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1.
Front Immunol ; 15: 1429946, 2024.
Article in English | MEDLINE | ID: mdl-38947318

ABSTRACT

Introduction: Chronic obstructive pulmonary disease (COPD) is currently listed as the 3rd leading cause of death in the United States. Accumulating data shows the association between COPD occurrence and the usage of electronic nicotine delivery systems (ENDS) in patients. However, the underlying pathogenesis mechanisms of COPD have not been fully understood. Methods: In the current study, bENaC-overexpressing mice (bENaC mice) were subjected to whole-body ENDS exposure. COPD related features including emphysema, mucus accumulation, inflammation and fibrosis are examined by tissue staining, FACS analysis, cytokine measurement. Cell death and ferroptosis of alveolar epithelial cells were further evaluated by multiple assays including staining, FACS analysis and lipidomics. Results: ENDS-exposed mice displayed enhanced emphysema and mucus accumulation, suggesting that ENDS exposure promotes COPD features. ENDS exposure also increased immune cell number infiltration in bronchoalveolar lavage and levels of multiple COPD-related cytokines in the lungs, including CCL2, IL-4, IL-13, IL-10, M-CSF, and TNF-α. Moreover, we observed increased fibrosis in ENDS-exposed mice, as evidenced by elevated collagen deposition and a-SMA+ myofibroblast accumulation. By investigating possible mechanisms for how ENDS promoted COPD, we demonstrated that ENDS exposure induced cell death of alveolar epithelial cells, evidenced by TUNEL staining and Annexin V/PI FACS analysis. Furthermore, we identified that ENDS exposure caused lipid dysregulations, including TAGs (9 species) and phospholipids (34 species). As most of these lipid species are highly associated with ferroptosis, we confirmed ENDS also enhanced ferroptosis marker CD71 in both type I and type II alveolar epithelial cells. Discussion: Overall, our data revealed that ENDS exposure exacerbates features of COPD in bENaC mice including emphysema, mucus accumulation, abnormal lung inflammation, and fibrosis, which involves the effect of COPD development by inducing ferroptosis in the lung.


Subject(s)
E-Cigarette Vapor , Ferroptosis , Nicotine , Pulmonary Disease, Chronic Obstructive , Animals , Pulmonary Disease, Chronic Obstructive/chemically induced , Pulmonary Disease, Chronic Obstructive/pathology , Pulmonary Disease, Chronic Obstructive/metabolism , Pulmonary Disease, Chronic Obstructive/etiology , Mice , Nicotine/adverse effects , Nicotine/toxicity , Nicotine/administration & dosage , E-Cigarette Vapor/adverse effects , Disease Models, Animal , Cytokines/metabolism , Mice, Inbred C57BL , Electronic Nicotine Delivery Systems , Male , Mice, Transgenic
2.
Chem Biomed Imaging ; 1(3): 268-285, 2023 Jun 26.
Article in English | MEDLINE | ID: mdl-37388961

ABSTRACT

Chronic lung diseases, such as idiopathic pulmonary fibrosis (IPF) and chronic obstructive pulmonary disease (COPD), are major leading causes of death worldwide and are generally associated with poor prognoses. The heterogeneous distribution of collagen, mainly type I collagen associated with excessive collagen deposition, plays a pivotal role in the progressive remodeling of the lung parenchyma to chronic exertional dyspnea for both IPF and COPD. To address the pressing need for noninvasive early diagnosis and drug treatment monitoring of pulmonary fibrosis, we report the development of human collagen-targeted protein MRI contrast agent (hProCA32.collagen) to specifically bind to collagen I overexpressed in multiple lung diseases. When compared to clinically approved Gd3+ contrast agents, hProCA32.collagen exhibits significantly better r1 and r2 relaxivity values, strong metal binding affinity and selectivity, and transmetalation resistance. Here, we report the robust detection of early and late-stage lung fibrosis with stage-dependent MRI signal-to-noise ratio (SNR) increase, with good sensitivity and specificity, using a progressive bleomycin-induced IPF mouse model. Spatial heterogeneous mapping of usual interstitial pneumonia (UIP) patterns with key features closely mimicking human IPF, including cystic clustering, honeycombing, and traction bronchiectasis, were noninvasively detected by multiple MR imaging techniques and verified by histological correlation. We further report the detection of fibrosis in the lung airway of an electronic cigarette-induced COPD mouse model, using hProCA32.collagen-enabled precision MRI (pMRI), and validated by histological analysis. The developed hProCA32.collagen is expected to have strong translational potential for the noninvasive detection and staging of lung diseases, and facilitating effective treatment to halt further chronic lung disease progression.

3.
J Cancer ; 12(21): 6543-6552, 2021.
Article in English | MEDLINE | ID: mdl-34659545

ABSTRACT

Aberrant expression of P68 RNA helicase (p68), a prototypical member of the DEAD box family of RNA helicases, contributes to tumor development and progression. P68 tyrosine phosphorylation induced by PDGF signaling facilitates cancer metastasis by promoting EMT. In this report, we show that p68 promotes breast cancer cell EMT and cell migration by upregulation of PDGF receptor ß (PDGFR-ß). Knockdown of p68 in MDA-MB-231 and BT549 cells significantly decreases PDGFR-ß both in mRNA and protein levels. P68 promotes EMT and cell migration in response to PDGF-BB stimulation via upregulation of PDGFR-ß, suggesting that p68 enhances PDGF signaling by a positive feedback loop in cancer cells. Furthermore, our study reveals that p68 mediates the effects of PDGFR-ß in regulation of androgen receptor (AR) in breast cancer cells. We demonstrate that p68 and PDGFR-ß co-regulate AR expression and promote androgen-mediated proliferation in breast cancer cells. Our studies uncover an important pathway of p68-PDGFR-ß axis in promoting breast cancer progression.

4.
iScience ; 24(10): 103165, 2021 Oct 22.
Article in English | MEDLINE | ID: mdl-34693222

ABSTRACT

Persistent activation of fibroblasts and resistance of myofibroblasts to turnover play important roles in organ-tissue fibrosis development and progression. The mechanism that mediates apoptosis resistance of myofibroblasts is not understood. Here, we report that myofibroblasts express and secrete PKM2. Extracellular PKM2 (EcPKM2) facilitates progression of fibrosis by protecting myofibroblasts from apoptosis. EcPKM2 upregulates arginase-1 expression in myofibroblasts and therefore facilitates proline biosynthesis and subsequent collagen production. EcPKM2 interacts with integrin αvß3 on myofibroblasts to activate FAK-PI3K signaling axis. Activation of FAK-PI3K by EcPKM2 activates downstream NF-κB survival pathway to prevent myofibroblasts from apoptosis. On the other hand, activation of FAK- PI3K by EcPKM2 suppresses PTEN to subsequently upregulate arginase-1 in myofibroblasts. Our studies uncover an important mechanism for organ fibrosis progression. More importantly, an antibody disrupting the interaction between PKM2 and integrin αvß3 is effective in reversing fibrosis, suggesting a possible therapeutic strategy and target for treatment of organ fibrosis.

5.
Theranostics ; 11(19): 9331-9341, 2021.
Article in English | MEDLINE | ID: mdl-34646373

ABSTRACT

Rationale: Fibrosis is a pathologic condition of abnormal accumulation of collagen fibrils. Collagen is a major extracellular matrix (ECM) protein synthesized and secreted by myofibroblasts, composing mainly (Gly-X-Y)n triplet repeats with >30% Gly residue. During fibrosis progression, myofibroblasts must upregulate glycine metabolism to meet the high demands of amino acids for collagen synthesis. Method: Expression of PKM2 in myofibroblasts was analyzed in cultured fibroblasts and fibrosis disease tissues. Functional roles of PKM2 and PKM2 activator in biosynthesis of serine → glycine and production of collagen from glycolysis intermediates were assayed in cultured activated LX-2 and human primary lung fibroblast cells. Mouse models of Liver, lung, and pancreas fibrosis were employed to analyze treatment effects of PKM2 activator in organ tissue fibrosis. Results: We report here that myofibroblast differentiation upregulates pyruvate kinase M2 (PKM2) and promotes dimerization of PKM2. Dimer PKM2 slows the flow rate of glycolysis and channels glycolytic intermediates to de novo glycine synthesis, which facilitates collagen synthesis and secretion in myofibroblasts. Our results show that PKM2 activator that converts PKM2 dimer to tetramer, inhibits fibrosis progression in mouse models of liver, lung, and pancreatic fibrosis. Furthermore, metabolism alteration by dimer PKM2 increases NADPH production, which consequently protects myofibroblasts from apoptosis. Conclusion: Our study uncovers a novel role of PKM2 in tissue/organ fibrosis, suggesting a possible strategy for treatment of fibrotic diseases using PKM2 activator.


Subject(s)
Fibrosis/metabolism , Glycine/metabolism , Pyruvate Kinase/metabolism , Animals , Apoptosis , Cell Differentiation , Collagen/metabolism , Extracellular Matrix/metabolism , Female , Fibroblasts/metabolism , Fibrosis/physiopathology , Glycine/physiology , Glycolysis/drug effects , Humans , Liver/pathology , Lung/pathology , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Myofibroblasts/metabolism , Myofibroblasts/physiology , Pancreas/pathology , Pyruvate Kinase/physiology , Signal Transduction
6.
Commun Biol ; 4(1): 1087, 2021 09 16.
Article in English | MEDLINE | ID: mdl-34531529

ABSTRACT

Chronic Liver Diseases (CLD) are characterized by abnormal accumulation of collagen fibrils, neo-angiogenesis, and sinusoidal remodeling. Collagen deposition along with intrahepatic angiogenesis and sinusoidal remodeling alters sinusoid structure resulting in portal hypertension, liver failure, and other complications. Efforts were made to develop treatments for CLDs. However, the success of such treatments is limited and unpredictable. We report a strategy for CLD treatment by induction of integrin αvß3 mediated cell apoptosis using a rationally designed protein (ProAgio). ProAgio is designed to target integrin αvß3 at a novel site. Integrin αvß3 is highly expressed in activated Hepatic Stellate Cells (HSC), angiogenic endothelium, and capillarized Liver Sinusoidal Endothelial Cells (LSEC). ProAgio induces apoptosis of these disease causative cells. Tests with liver fibrosis mouse models demonstrate that ProAgio reverses liver fibrosis and relieves blood flow resistance by depleting activated HSC and capillarized LSEC. Our studies demonstrate an effective approach for CLD treatment.


Subject(s)
Apoptosis , Integrin alphaVbeta3/chemistry , Liver Diseases/therapy , Protein Engineering , Animals , Chronic Disease/therapy , Disease Models, Animal , Mice
7.
Front Pharmacol ; 12: 726586, 2021.
Article in English | MEDLINE | ID: mdl-34393802

ABSTRACT

Although a few studies show that the use of electronic nicotine delivery systems (ENDS) may ameliorate objective and subjective outcomes in COPD smokers who switched to electronic cigarettes, it is unclear whether e-cigarette exposure alters lung pathological features and inflammatory response in COPD. Here, we employed ßENaC-overexpressing mice bearing COPD-like pulmonary abnormality, and exposed them to ENDS. We found that ENDS exposure aggravated airspace enlargement and mucus production in ßENaC-overexpressing mice, which was associated with increased MMP12 and Muc5ac, respectively. ENDS exposure to mice significantly increased the numbers of macrophages, particularly in M2 macrophages in bronchoalveolar lavage (BAL) fluid, despite ENDS did not induce M2 macrophage polarization in a cultured murine macrophage cell line (RAW264.7). There were no changes in neutrophils in BAL fluid by ENDS exposure. Multiple cytokine productions were increased including M-CSF, IL-1r α , IL-10, and TGF-ß1, in BAL fluid from mice when exposed to ENDS. The Sirius Red staining and hydroxyproline assay showed ENDS-exposed mice displayed enhanced fibrotic phenotypes compared to control mice. In conclusion, ENDS exposure enhances airspace enlargement, mucus secretion, and fibrogenesis in COPD mice. This is associated with increased MMP12, inflammatory responses, and M2 macrophage phenotype. This study provides pre-clinical data implicating that electronic cigarette exposure is not safe in COPD patients who want to replace traditional cigarettes with ENDS.

8.
J Exp Med ; 218(4)2021 04 05.
Article in English | MEDLINE | ID: mdl-33561195

ABSTRACT

Fibrotic tumor stroma plays an important role in facilitating triple-negative breast cancer (TNBC) progression and chemotherapeutic resistance. We previously reported a rationally designed protein (ProAgio) that targets integrin αvß3 at a novel site. ProAgio induces apoptosis via the integrin. Cancer-associated fibroblasts (CAFs) and angiogenic endothelial cells (aECs) in TNBC tumor express high levels of integrin αvß3. ProAgio effectively induces apoptosis in CAFs and aECs. The depletion of CAFs by ProAgio reduces intratumoral collagen and decreases growth factors released from CAFs in the tumor, resulting in decreased cancer cell proliferation and apoptotic resistance. ProAgio also eliminates leaky tumor angiogenic vessels, which consequently reduces tumor hypoxia and improves drug delivery. The depletion of CAFs and reduction in hypoxia by ProAgio decreases lysyl oxidase (LOX) secretion, which may play a role in the reduction of metastasis. ProAgio stand-alone or in combination with a chemotherapeutic agent provides survival benefit in TNBC murine models, highlighting the therapeutic potential of ProAgio as a treatment strategy.


Subject(s)
Angiogenesis Inhibitors/pharmacology , Cancer-Associated Fibroblasts/drug effects , Neovascularization, Pathologic/drug therapy , Triple Negative Breast Neoplasms/blood supply , Triple Negative Breast Neoplasms/drug therapy , Angiogenesis Inhibitors/therapeutic use , Animals , Antineoplastic Agents/therapeutic use , Apoptosis/drug effects , Cell Line, Tumor , Doxorubicin/therapeutic use , Endothelial Cells/drug effects , Endothelial Cells/metabolism , Female , Humans , Hypoxia/drug therapy , Integrin alphaVbeta3/antagonists & inhibitors , Mice , Mice, Inbred BALB C , Mice, Nude , Neovascularization, Pathologic/metabolism , Paclitaxel/therapeutic use , Protein-Lysine 6-Oxidase/metabolism , Signal Transduction/drug effects , Treatment Outcome , Triple Negative Breast Neoplasms/pathology , Tumor Burden/drug effects , Xenograft Model Antitumor Assays
9.
Cell Mol Gastroenterol Hepatol ; 11(1): 161-179, 2021.
Article in English | MEDLINE | ID: mdl-32810598

ABSTRACT

BACKGROUND & AIMS: Pancreatic ductal adenocarcinoma (PDAC) is resistant to most therapeutics owing to dense fibrotic stroma orchestrated by cancer-associated pancreatic stellate cells (CAPaSC). CAPaSC also support cancer cell growth, metastasis, and resistance to apoptosis. Currently, there is no effective therapy for PDAC that specifically targets CAPaSC. We previously reported a rationally designed protein, ProAgio, that targets integrin αvß3 at a novel site and induces apoptosis in integrin αvß3-expressing cells. Because both CAPaSC and angiogenic endothelial cells express high levels of integrin αvß3, we aimed to analyze the effects of ProAgio in PDAC tumor. METHODS: Expression of integrin αvß3 was examined in both patient tissue and cultured cells. The effects of ProAgio on CAPaSC were analyzed using an apoptosis assay kit. The effects of ProAgio in PDAC tumor were studied in 3 murine tumor models: subcutaneous xenograft, genetic engineered (KrasG12D; p53R172H; Pdx1-Cre, GEM-KPC) mice, and an orthotopic KrasG12D; p53R172H; Pdx1-Cre (KPC) model. RESULTS: ProAgio induces apoptosis in CAPaSC. ProAgio treatment significantly prolonged survival of a genetically engineered mouse-KPC and orthotopic KPC mice alone or in combination with gemcitabine (Gem). ProAgio specifically induced apoptosis in CAPaSC, resorbed collagen, and opened collapsed tumor vessels without an increase in angiogenesis in PDAC tumor, enabling drug delivery into the tumor. ProAgio decreased intratumoral insulin-like growth factor 1 levels as a result of depletion of CAPaSC and consequently decreased cytidine deaminase, a Gem metabolism enzyme in cancer cells, and thereby reduced resistance to Gem-induced apoptosis. CONCLUSIONS: Our study suggests that ProAgio is an effective PDAC treatment agent because it specifically depletes CAPaSC and eliminates tumor angiogenesis, thereby enhancing drug delivery and Gem efficacy in PDAC tumors.


Subject(s)
Antineoplastic Agents/pharmacology , Carcinoma, Pancreatic Ductal/drug therapy , Integrin alphaVbeta3/antagonists & inhibitors , Pancreatic Neoplasms/drug therapy , Pancreatic Stellate Cells/drug effects , Animals , Antineoplastic Agents/therapeutic use , Apoptosis/drug effects , Carcinoma, Pancreatic Ductal/genetics , Carcinoma, Pancreatic Ductal/pathology , Cell Line, Tumor , Disease Models, Animal , Humans , Integrin alphaVbeta3/analysis , Integrin alphaVbeta3/metabolism , Mice , Mice, Transgenic , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/pathology , Pancreatic Stellate Cells/pathology , Primary Cell Culture , Xenograft Model Antitumor Assays
10.
iScience ; 23(11): 101684, 2020 Nov 20.
Article in English | MEDLINE | ID: mdl-33196019

ABSTRACT

Cancer cells alter their nutrition metabolism to cope the stressful environment. One important metabolism adjustment is that cancer cells activate glutaminolysis in response to the reduced carbon from glucose entering into the TCA cycle due to inactivation of several enzymes in glycolysis. An important question is how the cancer cells coordinate the changes of glycolysis and glutaminolysis. In this report, we demonstrate that the pyruvate kinase inactive dimer PKM2 facilitates activation of glutaminolysis. Our experiments show that growth stimulations promote PKM2 dimer. The dimer PKM2 plays a role in regulation of glutaminolysis by upregulation of mitochondrial glutaminase I (GLS-1). PKM2 dimer regulates the GLS-1 expression by controlling internal ribosome entry site (IRES)-dependent c-myc translation. Growth stimulations promote PKM2 interacting with c-myc IRES-RNA, thus facilitating c-myc IRES-dependent translation. Our study reveals an important linker that coordinates the metabolism adjustment in cancer cells.

11.
Nat Commun ; 10(1): 4777, 2019 10 29.
Article in English | MEDLINE | ID: mdl-31664017

ABSTRACT

Early diagnosis and noninvasive detection of liver fibrosis and its heterogeneity remain as major unmet medical needs for stopping further disease progression toward severe clinical consequences. Here we report a collagen type I targeting protein-based contrast agent (ProCA32.collagen1) with strong collagen I affinity. ProCA32.collagen1 possesses high relaxivities per particle (r1 and r2) at both 1.4 and 7.0 T, which enables the robust detection of early-stage (Ishak stage 3 of 6) liver fibrosis and nonalcoholic steatohepatitis (Ishak stage 1 of 6 or 1 A Mild) in animal models via dual contrast modes. ProCA32.collagen1 also demonstrates vasculature changes associated with intrahepatic angiogenesis and portal hypertension during late-stage fibrosis, and heterogeneity via serial molecular imaging. ProCA32.collagen1 mitigates metal toxicity due to lower dosage and strong resistance to transmetallation and unprecedented metal selectivity for Gd3+ over physiological metal ions with strong translational potential in facilitating effective treatment to halt further chronic liver disease progression.


Subject(s)
Contrast Media/chemistry , Gadolinium/chemistry , Hypertension, Portal/diagnostic imaging , Liver/diagnostic imaging , Magnetic Resonance Imaging/methods , Chronic Disease , Early Diagnosis , Humans
12.
Mol Ther Oncolytics ; 14: 188-195, 2019 Sep 27.
Article in English | MEDLINE | ID: mdl-31312717

ABSTRACT

Despite reports of successful clinical cases, many tumors appear to resist infection by oncolytic viruses (OVs). To circumvent this problem, an armed vesicular stomatitis virus was constructed by inserting a transgene to express Smac/DIABLO during virus infection (VSV-S). Endogenous Smac in HeLa cells was diminished during wtVSV infection, whereas the Smac level was enhanced during VSV-S infection. Apoptosis was readily induced by VSV-S, but not wtVSV, infection. More importantly, the tumor volume was reduced to a larger extent when xenografts of 4T1 cells in BALB/c mice and OV-resistant T-47D cells in nude mice were intratumorally injected with VSV-S. VSV-S represents a novel mechanism to overcome tumor resistance, resulting in more significant tumor regression due to enhanced apoptosis.

14.
Neurotherapeutics ; 15(3): 770-784, 2018 07.
Article in English | MEDLINE | ID: mdl-29869055

ABSTRACT

Ischemic stroke remains a serious threat to human life. Generation of neuronal and vascular cells is an endogenous regenerative mechanism in the adult brain, which may contribute to tissue repair after stroke. However, the regenerative activity is typically insufficient for significant therapeutic effects after brain injuries. Pyruvate kinase isoform M2 (PKM2) is a key regulator for energy metabolism. PKM2 also has nonmetabolic roles involving regulations of gene expression, cell proliferation, and migration in cancer cells as well as noncancerous cells. In a focal ischemic stroke mouse model, recombinant PKM2 (rPKM2) administration (160 ng/kg, intranasal delivery) at 1 h after stroke showed the significant effect of a reduced infarct volume of more the 60%. Delayed treatment of rPKM2, however, lost the acute neuroprotective effect. We then tested a novel hypothesis that delayed treatment of PKM2 might show proregenerative effects for long-term functional recovery and this chronic action could be mediated by its downstream STAT3 signaling. rPKM2 (160 ng/kg) was delivered to the brain using noninvasive intranasal administration 24 h after the stroke and repeated every other day. Western blot analysis revealed that, 7 days after the stroke, the levels of PKM2 and phosphorylated STAT3 and the expression of angiogenic factors VEGF, Ang-1, and Tie-2 in the peri-infarct region were significantly increased in the rPKM2 treatment group compared with those of the stroke vehicle group. To label proliferating cells, 5-bromo-2'-deoxyuridine (BrdU, 50 mg/kg, i.p.) was injected every day starting 3 days after stroke. At 14 days after stroke, immunohistochemistry showed that rPKM2 increased cell homing of doublecortin (DCX)-positive neuroblasts to the ischemic cortex. In neural progenitor cell (NPC) cultures, rPKM2 (0.4-4 nM) increased the expression of integrin ß1 and the activation/phosphorylation of focal adhesion kinase (FAK). A mediator role of FAK in PKM2-promoted cell migration was verified in FAK-knockout fibroblast cultures. In the peri-infarct region of the brain, increased numbers of Glut-1/BrdU and NeuN/BrdU double-positive cells indicated enhanced angiogenesis and neurogenesis, respectively, compared to stroke vehicle mice. Using Laser Doppler imaging, we observed better recovery of the local blood flow in the peri-infarct region of rPKM2-treated mice 14 days after stroke. Meanwhile, rPKM2 improved the sensorimotor functional recovery measured by the adhesive removal test. Inhibiting the STAT3 phosphorylation/activation by the STAT3 inhibitor, BP-1-102 (3 mg/kg/day, o.g.), abolished all beneficial effects of rPKM2 in the stroke mice. Taken together, this investigation provides the first evidence demonstrating that early treatment of rPKM2 shows an acute neuroprotective effect against ischemic brain damage, whereas delayed rPKM2 treatment promotes regenerative activities in the poststroke brain leading to better functional recovery. The underlying mechanism involves activation of the STAT3 and FAK signals in the poststroke brain.


Subject(s)
Focal Adhesion Protein-Tyrosine Kinases/genetics , Infarction, Middle Cerebral Artery/drug therapy , Neovascularization, Physiologic/drug effects , Neurogenesis/drug effects , Pyruvate Kinase , Recovery of Function/drug effects , STAT3 Transcription Factor/genetics , Animals , Cell Movement/drug effects , Cells, Cultured , Cerebrovascular Circulation/drug effects , Disease Models, Animal , Doublecortin Protein , Fibroblasts/drug effects , Focal Adhesion Protein-Tyrosine Kinases/metabolism , Infarction, Middle Cerebral Artery/physiopathology , Mice , Mice, Inbred C57BL , Phosphopyruvate Hydratase/metabolism , Pyruvate Kinase/pharmacology , Pyruvate Kinase/therapeutic use , STAT3 Transcription Factor/metabolism , Stem Cells/drug effects , Up-Regulation/drug effects
15.
Nat Commun ; 7: 11675, 2016 05 31.
Article in English | MEDLINE | ID: mdl-27241473

ABSTRACT

Integrin αvß3 expression is altered in various diseases and has been proposed as a drug target. Here we use a rational design approach to develop a therapeutic protein, which we call ProAgio, that binds to integrin αvß3 outside the classical ligand-binding site. We show ProAgio induces apoptosis of integrin αvß3-expressing cells by recruiting and activating caspase 8 to the cytoplasmic domain of integrin αvß3. ProAgio also has anti-angiogenic activity and strongly inhibits growth of tumour xenografts, but does not affect the established vasculature. Toxicity analyses demonstrate that ProAgio is not toxic to mice. Our study reports a new integrin-targeting agent with a unique mechanism of action, and provides a template for the development of integrin-targeting therapeutics.


Subject(s)
Angiogenesis Inhibitors/pharmacology , Apoptosis/drug effects , Drug Design , Integrin alphaVbeta3/metabolism , Neovascularization, Pathologic/drug therapy , Amino Acid Sequence/genetics , Angiogenesis Inhibitors/chemistry , Angiogenesis Inhibitors/therapeutic use , Animals , Binding Sites/genetics , Cell Line , Female , Humans , Ligands , Male , Mice , Mice, Inbred BALB C , Mice, Nude , Molecular Docking Simulation , Molecular Targeted Therapy/methods , Neoplasms/blood supply , Neoplasms/drug therapy , Neoplasms/pathology , Neovascularization, Pathologic/pathology , Protein Binding/genetics , Protein Engineering/methods , Protein Structure, Tertiary , Signal Transduction/drug effects , Xenograft Model Antitumor Assays
16.
Wound Repair Regen ; 24(2): 328-36, 2016 03.
Article in English | MEDLINE | ID: mdl-26808610

ABSTRACT

Neutrophils infiltration/activation following wound induction marks the early inflammatory response in wound repair. However, the role of the infiltrated/activated neutrophils in tissue regeneration/proliferation during wound repair is not well understood. Here, we report that infiltrated/activated neutrophils at wound site release pyruvate kinase M2 (PKM2) by its secretive mechanisms during early stages of wound repair. The released extracellular PKM2 facilitates early wound healing by promoting angiogenesis at wound site. Our studies reveal a new and important molecular linker between the early inflammatory response and proliferation phase in tissue repair process.


Subject(s)
Neovascularization, Physiologic , Neutrophils/metabolism , Pyruvate Kinase/metabolism , Wound Healing/physiology , Wounds and Injuries/enzymology , Wounds and Injuries/pathology , Animals , Cell Proliferation , Disease Models, Animal , Extracellular Matrix/pathology , Immunohistochemistry , Inflammation/enzymology , Inflammation/pathology , Mice , Mice, Inbred C57BL , Neutrophils/enzymology
17.
Sci Rep ; 5: 16214, 2015 Nov 18.
Article in English | MEDLINE | ID: mdl-26577829

ABSTRACT

Gastrin-releasing peptide receptor (GRPR) is differentially expressed on the surfaces of various diseased cells, including prostate and lung cancer. However, monitoring temporal and spatial expression of GRPR in vivo by clinical MRI is severely hampered by the lack of contrast agents with high relaxivity, targeting capability and tumor penetration. Here, we report the development of a GRPR-targeted MRI contrast agent by grafting the GRPR targeting moiety into a scaffold protein with a designed Gd(3+) binding site (ProCA1.GRPR). In addition to its strong binding affinity for GRPR (Kd = 2.7 nM), ProCA1.GRPR has high relaxivity (r1 = 42.0 mM(-1)s(-1) at 1.5 T and 25 °C) and strong Gd(3+) selectivity over physiological metal ions. ProCA1.GRPR enables in vivo detection of GRPR expression and spatial distribution in both PC3 and H441 tumors in mice using MRI. ProCA1.GRPR is expected to have important preclinical and clinical implications for the early detection of cancer and for monitoring treatment effects.


Subject(s)
Contrast Media , Magnetic Resonance Imaging , Molecular Imaging , Neoplasms/diagnosis , Neoplasms/metabolism , Receptors, Bombesin/metabolism , Animals , Binding Sites , Biomarkers , Cell Line, Tumor , Contrast Media/chemistry , Contrast Media/metabolism , Contrast Media/pharmacokinetics , Disease Models, Animal , Gene Expression , Heterografts , Humans , Ligands , Magnetic Resonance Imaging/methods , Mice , Models, Molecular , Molecular Conformation , Molecular Imaging/methods , Neoplasms/genetics , Protein Binding , Rats , Receptors, Bombesin/chemistry , Receptors, Bombesin/genetics , Tissue Distribution
18.
Proc Natl Acad Sci U S A ; 112(21): 6607-12, 2015 May 26.
Article in English | MEDLINE | ID: mdl-25971726

ABSTRACT

With available MRI techniques, primary and metastatic liver cancers that are associated with high mortality rates and poor treatment responses are only diagnosed at late stages, due to the lack of highly sensitive contrast agents without Gd(3+) toxicity. We have developed a protein contrast agent (ProCA32) that exhibits high stability for Gd(3+) and a 10(11)-fold greater selectivity for Gd(3+) over Zn(2+) compared with existing contrast agents. ProCA32, modified from parvalbumin, possesses high relaxivities (r1/r2: 66.8 mmol(-1)⋅s(-1)/89.2 mmol(-1)⋅s(-1) per particle). Using T1- and T2-weighted, as well as T2/T1 ratio imaging, we have achieved, for the first time (to our knowledge), robust MRI detection of early liver metastases as small as ∼0.24 mm in diameter, much smaller than the current detection limit of 10-20 mm. Furthermore, ProCA32 exhibits appropriate in vivo preference for liver sinusoidal spaces and pharmacokinetics for high-quality imaging. ProCA32 will be invaluable for noninvasive early detection of primary and metastatic liver cancers as well as for monitoring treatment and guiding therapeutic interventions, including drug delivery.


Subject(s)
Contrast Media , Liver Neoplasms, Experimental/diagnosis , Liver Neoplasms, Experimental/metabolism , Magnetic Resonance Imaging/methods , Melanoma, Experimental/diagnosis , Melanoma, Experimental/metabolism , Parvalbumins , Animals , Cell Line, Tumor , Contrast Media/chemistry , Contrast Media/pharmacokinetics , Female , Gadolinium , Limit of Detection , Liver Neoplasms, Experimental/secondary , Mice , Mice, Inbred C57BL , Models, Molecular , Parvalbumins/chemistry , Parvalbumins/pharmacokinetics , Protein Engineering , Protein Stability , Recombinant Proteins/chemistry , Recombinant Proteins/pharmacokinetics
19.
J Cell Biochem ; 116(8): 1595-601, 2015 Aug.
Article in English | MEDLINE | ID: mdl-25649741

ABSTRACT

1-(3,5-Dimethoxyphenyl)-4-[(6-fluoro-2-methoxyquinoxalin-3-yl)aminocarbonyl] piperazine (RX-5902) exhibits strong growth inhibition in various human cancer cell lines with IC50 values ranging between 10 and 20 nM. In this study, we demonstrate that p68 RNA helicase is a cellular target of RX-5902 by the drug affinity responsive target stability (DARTS) method, and confirmed the direct binding of (3) H-labeled RX-5902 to Y593 phospho-p68 RNA helicase. We further demonstrated RX-5902 inhibited the ß-catenin dependent ATPase activity of p68 RNA helicase in an in vitro system. Furthermore, we showed that treatment of cancer cells with RX-5902 resulted in the downregulation of the expression of certain genes, which are known to be regulated by the ß-catenin pathway, such as c-Myc, cyclin D1 and p-c-Jun. Therefore, our study indicates that the inhibition of Y593 phospho-p68 helicase - ß-catenin interaction by direct binding of RX-5902 to Y593 phospho-p68 RNA helicase may contribute to the anti-cancer activity of this compound.


Subject(s)
Antineoplastic Agents/pharmacology , DEAD-box RNA Helicases/metabolism , Neoplasms/drug therapy , Piperazines/pharmacology , Quinoxalines/pharmacology , beta Catenin/metabolism , Binding Sites/drug effects , Cell Line, Tumor , DEAD-box RNA Helicases/chemistry , Humans , Neoplasms/metabolism , Phosphorylation , Protein Binding/drug effects , Signal Transduction/drug effects
20.
J Biol Chem ; 289(37): 25812-21, 2014 Sep 12.
Article in English | MEDLINE | ID: mdl-25070887

ABSTRACT

It is long known that pyruvate kinase isoform M2 (PKM2) is released into the circulation of cancer patients. The PKM2 levels in patients have been suggested as a diagnostic marker for many types of cancers. However, it is not known how PKM2 is released in the blood, and whether the circulating PKM2 has any physiological function(s) in tumor progression. In this report, we demonstrate that PKM2 in the blood facilitates tumor growth by promoting tumor angiogenesis. Our experiments show that PKM2 promotes tumor angiogenesis by increasing endothelial cell proliferation, migration, and cell-ECM adhesion. Only the dimeric PKM2 possess the activity in promoting tumor angiogenesis, which is consistent with the observations that PKM2 in circulation of cancer patients is a dimer form.


Subject(s)
Carrier Proteins/blood , Cell Proliferation/genetics , Membrane Proteins/blood , Neovascularization, Pathologic/pathology , Protein Isoforms/blood , Thyroid Hormones/blood , Animals , Cell Adhesion/genetics , Cell Line, Tumor , Cell Movement/genetics , Glycolysis/genetics , Human Umbilical Vein Endothelial Cells , Humans , Mice , Neoplasms/blood , Neoplasms/pathology , Neoplastic Cells, Circulating/metabolism , Neovascularization, Pathologic/blood , Xenograft Model Antitumor Assays , Thyroid Hormone-Binding Proteins
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