ABSTRACT
Reactive oxygen species (ROS) are implicated in triggering cell signalling events and pathways to promote and maintain tumorigenicity. Chemotherapy and radiation can induce ROS to elicit cell death allows for targeting ROS pathways for effective anti-cancer therapeutics. Coenzyme Q10 is a critical cofactor in the electron transport chain with complex biological functions that extend beyond mitochondrial respiration. This study demonstrates that delivery of oxidized Coenzyme Q10 (ubidecarenone) to increase mitochondrial Q-pool is associated with an increase in ROS generation, effectuating anti-cancer effects in a pancreatic cancer model. Consequent activation of cell death was observed in vitro in pancreatic cancer cells, and both human patient-derived organoids and tumour xenografts. The study is a first to demonstrate the effectiveness of oxidized ubidecarenone in targeting mitochondrial function resulting in an anti-cancer effect. Furthermore, these findings support the clinical development of proprietary formulation, BPM31510, for treatment of cancers with high ROS burden with potential sensitivity to ubidecarenone.
Subject(s)
Apoptosis , Mitochondria/metabolism , Pancreatic Neoplasms/pathology , Reactive Oxygen Species/metabolism , Ubiquinone/analogs & derivatives , Animals , Cell Line, Tumor , Cell Proliferation , Cell Respiration , Cell Survival , Electron Transport Complex II/metabolism , Glycerol-3-Phosphate Dehydrogenase (NAD+) , Humans , Membrane Potential, Mitochondrial , Mice, Nude , Organoids/pathology , Oxidative Stress , Oxygen Consumption , Pancreatic Neoplasms/metabolism , Substrate Specificity , Ubiquinone/metabolismABSTRACT
Metabolic reprogramming in cancer cells, vs. non-cancer cells, elevates levels of reactive oxygen species (ROS) leading to higher oxidative stress. The elevated ROS levels suggest a vulnerability to excess prooxidant loads leading to selective cell death, a therapeutically exploitable difference. Co-enzyme Q10 (CoQ10) an endogenous mitochondrial resident molecule, plays an important role in mitochondrial redox homeostasis, membrane integrity, and energy production. BPM31510 is a lipid-drug conjugate nanodispersion specifically formulated for delivery of supraphysiological concentrations of ubidecarenone (oxidized CoQ10) to the cell and mitochondria, in both in vitro and in vivo model systems. In this study, we sought to investigate the therapeutic potential of ubidecarenone in the highly treatment-refractory glioblastoma. Rodent (C6) and human (U251) glioma cell lines, and non-tumor human astrocytes (HA) and rodent NIH3T3 fibroblast cell lines were utilized for experiments. Tumor cell lines exhibited a marked increase in sensitivity to ubidecarenone vs. non-tumor cell lines. Further, elevated mitochondrial superoxide production was noted in tumor cells vs. non-tumor cells hours before any changes in proliferation or the cell cycle could be detected. In vitro co-culture experiments show ubidecarenone differentially affecting tumor cells vs. non-tumor cells, resulting in an equilibrated culture. In vivo activity in a highly aggressive orthotopic C6 glioma model demonstrated a greater than 25% long-term survival rate. Based on these findings we conclude that high levels of ubidecarenone delivered using BPM31510 provide an effective therapeutic modality targeting cancer-specific modulation of redox mechanisms for anti-cancer effects.
Subject(s)
Drug Delivery Systems , Glioma/pathology , Lipids/chemistry , Nanoparticles/chemistry , Pharmaceutical Preparations/chemistry , Ubiquinone/analogs & derivatives , Animals , Cell Cycle Checkpoints/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Female , Glioma/drug therapy , Humans , Mice , NIH 3T3 Cells , Oxidation-Reduction , Rats, Wistar , Superoxides/metabolism , Ubiquinone/administration & dosage , Ubiquinone/pharmacology , Ubiquinone/therapeutic useABSTRACT
Mechanisms governing the distinct temporal dynamics that characterize post-natal angiogenesis and lymphangiogenesis elicited by cutaneous wounds and inflammation remain unclear. RhoB, a stress-induced small GTPase, modulates cellular responses to growth factors, genotoxic stress and neoplastic transformation. Here we show, using RhoB null mice, that loss of RhoB decreases pathological angiogenesis in the ischaemic retina and reduces angiogenesis in response to cutaneous wounding, but enhances lymphangiogenesis following both dermal wounding and inflammatory challenge. We link these unique and opposing roles of RhoB in blood versus lymphatic vasculatures to the RhoB-mediated differential regulation of sprouting and proliferation in primary human blood versus lymphatic endothelial cells. We demonstrate that nuclear RhoB-GTP controls expression of distinct gene sets in each endothelial lineage by regulating VEZF1-mediated transcription. Finally, we identify a small-molecule inhibitor of VEZF1-DNA interaction that recapitulates RhoB loss in ischaemic retinopathy. Our findings establish the first intra-endothelial molecular pathway governing the phased response of angiogenesis and lymphangiogenesis following injury.
Subject(s)
Kruppel-Like Transcription Factors/genetics , Kruppel-Like Transcription Factors/metabolism , Lymphangiogenesis , Neovascularization, Pathologic , Retinal Diseases/physiopathology , rhoB GTP-Binding Protein/physiology , Animals , Animals, Newborn , Cell Lineage/genetics , DNA-Binding Proteins , Endothelial Cells/pathology , Endothelial Cells/physiology , Female , Gene Expression Regulation , Inflammation/genetics , Inflammation/physiopathology , Lymphangiogenesis/genetics , Male , Mice , Neovascularization, Pathologic/genetics , Retinal Diseases/genetics , Retinal Diseases/pathology , Transcription Factors , Wound Healing/genetics , Wound Healing/physiology , rhoB GTP-Binding Protein/geneticsABSTRACT
Tumors are composed of cancer cells but also a larger number of diverse stromal cells in the tumor microenvironment. Stromal cells provide essential supports to tumor pathophysiology but the distinct characteristics of their signaling networks are not usually considered in developing drugs to target tumors. This oversight potentially confounds proof-of-concept studies and increases drug development risks. Here, we show in established murine and human models of breast cancer how differential regulation of Akt by the small GTPase RhoB in cancer cells or stromal endothelial cells determines their dormancy versus outgrowth when angiogenesis becomes critical. In cancer cells in vitro or in vivo, RhoB functions as a tumor suppressor that restricts EGF receptor (EGFR) cell surface occupancy as well as Akt signaling. However, after activation of the angiogenic switch, RhoB functions as a tumor promoter by sustaining endothelial Akt signaling, growth, and survival of stromal endothelial cells that mediate tumor neoangiogenesis. Altogether, the positive impact of RhoB on angiogenesis and progression supercedes its negative impact in cancer cells themselves. Our findings elucidate the dominant positive role of RhoB in cancer. More generally, they illustrate how differential gene function effects on signaling pathways in the tumor stromal component can complicate the challenge of developing therapeutics to target cancer pathophysiology.
Subject(s)
Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cell Transformation, Neoplastic/metabolism , Endothelial Cells/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Stromal Cells/metabolism , rhoB GTP-Binding Protein/metabolism , Animals , Carcinoma, Ductal, Breast/metabolism , Carcinoma, Ductal, Breast/pathology , Female , Flow Cytometry , Gene Expression Regulation, Neoplastic , Humans , Immunoblotting , Immunohistochemistry , Immunoprecipitation , In Situ Hybridization , Mice , Mice, Transgenic , Neovascularization, Pathologic/metabolism , Real-Time Polymerase Chain Reaction , Tumor Microenvironment/physiologyABSTRACT
RhoB is an early-response gene whose expression is elevated by multiple cellular stresses; this gene plays an important role in cancer, macrophage motility, and apoptosis. These factors are essential for the onset of type 1 diabetes mellitus and related complications. This study explores the role of RhoB in ß-cell depletion and hyperglycemia-associated complications and tests whether the pleiotropic effect of statins on glycemic control is RhoB dependent. We induced ß-cell depletion in RhoB(+/+), RhoB(+/-), and RhoB(-/-) mice with streptozotocin (STZ). Diabetic status was assessed by glucose tolerance and pancreatic islet loss. RhoB(-/-) mice showed a significant reduction in the severity of STZ-induced diabetes; only 13% of the STZ-treated RhoB-null animals became hyperglycemic, as opposed to 61% of the wild-type controls. Diabetes-related complications, such as wound healing rate and onset of nephropathy, were also assessed. Hyperglycemic RhoB(-/-) mice had fewer signs of nephropathy and showed faster wound healing than RhoB(+/+) animals. After assessing the diabetic status of mice treated simultaneously with STZ and simvastatin, we conclude that the effect of statins in improving glycemic control is RhoB independent. We propose that RhoB is a modifier of diabetes, important for the induction of ß-cell loss. Suppression of RhoB expression may have potential application in the treatment of diabetes and associated complications.
Subject(s)
Diabetes Mellitus, Experimental/genetics , Diabetes Mellitus, Type 1/genetics , Insulin-Secreting Cells/enzymology , rhoB GTP-Binding Protein/genetics , Animals , Diabetes Mellitus, Experimental/complications , Diabetes Mellitus, Experimental/pathology , Diabetes Mellitus, Type 1/complications , Diabetes Mellitus, Type 1/pathology , Diabetic Nephropathies/genetics , Hydroxymethylglutaryl-CoA Reductase Inhibitors/therapeutic use , Hyperglycemia/drug therapy , Hyperglycemia/enzymology , Insulin-Secreting Cells/pathology , Mice , Mice, Mutant Strains , Wound Healing/geneticsABSTRACT
Bacterial diarrheagenic heat-stable enterotoxins induce colon cancer cell cytostasis by targeting guanylyl cyclase C (GCC) signaling. Anticancer actions of these toxins are mediated by cyclic guanosine 3',5'-monophosphate (cGMP)-dependent influx of Ca2+ through cyclic nucleotide-gated channels. However, prolonged stimulation of GCC produces resistance in tumor cells to heat-stable enterotoxin-induced cytostasis. Resistance reflects rapid (tachyphylaxis) and slow (bradyphylaxis) mechanisms of desensitization induced by cGMP. Tachyphylaxis is mediated by cGMP-dependent protein kinase, which limits the conductance of cyclic nucleotide-gated channels, reducing the influx of Ca2+ propagating the antiproliferative signal from the membrane to the nucleus. In contrast, bradyphylaxis is mediated by cGMP-dependent allosteric activation of phosphodiesterase 5, which shapes the amplitude and duration of heat-stable enterotoxin-dependent cyclic nucleotide accumulation required for cytostasis. Importantly, interruption of tachyphylaxis and bradyphylaxis restores cancer cell cytostasis induced by heat-stable enterotoxins. Thus, regimens that incorporate cytostatic bacterial enterotoxins and inhibitors of cGMP-mediated desensitization offer a previously unrecognized therapeutic paradigm for treatment and prevention of colorectal cancer.
Subject(s)
Bacterial Toxins/pharmacology , Colonic Neoplasms/drug therapy , Colonic Neoplasms/metabolism , Cyclic GMP-Dependent Protein Kinases/metabolism , Cyclic GMP/metabolism , Enterotoxins/pharmacology , Phosphoric Diester Hydrolases/metabolism , 3',5'-Cyclic-GMP Phosphodiesterases , Calcium/metabolism , Cell Line, Tumor , Colonic Neoplasms/enzymology , Colonic Neoplasms/pathology , Cyclic GMP/analogs & derivatives , Cyclic GMP/pharmacology , Cyclic Nucleotide Phosphodiesterases, Type 5 , Escherichia coli Proteins , Guanylate Cyclase/metabolism , Humans , Receptors, Enterotoxin , Receptors, Guanylate Cyclase-Coupled , Receptors, Peptide/metabolism , Signal TransductionABSTRACT
Guanylyl cyclase C and accumulation of cGMP induced by bacterial heat-stable enterotoxins (STs) promote colon cancer cell cytostasis, serving as a tumor suppressor in intestine. Conversely, capacitative calcium entry through store-operated calcium channels (SOCs) is a key signaling mechanism that promotes colon cancer cell proliferation. The present study revealed that proliferative signaling by capacitative calcium entry through SOCs opposes and is reciprocally coupled to cytostasis mediated by guanylyl cyclase C in T84 human colon carcinoma cells. Elimination of capacitative calcium entry employing 2-aminoethoxydiphenylborate (2-APB), a selective inhibitor of SOCs, potentiated cytostasis induced by ST. Opposition of ST-induced cytostasis by capacitative calcium entry reflects reciprocal inhibition of guanylyl cyclase C signaling. Calcium entry through SOCs induced by the calcium-ATPase inhibitor thapsigargin or the receptor agonists UTP or carbachol inhibited guanylyl cyclase C-dependent cGMP accumulation. This effect was mimicked by the calcium ionophore ionomycin and blocked by 2-APB and intracellular 1,2-bis(o-amino-5,5'-dibromophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester (BAPTA-AM), a chelator of calcium. Moreover, regulation by capacitative calcium entry reflected ligand-dependent sensitization of guanylyl cyclase C to inhibition by that cation. Although basal catalytic activity was refractory, ST-stimulated guanylyl cyclase C was inhibited by calcium, which antagonized binding of magnesium to allosteric sites required for receptor-effector coupling. These observations demonstrate that reciprocal regulation of guanylyl cyclase C signaling by capacitative calcium entry through SOCs represents one limb of a coordinated mechanism balancing colon cancer cell proliferation and cytostasis. They suggest that combining guanylyl cyclase C agonists and SOC inhibitors offers a novel paradigm for cGMP-directed therapy and prevention for colorectal tumors.
Subject(s)
Bacterial Toxins/pharmacology , Calcium Channels/physiology , Colonic Neoplasms/pathology , Enterotoxins/pharmacology , Calcium/metabolism , Cell Line, Tumor , Cell Proliferation/drug effects , Colonic Neoplasms/drug therapy , Cyclic GMP/physiology , Escherichia coli Proteins , Guanylate Cyclase/physiology , Humans , Signal TransductionABSTRACT
Calcium and guanosine-3',5'-cyclic monophosphate (cGMP) are second messenger molecules that regulate opposing physiological functions, reflected in the reciprocal regulation of their intracellular concentrations, in many systems. Indeed, cGMP and Ca2+ constitute discrete points of integration between multiple cell signaling cascades in both convergent and parallel pathways. This chapter describes the molecular mechanisms regulating intracellular Ca2+ and cGMP, and their integration in specific cellular responses.
Subject(s)
Calcium Signaling/physiology , Calcium/physiology , Cyclic GMP/physiology , Animals , HumansABSTRACT
The purpose of the present study was to characterize different beta-adrenoceptors (beta-ARs) and determine their role in the spontaneously tonic smooth muscle of the internal anal sphincter (IAS). The beta-AR subtypes in the opossum IAS were investigated by functional in vitro, radioligand binding, Western blot, and reverse transcription-polymerase chain reaction (RT-PCR) studies. ZD 7114 [(S)-4-[2-hydroxy-3-phenoxypropylaminoethoxy]-N-(2-methoxyethyl)phenoxyacetamide], a selective beta(3)-AR agonist, caused a potent and concentration-dependent relaxation of the IAS smooth muscle that was antagonized by the beta(3)-AR antagonist SR 59230A [1-(2-ethylphenoxy)-3-[[(1S)-1,2,3,4-tetrahydro-1-naphthalenyl]amino]-(2S)-2-propanol hydrochloride]. Conversely, the IAS smooth muscle relaxation caused by beta(1)- and beta(2)-AR agonists (xamoterol and procaterol, respectively) was selectively antagonized by their respective antagonists CGP 20712 [(+/-)-2-hydroxy-5-[2-[[2-hydroxy-3-[4-[1-methyl-4-(trifluoromethyl)-1H-imidazol-2-yl]phenoxy]propyl]amino]ethoxy]-benzamide methanesulfonate salt] and ICI 118551. Saturation binding of [(125)I]iodocyanopindolol to beta-AR subtypes revealed the presence of a high-affinity site (K(d1) = 96.4 +/- 8.7 pM; B(max1) = 12.5 +/- 0.6 fmol/mg protein) and a low-affinity site (K(d2) = 1.96 +/- 1.7 nM; B(max2) = 58.7 +/- 4.3 fmol/mg protein). Competition binding with selective beta-AR antagonists revealed that the high-affinity site correspond to beta(1)/beta(2)-AR and the low affinity site to beta(3)-AR. Receptor binding data suggest the predominant presence of beta(3)-AR over beta(1)/beta(2)-AR. Western blot studies identified beta(1)-, beta(2)-, and beta(3)-AR subtypes. The presence of beta(1)-, beta(2)-, and beta(3)-ARs was further demonstrated by mRNA analysis using RT-PCR. The studies demonstrate a comprehensive functional and molecular characterization of beta(1)-, beta(2)-, and beta(3)-ARs in IAS smooth muscle. These studies may have important implications in anorectal and other gastrointestinal motility disorders.
Subject(s)
Anal Canal/chemistry , Anal Canal/drug effects , Opossums/physiology , Receptors, Adrenergic, beta/chemistry , Receptors, Adrenergic, beta/drug effects , Adrenergic beta-Agonists/pharmacology , Adrenergic beta-Antagonists , Animals , Blotting, Western , In Vitro Techniques , Iodocyanopindolol , Isometric Contraction/drug effects , Male , Membranes/metabolism , Muscle Contraction/drug effects , Muscle Relaxation/drug effects , Muscle, Smooth/drug effects , Phenoxyacetates/pharmacology , Phenoxypropanolamines , Procaterol/pharmacology , RNA, Messenger/biosynthesis , Radioligand Assay , Receptors, Adrenergic, beta/biosynthesis , Reverse Transcriptase Polymerase Chain Reaction , Xamoterol/pharmacologyABSTRACT
Nitric oxide (NO), the principal endogenous ligand for soluble guanylate cyclase (sGC), stimulates that enzyme and accumulation of intracellular cGMP, which mediates many of the (patho) physiological effects of NO. Previous studies demonstrated that 2-substituted adenine nucleotides, including 2-methylthioATP (2MeSATP) and 2-chloroATP (2ClATP), allosterically inhibit guanylate cyclase C, the membrane-bound receptor for the Escherichia coli heat-stable enterotoxin in the intestine. The present study examined the effects of 2-substituted adenine nucleotides on crude and purified sGC. 2-Substituted nucleotides inhibited basal and NO-activated crude and purified sGC, when Mg2+ served as the substrate cation cofactor. Similarly, 2-substituted adenine nucleotides inhibited those enzymes when Mn2+, which activates sGC in a ligand-independent fashion, served as the substrate cation cofactor. Inhibition of sGC by 2-substituted nucleotides was associated with a decrease in Vmax, consistent with a noncompetitive mechanism. In contrast to guanylate cyclase C, 2-substituted nucleotides inhibited sGC by a guanine nucleotide-independent mechanism. These studies demonstrate that 2-substituted adenine nucleotides allosterically inhibit basal and ligand-stimulated sGC. They support the suggestion that allosteric inhibition by adenine nucleotides is a general characteristic of the family of guanylate cyclases. This allosteric inhibition is mediated by direct interaction of adenine nucleotides with sGC, likely at the catalytic domain in a region outside the substrate-binding site.
Subject(s)
Adenine Nucleotides/chemistry , Guanylate Cyclase/antagonists & inhibitors , Guanylate Cyclase/chemistry , Adenine Nucleotides/metabolism , Allosteric Regulation , Cell Line , Humans , Kinetics , Manganese/metabolism , Nitric Oxide/metabolismABSTRACT
Cyclic GMP (cGMP) and Ca(2+) regulate opposing mechanisms in (patho)physiological processes reflected in the reciprocal regulation of their intracellular concentrations. Although mechanisms by which cGMP regulates [Ca(2+)](i) have been described, those by which Ca(2+) regulates [cGMP](i) are less well understood. In the present study, Ca(2+) inhibited purified sGC activated by sodium nitroprusside (SNP), a precursor of nitric oxide (NO), employing Mg-GTP as substrate in a concentration-dependent fashion, but was without effect on basal enzyme activity. Ca(2+) inhibited sGC stimulated by protoporphyrin IX or YC-1 suggesting that inhibition was not NO-dependent. In contrast, Ca(2+) was without effect on sGC activated by SNP employing Mn-GTP as substrate, demonstrating that inhibition did not reflect displacement of heme from sGC. Ligand activation of sGC unmasked negative allosteric sites of high (K(i) similar 10(-7) M) and low (K(i) approximately 10(-5) M) affinity for Ca(2+) that mediated noncompetitive and uncompetitive inhibition, respectively. Free Mg(2+) in excess of substrate did not alter the concentration-response relationship of Ca(2+) inhibition at high affinity sites, but produced a rightward shift in that relationship at low affinity sites. Similarly, Ca(2+) inhibition at high affinity sites was noncompetitive, whereas inhibition at low affinity sites was competitive, with respect to free Mg(2+). Purified sGC specifically bound (45)Ca(2+) in the presence of a 1000-fold excess of Mg(2+) and in the absence of activating ligands. These data suggest that sGC is a constitutive Ca(2+) binding protein whose allosteric function is conditionally dependent upon ligand activation.