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1.
J Exp Med ; 218(4)2021 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-33561195

RESUMO

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.

2.
iScience ; 23(11): 101684, 2020 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-33196019

RESUMO

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.

3.
Cell Mol Gastroenterol Hepatol ; 11(1): 161-179, 2020 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-32810598

RESUMO

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.

4.
Nat Commun ; 10(1): 4777, 2019 10 29.
Artigo em Inglês | MEDLINE | ID: mdl-31664017

RESUMO

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.


Assuntos
Meios de Contraste/química , Gadolínio/química , Hipertensão Portal/diagnóstico por imagem , Fígado/diagnóstico por imagem , Imagem por Ressonância Magnética/métodos , Doença Crônica , Diagnóstico Precoce , Humanos
5.
Mol Ther Oncolytics ; 14: 188-195, 2019 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-31312717

RESUMO

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.

7.
Neurotherapeutics ; 15(3): 770-784, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29869055

RESUMO

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.


Assuntos
Proteína-Tirosina Quinases de Adesão Focal/genética , Infarto da Artéria Cerebral Média/tratamento farmacológico , Neovascularização Fisiológica/efeitos dos fármacos , Neurogênese/efeitos dos fármacos , Piruvato Quinase , Recuperação de Função Fisiológica/efeitos dos fármacos , Fator de Transcrição STAT3/genética , Animais , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Circulação Cerebrovascular/efeitos dos fármacos , Modelos Animais de Doenças , Fibroblastos/efeitos dos fármacos , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Infarto da Artéria Cerebral Média/fisiopatologia , Camundongos , Camundongos Endogâmicos C57BL , Fosfopiruvato Hidratase/metabolismo , Piruvato Quinase/farmacologia , Piruvato Quinase/uso terapêutico , Fator de Transcrição STAT3/metabolismo , Células-Tronco/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos
8.
Nat Commun ; 7: 11675, 2016 05 31.
Artigo em Inglês | MEDLINE | ID: mdl-27241473

RESUMO

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.


Assuntos
Inibidores da Angiogênese/farmacologia , Apoptose/efeitos dos fármacos , Desenho de Fármacos , Integrina alfaVbeta3/metabolismo , Neovascularização Patológica/tratamento farmacológico , Sequência de Aminoácidos/genética , Inibidores da Angiogênese/química , Inibidores da Angiogênese/uso terapêutico , Animais , Sítios de Ligação/genética , Linhagem Celular , Feminino , Humanos , Ligantes , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Simulação de Acoplamento Molecular , Terapia de Alvo Molecular/métodos , Neoplasias/irrigação sanguínea , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Neovascularização Patológica/patologia , Ligação Proteica/genética , Engenharia de Proteínas/métodos , Estrutura Terciária de Proteína , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
9.
Wound Repair Regen ; 24(2): 328-36, 2016 03.
Artigo em Inglês | MEDLINE | ID: mdl-26808610

RESUMO

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.


Assuntos
Neovascularização Fisiológica , Neutrófilos/metabolismo , Piruvato Quinase/metabolismo , Cicatrização/fisiologia , Ferimentos e Lesões/enzimologia , Ferimentos e Lesões/patologia , Animais , Proliferação de Células , Modelos Animais de Doenças , Matriz Extracelular/patologia , Imuno-Histoquímica , Inflamação/enzimologia , Inflamação/patologia , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos/enzimologia
10.
Sci Rep ; 5: 16214, 2015 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-26577829

RESUMO

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.


Assuntos
Meios de Contraste , Imagem por Ressonância Magnética , Imagem Molecular , Neoplasias/diagnóstico , Neoplasias/metabolismo , Receptores da Bombesina/metabolismo , Animais , Sítios de Ligação , Biomarcadores , Linhagem Celular Tumoral , Meios de Contraste/química , Meios de Contraste/metabolismo , Meios de Contraste/farmacocinética , Modelos Animais de Doenças , Expressão Gênica , Xenoenxertos , Humanos , Ligantes , Imagem por Ressonância Magnética/métodos , Camundongos , Modelos Moleculares , Conformação Molecular , Imagem Molecular/métodos , Neoplasias/genética , Ligação Proteica , Ratos , Receptores da Bombesina/química , Receptores da Bombesina/genética , Distribuição Tecidual
11.
Proc Natl Acad Sci U S A ; 112(21): 6607-12, 2015 May 26.
Artigo em Inglês | MEDLINE | ID: mdl-25971726

RESUMO

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.


Assuntos
Meios de Contraste , Neoplasias Hepáticas Experimentais/diagnóstico , Neoplasias Hepáticas Experimentais/metabolismo , Imagem por Ressonância Magnética/métodos , Melanoma Experimental/diagnóstico , Melanoma Experimental/metabolismo , Parvalbuminas , Animais , Linhagem Celular Tumoral , Meios de Contraste/química , Meios de Contraste/farmacocinética , Feminino , Gadolínio , Limite de Detecção , Neoplasias Hepáticas Experimentais/secundário , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Parvalbuminas/química , Parvalbuminas/farmacocinética , Engenharia de Proteínas , Estabilidade Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacocinética
12.
J Cell Biochem ; 116(8): 1595-601, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25649741

RESUMO

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.


Assuntos
Antineoplásicos/farmacologia , RNA Helicases DEAD-box/metabolismo , Neoplasias/tratamento farmacológico , Piperazinas/farmacologia , Quinoxalinas/farmacologia , beta Catenina/metabolismo , Sítios de Ligação/efeitos dos fármacos , Linhagem Celular Tumoral , RNA Helicases DEAD-box/química , Humanos , Neoplasias/metabolismo , Fosforilação , Ligação Proteica/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos
13.
J Biol Chem ; 289(37): 25812-21, 2014 Sep 12.
Artigo em Inglês | MEDLINE | ID: mdl-25070887

RESUMO

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.


Assuntos
Proteínas de Transporte/sangue , Proliferação de Células/genética , Proteínas de Membrana/sangue , Neovascularização Patológica/patologia , Isoformas de Proteínas/sangue , Hormônios Tireóideos/sangue , Animais , Adesão Celular/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Glicólise/genética , Células Endoteliais da Veia Umbilical Humana , Humanos , Camundongos , Neoplasias/sangue , Neoplasias/patologia , Células Neoplásicas Circulantes/metabolismo , Neovascularização Patológica/sangue , Ensaios Antitumorais Modelo de Xenoenxerto
14.
Med Res Rev ; 34(5): 1070-99, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24615853

RESUMO

Magnetic resonance imaging (MRI) is the leading imaging technique for disease diagnostics, providing high resolution, three-dimensional images noninvasively. MRI contrast agents are designed to improve the contrast and sensitivity of MRI. However, current clinically used MRI contrast agents have relaxivities far below the theoretical upper limit, which largely prevent advancing molecular imaging of biomarkers with desired sensitivity and specificity. This review describes current progress in the development of a new class of protein-based MRI contrast agents (ProCAs) with high relaxivity using protein design to optimize the parameters that govern relaxivity. Further, engineering with targeting moiety allows these contrast agents to be applicable for molecular imaging of prostate cancer biomarkers by MRI. The developed protein-based contrast agents also exhibit additional in vitro and in vivo advantages for molecular imaging of disease biomarkers, such as high metal-binding stability and selectivity, reduced toxicity, proper blood circulation time, and higher permeability in tumor tissue in addition to improved relaxivities.


Assuntos
Biomarcadores Tumorais/análise , Meios de Contraste , Gadolínio/administração & dosagem , Imagem por Ressonância Magnética/métodos , Relação Dose-Resposta a Droga , Gadolínio/química
15.
J Biol Inorg Chem ; 19(2): 259-70, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24366655

RESUMO

Epidermal growth factor receptor (EGFR) and HER2 are major prognosis biomarkers and drug targets overexpressed in various types of cancer cells. There is a pressing need to develop MRI contrast agents capable of enhancing the contrast between normal tissues and tumors with high relaxivity, capable of targeting tumors, and with high intratumoral distribution and minimal toxicity. In this review, we first discuss EGFR signaling and its role in tumor progression as a major drug target. We then report our progress in the development of protein contrast agents with significant improvement of both r1 and r2 relaxivities, pharmacokinetics, in vivo retention time, and in vivo dose efficiency. Finally, we report our effort in the development of EGFR-targeted protein contrast agents with the capability to cross the endothelial boundary and with good tissue distribution across the entire tumor mass. The noninvasive capability of MRI to visualize spatially and temporally the intratumoral distribution as well as quantify the levels of EGFR and HER2 would greatly improve our ability to track changes of the biomarkers during tumor progression, monitor treatment efficacy, aid in patient selection, and further develop novel targeted therapies for clinical application.


Assuntos
Meios de Contraste , Receptores ErbB/metabolismo , Imagem por Ressonância Magnética/métodos , Imagem Molecular/métodos , Neoplasias/diagnóstico , Neoplasias/metabolismo , Receptor ErbB-2/metabolismo , Biomarcadores Tumorais/metabolismo , Humanos , Neoplasias/patologia
17.
J Biol Chem ; 288(22): 15971-9, 2013 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-23576436

RESUMO

Pyruvate kinase isoform M2 (PKM2) is an enzyme-catalyzing conversion of phosphoenolpyruvate to pyruvate in the glycolysis pathway. It was demonstrated that PKM2 interacts with tyrosine phosphopeptide, and the interaction with the tyrosine phosphopeptide affects the pyruvate kinase activity of PKM2. Our experiments suggest that PKM2 is also an active protein kinase (Gao, X., Wang, H., Yang, J. J., Liu, X., and Liu, Z. R. (2012) Mol. Cell 45, 598-609). We report here that growth signals reciprocally regulate the pyruvate kinase and protein kinase activities of PKM2 by different mechanisms. On the one hand, growth signals induce protein tyrosine phosphorylations. The tyrosine-phosphorylated protein(s) regulates the conversion of pyruvate kinase and protein kinase of PKM2 by directly interacting with PKM2. Binding of the tyrosyl-phosphorylated proteins at the fructose 1,6-bisphosphate-binding site converts the tetrameric PKM2 to a dimer. On the other hand, growth stimulations also lead to PKM2 phosphorylation, which consequently regulates the conversion of protein kinase and pyruvate kinase activities. Growth factor stimulations significantly increase the dimer/tetramer PKM2 ratio in cells and consequently activate the protein kinase activity of PKM2. Our study suggests that the conversion between the pyruvate kinase and protein kinase activities of PKM2 may be an important mechanism mediating the effects of growth signals in promoting cell proliferation.


Assuntos
Proliferação de Células , Multimerização Proteica/fisiologia , Proteínas Tirosina Quinases/metabolismo , Piruvato Quinase/metabolismo , Transdução de Sinais/fisiologia , Sítios de Ligação , Linhagem Celular , Humanos , Fosforilação/fisiologia , Proteínas Tirosina Quinases/genética , Piruvato Quinase/genética
18.
Nat Commun ; 4: 1354, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23322042

RESUMO

p68 RNA helicase is a prototypical RNA helicase. Here we present evidence to show that, by interacting with Ca-calmodulin, p68 has a role in cancer metastasis and cell migration. A peptide fragment that spans the IQ motif of p68 strongly inhibits cancer metastasis in two different animal models. The peptide interrupts p68 and Ca-calmodulin interaction and inhibits cell migration. Our results demonstrate that the p68-Ca-calmodulin interaction is essential for the formation of lamellipodia and filopodia in migrating cells. p68 interacts with microtubules in the presence of Ca-calmodulin. Our experiments show that interaction with microtubules stimulates p68 ATPase activity. Further, microtubule gliding assays demonstrate that p68, in the presence of Ca-calmodulin, can function as a microtubule motor. This motor activity may allow p68 to transport Ca-calmodulin to the leading edge of migrating cells.


Assuntos
Cálcio/metabolismo , Calmodulina/metabolismo , Movimento Celular , RNA Helicases DEAD-box/metabolismo , Neoplasias/enzimologia , Neoplasias/patologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , RNA Helicases DEAD-box/química , Técnicas de Silenciamento de Genes , Humanos , Imunoprecipitação , Camundongos , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Modelos Biológicos , Proteínas Motores Moleculares/metabolismo , Dados de Sequência Molecular , Metástase Neoplásica , Neoplasias/tratamento farmacológico , Peptídeos/química , Peptídeos/metabolismo , Peptídeos/farmacologia , Peptídeos/uso terapêutico , Ligação Proteica/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
19.
BMC Cell Biol ; 13: 27, 2012 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-23110695

RESUMO

BACKGROUND: We previously demonstrated that p68 phosphorylation at threonine residues correlates with cancer cell apoptosis under the treatments of TNF-α and TRAIL (Yang, L. Mol Cancer Res Vol 3, pp 355-63 2005). RESULTS: In this report, we characterized the role of p68 phosphorylation in apoptosis induction under the treatment of oxaliplatin in the colon cancer cells. Our data suggest that oxaliplatin treatment activates p38 MAP kinase, which subsequently phosphorylates p68 at T564 and/or T446. The phosphorylation of p68, at least partially, mediates the effects of the drug on apoptosis induction, as mutations at these two sites greatly reduce the cancer cell death. CONCLUSION: Our studies reveal an important molecular mechanism that mediates the effects of anti-cancer drug, providing a potential strategy for improving cancer treatment.


Assuntos
Antineoplásicos/toxicidade , Apoptose/efeitos dos fármacos , RNA Helicases DEAD-box/metabolismo , Compostos Organoplatínicos/toxicidade , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Células HCT116 , Humanos , Oxaliplatina , Fosforilação
20.
Mol Cell ; 45(5): 598-609, 2012 Mar 09.
Artigo em Inglês | MEDLINE | ID: mdl-22306293

RESUMO

Pyruvate kinase isoform M2 (PKM2) is a glycolysis enzyme catalyzing conversion of phosphoenolpyruvate (PEP) to pyruvate by transferring a phosphate from PEP to ADP. We report here that PKM2 localizes to the cell nucleus. The levels of nuclear PKM2 correlate with cell proliferation. PKM2 activates transcription of MEK5 by phosphorylating stat3 at Y705. In vitro phosphorylation assays show that PKM2 is a protein kinase using PEP as a phosphate donor. ADP competes with the protein substrate binding, indicating that the substrate may bind to the ADP site of PKM2. Our experiments suggest that PKM2 dimer is an active protein kinase, while the tetramer is an active pyruvate kinase. Expression of a PKM2 mutant that exists as a dimer promotes cell proliferation, indicating that protein kinase activity of PKM2 plays a role in promoting cell proliferation. Our study reveals an important link between metabolism alteration and gene expression during tumor transformation and progression.


Assuntos
Regulação da Expressão Gênica , Piruvato Quinase/fisiologia , Transcrição Genética , Sítios de Ligação , Linhagem Celular Tumoral , Proliferação de Células , Humanos , MAP Quinase Quinase 5/genética , MAP Quinase Quinase 5/metabolismo , Fosforilação , Piruvato Quinase/genética , Piruvato Quinase/metabolismo , Fator de Transcrição STAT3/metabolismo
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