyrimidines/pharmacology , Animals , Cell Growth Processes/drug effects , Cell Hypoxia/physiology , Cell Line, Tumor , Cell Movement/drug effects , Dose-Response Relationship, Drug , Human Umbilical Vein Endothelial Cells , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Male , Mice , Neovascularization, Pathologic/drug therapy , Prostatic Neoplasms/blood supply , Prostatic Neoplasms/metabolism , Prostatic Neoplasms/pathology , Signal Transduction/drug effects , Topotecan/pharmacology , Vascular Endothelial Growth Factor A/metabolism , Xenograft Model Antitumor AssaysABSTRACT
Changes in the composition of nuclear matrix associated proteins contribute to alterations in nuclear structure, one of the major phenotypes of malignant cancer cells. The malignancy-induced changes in this structure lead to alterations in chromatin folding, the fidelity of genome replication and gene expression programs. The nuclear matrix forms a scaffold upon which the chromatin is organized into periodic loop domains called matrix attachment regions (MAR) by binding to various MAR binding proteins (MARBPs). Aberrant expression of MARBPs modulates the chromatin organization and disrupt transcriptional network that leads to oncogenesis. Dysregulation of nuclear matrix associated MARBPs has been reported in different types of cancers. Some of these proteins have tumor specific expression and are therefore considered as promising diagnostic or prognostic markers in few cancers. SMAR1 (scaffold/matrix attachment region binding protein 1), is one such nuclear matrix associated protein whose expression is drastically reduced in higher grades of breast cancer. SMAR1 gene is located on human chromosome 16q24.3 locus, the loss of heterozygosity (LOH) of which has been reported in several types of cancers. This review elaborates on the multiple roles of nuclear matrix associated protein SMAR1 in regulating various cellular target genes involved in cell growth, apoptosis and tumorigenesis.
Subject(s)
Cell Cycle Proteins/physiology , DNA-Binding Proteins/physiology , Gene Expression Regulation , Homeostasis , Neoplasms/etiology , Nuclear Proteins/physiology , Animals , Apoptosis , Cell Cycle , Cell Cycle Proteins/genetics , DNA-Binding Proteins/genetics , Genes, bcl-1 , Humans , Neoplasm Invasiveness , Nuclear Proteins/genetics , Signal Transduction , Transforming Growth Factor beta/physiologyABSTRACT
Hypoxia-inducible factor-1α (HIF-1α) is a critical regulatory protein of cellular response to hypoxia, and regulates the transcription of many genes involved in key aspects of cancer biology, including immortalization, maintenance of stem cell pools, cellular dedifferentiation, vascularization, and invasion/metastasis. HIF-1α has been implicated in the regulation of genes involved in angiogenesis, for example, VEGF and is associated with tumor progression. In the last decade, over expression of HIF-1α has been demonstrated in many common human cancers and emerging as a validated target for anticancer drug discovery. Here we report the discovery of newly designed and synthesized pyridylpyrimidine based potent and selective inhibitors of HIF-1α. P2630 has been found as potent antiproliferative, antiangiogenic and orally efficacious compound in PC-3 xenograft mice model.
Subject(s)
Antineoplastic Agents/pharmacology , Drug Discovery , Hypoxia-Inducible Factor 1, alpha Subunit/antagonists & inhibitors , Pyridines/pharmacology , Pyrimidines/pharmacology , Animals , Antineoplastic Agents/chemistry , Cells, Cultured , Drug Screening Assays, Antitumor , Humans , Mice , Mice, SCIDABSTRACT
BACKGROUND: Imaging tools such as scanning electron microscope (SEM) and atomic force microscope (AFM) can be used to produce high-resolution topographic images of biomedical specimens and hence are well suited for imaging alterations in cell morphology. We have studied the correlation of SMAR1 expression with cell surface smoothness in cell lines as well as in different grades of human breast cancer and mouse tumor sections. METHODS: We validated knockdown and overexpression of SMAR1 using RT-PCR as well as Western blotting in human embryonic kidney (HEK) 293, human breast cancer (MCF-7) and mouse melanoma (B16F1) cell lines. The samples were then processed for cell surface roughness studies using atomic force microscopy (AFM) and scanning electron microscopy (SEM). The same samples were used for microarray analysis as well. Tumors sections from control and SMAR1 treated mice as well as tissues sections from different grades of human breast cancer on poly L-lysine coated slides were used for AFM and SEM studies. RESULTS: Tumor sections from mice injected with melanoma cells showed pronounced surface roughness. In contrast, tumor sections obtained from nude mice that were first injected with melanoma cells followed by repeated injections of SMAR1-P44 peptide, exhibited relatively smoother surface profile. Interestingly, human breast cancer tissue sections that showed reduced SMAR1 expression exhibited increased surface roughness compared to the adjacent normal breast tissue. Our AFM data establishes that treatment of cells with SMAR1-P44 results into increase in cytoskeletal volume that is supported by comparative gene expression data showing an increase in the expression of specific cytoskeletal proteins compared to the control cells. Altogether, these findings indicate that tumor suppressor function of SMAR1 might be exhibited through smoothening of cell surface by regulating expression of cell surface proteins. CONCLUSION: Tumor suppressor protein SMAR1 might be used as a phenotypic differentiation marker between cancerous and non-cancerous cells.
Subject(s)
Cell Cycle Proteins/metabolism , Cells/chemistry , Cells/metabolism , DNA-Binding Proteins/metabolism , Nuclear Proteins/metabolism , Animals , Cell Cycle Proteins/genetics , Cell Line, Tumor , Cell Size , Cells/cytology , Cytoskeleton/genetics , Cytoskeleton/metabolism , DNA-Binding Proteins/genetics , Humans , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mice , Microscopy, Atomic Force , Microscopy, Electron, Scanning , Nuclear Proteins/genetics , Surface PropertiesABSTRACT
The use of pharmacologically active short peptide sequences is a better option in cancer therapeutics than the full-length protein. Here we report one such 44-mer peptide sequence of SMAR1 (TAT-SMAR1 wild type, P44) that retains the tumor suppressor activity of the full-length protein. The protein transduction domain of human immunodeficiency virus, type 1, Tat protein was used here to deliver the 33-mer peptide of SMAR1 into the cells. P44 peptide could efficiently activate p53 by mediating its phosphorylation at serine 15, resulting in the activation of p21 and in effect regulating cell cycle checkpoint. In vitro phosphorylation assays with point-mutated P44-derived peptides suggested that serine 347 of SMAR1 was indispensable for its activity and represented the substrate motif for the protein kinase C family of proteins. Using xenograft nude mice models, we further demonstrate that P44 was capable of inhibiting tumor growth by preventing cellular proliferation. P44 treatment to tumor-bearing mice prevented the formation of poorly organized tumor vasculature and an increase in hypoxia-inducible factor-1alpha expression, both being signatures of tumor progression. The chimeric TAT-SMAR1-derived peptide, P44, thus has a strong therapeutic potential as an anticancer drug.