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1.
Cytotherapy ; 24(7): 720-732, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35570170

RESUMO

BACKGROUND: Chimeric antigen receptor (CAR) T cell therapy has yielded impressive clinical results in hematological malignancies and is a promising approach for solid tumor treatment. However, toxicity, including cytokine-release syndrome (CRS) and neurotoxicity, is a concern hampering its broader use. METHODS: In selecting a lead CAR-T candidate against the oncofetal antigen glypican 3 (GPC3), we compared CARs bearing a low- and high-affinity single-chain variable fragment (scFv) binding to a similar epitope and cross-reactive with murine GPC3. RESULTS: Where the high-affinity CAR-T cells were toxic in vivo, the low-affinity CAR maintained cytotoxic function against antigen-positive tumor cells but did not show toxicity against normal tissues. High-affinity CAR-induced toxicity was caused by on-target, off-tumor binding, based on the observation that higher doses of the high-affinity CAR-T caused toxicity in non-tumor-bearing mice and accumulated in organs with low expression of GPC3. To explore another layer of controlling CAR-T toxicity, we developed a means to target and eliminate CAR-T cells using anti-TNF-α antibody therapy after CAR-T infusion. The antibody was shown to function by eliminating early antigen-activated, but not all, CAR-T cells, allowing a margin where the toxic response could be effectively decoupled from antitumor efficacy with only a minor loss in tumor control. By exploring additional traits of the CAR-T cells after activation, we identified a mechanism whereby we could use approved therapeutics and apply them as an exogenous kill switch that eliminated early activated CAR-T following antigen engagement in vivo. CONCLUSIONS: By combining the reduced-affinity CAR with this exogenous control mechanism, we provide evidence that we can modulate and control CAR-mediated toxicity.


Assuntos
Glipicanas , Receptores de Antígenos Quiméricos , Animais , Linhagem Celular Tumoral , Glipicanas/metabolismo , Imunoterapia Adotiva/métodos , Camundongos , Receptores de Antígenos de Linfócitos T , Receptores de Antígenos Quiméricos/genética , Receptores de Antígenos Quiméricos/metabolismo , Linfócitos T , Inibidores do Fator de Necrose Tumoral , Ensaios Antitumorais Modelo de Xenoenxerto
4.
Mol Cancer Ther ; 22(2): 254-263, 2023 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-36722141

RESUMO

Antibody-drug conjugates (ADC) delivering pyrrolobenzodiazepine (PBD) DNA cross-linkers are currently being evaluated in clinical trials, with encouraging results in Hodgkin and non-Hodgkin lymphomas. The first example of an ADC delivering a PBD DNA cross-linker (loncastuximab tesirine) has been recently approved by the FDA for the treatment of relapsed and refractory diffuse large B-cell lymphoma. There has also been considerable interest in mono-alkylating PBD analogs. We conducted a head-to-head comparison of a conventional PBD bis-imine and a novel PBD mono-imine. Key Mitsunobu chemistry allowed clean and convenient access to the mono-imine class. Extensive DNA-binding studies revealed that the mono-imine mediated a type of DNA interaction that is described as "pseudo cross-linking," as well as alkylation. The PBD mono-imine ADC demonstrated robust antitumor activity in mice bearing human tumor xenografts at doses 3-fold higher than those that were efficacious for the PBD bis-imine ADC. A single-dose toxicology study in rats demonstrated that the MTD of the PBD mono-alkylator ADC was approximately 3-fold higher than that of the ADC bearing a bis-imine payload, suggesting a comparable therapeutic index for this molecule. However, although both ADCs caused myelosuppression, renal toxicity was observed only for the bis-imine, indicating possible differences in toxicologic profiles that could influence tolerability and therapeutic index. These data show that mono-amine PBDs have physicochemical and pharmacotoxicologic properties distinct from their cross-linking analogs and support their potential utility as a novel class of ADC payload.


Assuntos
Imunoconjugados , Linfoma não Hodgkin , Humanos , Animais , Camundongos , Ratos , Alquilação , DNA , Iminas , Imunoconjugados/farmacologia
5.
J Biol Chem ; 286(27): 24089-100, 2011 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-21576240

RESUMO

The development of intratumoral hypoxia, a hallmark of rapidly progressing solid tumors, renders tumor cells resistant to chemotherapy and radiation therapy. We have recently shown that inhibition of aldose reductase (AR), an enzyme that catalyzes the reduction of lipid aldehydes and their glutathione conjugates, prevents human colon cancer cell growth in culture as well as in nude mouse xenografts by inhibiting the NF-κB-dependent activation of oxidative stress-mediated inflammatory and carcinogenic markers. However, the role of AR in mediating hypoxic stress signals is not known. We therefore investigated the molecular mechanisms by which AR inhibition prevents the hypoxia-induced human colon cancer cells growth and invasion. Our results indicate that AR inhibition by the pharmacological inhibitor fidarestat or ablation by AR-specific siRNA prevents hypoxia-induced proliferation of HT29, SW480, and Caco-2 colon cancer cells. Furthermore, hypoxia-induced increase in the level of HIF-1α in colon cancer cells was significantly decreased by AR inhibition. During hypoxic conditions, treatment of HT29 cells with the AR inhibitor fidarestat significantly decreased the expression of vascular endothelial growth factor, a down target of HIF-1α, at both mRNA and protein levels and also prevented the activation of PI3K/AKT, GSK3ß, Snail, and lysyl oxidase. Furthermore, inhibition of hypoxia-induced HIF-1α protein accumulation by AR inhibition was abolished in the presence of MG132, a potent inhibitor of the 26 S proteasome. In addition, AR inhibition also prevented the hypoxia-induced inflammatory molecules such as Cox-2 and PGE2 and expression of extracellular matrix proteins such as MMP2, vimentin, uPAR, and lysyl oxidase 2. In conclusion, our results indicate that AR mediates hypoxic signals, leading to tumor progression and invasion.


Assuntos
Aldeído Redutase/metabolismo , Neoplasias do Colo/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Fator A de Crescimento do Endotélio Vascular/biossíntese , Aldeído Redutase/antagonistas & inibidores , Aldeído Redutase/genética , Células CACO-2 , Hipóxia Celular/efeitos dos fármacos , Hipóxia Celular/genética , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Inibidores de Cisteína Proteinase/farmacologia , Dinoprostona/genética , Dinoprostona/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/genética , Quinase 3 da Glicogênio Sintase/genética , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Imidazolidinas/farmacologia , Leupeptinas/farmacologia , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 2 da Matriz/metabolismo , Invasividade Neoplásica , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Complexo de Endopeptidases do Proteassoma/genética , Inibidores de Proteassoma , Proteína-Lisina 6-Oxidase/genética , Proteína-Lisina 6-Oxidase/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RNA Neoplásico/biossíntese , RNA Neoplásico/genética , Fator A de Crescimento do Endotélio Vascular/genética , Vimentina/genética , Vimentina/metabolismo
6.
Carcinogenesis ; 32(8): 1259-67, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21642355

RESUMO

Colon cancer is the third most common cause of cancer and is the second leading cause of cancer deaths in the USA. Although inhibition of aldose reductase (AR) is known to prevent human colon cancer cell growth in nude mice xenografts, the role of AR in the regulation of cancer metastasis is not known. We now demonstrate the mechanisms by which AR regulates colon cancer metastasis in vitro and in vivo. Inhibition of AR prevented the epidermal growth factor (EGF) or fibroblast growth factor (FGF)-induced migration and invasion of human colon cancer (HT29; KM20) cells by >70% and also inhibited (>80%) the adhesion of the cancer cells to endothelial cells. Treatment of endothelial cells with AR inhibitors significantly (∼85%) downregulated the EGF or FGF-induced expression of Inter-Cellular Adhesion Molecule-1, Vascular cell adhesion molecule-1 and vascular endothelial-cadherin. Furthermore, liver metastasis of green fluorescent protein-labeled KM20 cells injected into the spleen of athymic nude mice was significantly (>65%) prevented by AR inhibitor, fidarestat or ARsiRNA delivered systemically into the mice. Similar results were observed with HT29 cells. AR inhibition or ablation also prevented (70-90%) the increase in the levels of matrix metalloproteinase-2, cyclin D1, CD31, CD34 and the activation of nuclear factor-kappa-binding protein in metastatic liver. Thus, our results indicate that AR regulates cancer cell adhesion, invasion and migration events which initiate metastasis and therefore, AR inhibition could be a novel therapeutic approach for the prevention of colon cancer metastasis.


Assuntos
Aldeído Redutase/antagonistas & inibidores , Movimento Celular/efeitos dos fármacos , Neoplasias do Colo/enzimologia , Neoplasias do Colo/prevenção & controle , Imidazolidinas/uso terapêutico , Neoplasias Hepáticas Experimentais/enzimologia , Neoplasias Hepáticas Experimentais/prevenção & controle , Aldeído Redutase/genética , Aldeído Redutase/metabolismo , Animais , Western Blotting , Adesão Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Neoplasias do Colo/patologia , Humanos , Técnicas Imunoenzimáticas , Neoplasias Hepáticas Experimentais/secundário , Masculino , Camundongos , Camundongos Nus , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa
7.
Angiogenesis ; 14(2): 209-21, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21409599

RESUMO

We have recently shown that aldose reductase (AR, EC 1.1.1.21) a nicotinamide adenine dinucleotide phosphate-dependent aldo-keto reductase, known to be involved in oxidative stress-signaling, prevents human colon cancer cell growth in culture as well as in nude mice xenografts. Inhibition of AR also prevents azoxymethane-induced aberrant crypt foci formation in mice. In order to understand the chemopreventive mechanism(s) of AR inhibition in colon cancer, we have investigated the role of AR in the mediation of angiogenic signals in vitro and in vivo models. Our results show that inhibition of AR significantly prevented the VEGF- and FGF -induced proliferation and expression of proliferative marker Ki67 in the human umbilical vein endothelial cells (HUVEC). Further, AR inhibition or ablation with siRNA prevented the VEGF- and FGF -induced invasion and migration in HUVEC. AR inhibition also prevented the VEGF- and FGF- induced secretion/expression of IL-6, MMP2, MMP9, ICAM, and VCAM. The anti-angiogenic feature of AR inhibition in HUVEC was associated with inactivation of PI3 K/AKT and NF-κB (p65) and suppression of VEGF receptor 2 protein levels. Most importantly, matrigel plug model of angiogenesis in rats showed that inhibition of AR prevented infiltration of blood cells, invasion, migration and formation of capillary like structures, and expression of blood vessels markers CD31 and vWF. Thus, our results demonstrate that AR inhibitors could be novel agents to prevent angiogenesis.


Assuntos
Aldeído Redutase/antagonistas & inibidores , Neovascularização Patológica/enzimologia , Aldeído Redutase/metabolismo , Indutores da Angiogênese/farmacologia , Animais , Capilares/efeitos dos fármacos , Capilares/crescimento & desenvolvimento , Moléculas de Adesão Celular/metabolismo , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Colágeno , Combinação de Medicamentos , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/enzimologia , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Humanos , Imidazolidinas/farmacologia , Interleucina-6/metabolismo , Laminina , Metaloproteinases da Matriz/metabolismo , Camundongos , NF-kappa B/metabolismo , Neovascularização Fisiológica/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/metabolismo , Proteoglicanas , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/patologia , Veias Umbilicais/citologia
8.
Carcinogenesis ; 30(5): 799-807, 2009 May.
Artigo em Inglês | MEDLINE | ID: mdl-19028703

RESUMO

Aldose reductase (AR; EC 1.1.1.21), an nicotinamide adenine dinucleotide phosphate-dependent aldo-keto reductase, has been shown to be involved in oxidative stress signaling initiated by inflammatory cytokines, chemokines and growth factors. Recently, we have shown that inhibition of this enzyme prevents the growth of colon cancer cells in vitro as well as in nude mice xenografts. Herein, we investigated the mediation of AR in the formation of colonic preneoplastic aberrant crypt foci (ACF) using azoxymethane (AOM)-induced colon cancer mice model. Male BALB/c mice were administrated with AOM without or with AR inhibitor, sorbinil and at the end of the protocol, all the mice were euthanized and colons were evaluated for ACF formation. Administration of sorbinil significantly lowered the number of AOM-induced ACF. Similarly, AR-null mice administered with AOM demonstrated significant resistance to ACF formation. Furthermore, inhibition of AR or knockout of AR gene in the mice significantly prevented AOM-induced expression of inducible nitric oxide synthase and cyclooxygenase-2 proteins as well as their messenger RNA. AR inhibition or knockdown also significantly decreased the phosphorylation of protein kinase C (PKC) beta2 and nuclear factor kappa binding protein as well as expression of preneoplastic marker proteins such as cyclin D1 and beta-catenin in mice colons. Our results suggest that AR mediates the formation of ACF in AOM-treated mice and thereby inhibition of AR could provide an effective chemopreventive approach for the treatment of colon cancer.


Assuntos
Aldeído Redutase/deficiência , Azoximetano/toxicidade , Neoplasias do Colo/induzido quimicamente , Neoplasias do Colo/prevenção & controle , Lesões Pré-Cancerosas/induzido quimicamente , Lesões Pré-Cancerosas/prevenção & controle , Aldeído Redutase/metabolismo , Animais , Neoplasias do Colo/patologia , Primers do DNA , Peroxidação de Lipídeos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Estresse Oxidativo/fisiologia , Lesões Pré-Cancerosas/patologia , Proteína Quinase C/metabolismo , Proteína Quinase C beta , Reação em Cadeia da Polimerase Via Transcriptase Reversa
9.
Mol Cancer Ther ; 18(1): 89-99, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30352801

RESUMO

Pyrrolobenzodiazepine dimers (PBD) form cross-links within the minor groove of DNA causing double-strand breaks (DSB). DNA repair genes such as BRCA1 and BRCA2 play important roles in homologous recombination repair of DSB. We hypothesized that PBD-based antibody-drug conjugates (ADC) will have enhanced killing of cells in which homologous recombination processes are defective by inactivation of BRCA1 or BRCA2 genes. To support this hypothesis, we found 5T4-PBD, a PBD-dimer conjugated to anti-5T4 antibody, elicited more potent antitumor activity in tumor xenografts that carry defects in DNA repair due to BRCA mutations compared with BRCA wild-type xenografts. To delineate the role of BRCA1/2 mutations in determining sensitivity to PBD, we used siRNA knockdown and isogenic BRCA1/2 knockout models to demonstrate that BRCA deficiency markedly increased cell sensitivity to PBD-based ADCs. To understand the translational potential of treating patients with BRCA deficiency using PBD-based ADCs, we conducted a "mouse clinical trial" on 23 patient-derived xenograft (PDX) models bearing mutations in BRCA1 or BRCA2 Of these PDX models, 61% to 74% had tumor stasis or regression when treated with a single dose of 0.3 mg/kg or three fractionated doses of 0.1 mg/kg of a PBD-based ADC. Furthermore, a suboptimal dose of PBD-based ADC in combination with olaparib resulted in significantly improved antitumor effects, was not associated with myelotoxicity, and was well tolerated. In conclusion, PBD-based ADC alone or in combination with a PARP inhibitor may have improved therapeutic window in patients with cancer carrying BRCA mutations.


Assuntos
Antineoplásicos Imunológicos/administração & dosagem , Benzodiazepinas/química , Imunoconjugados/administração & dosagem , Neoplasias Experimentais/tratamento farmacológico , Ftalazinas/administração & dosagem , Piperazinas/administração & dosagem , Inibidores de Poli(ADP-Ribose) Polimerases/administração & dosagem , Pirróis/química , Administração Intravenosa , Animais , Antineoplásicos Imunológicos/química , Antineoplásicos Imunológicos/farmacologia , Proteína BRCA1/genética , Proteína BRCA2/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sinergismo Farmacológico , Células HeLa , Humanos , Imunoconjugados/química , Imunoconjugados/farmacologia , Glicoproteínas de Membrana/antagonistas & inibidores , Camundongos , Mutação , Neoplasias Experimentais/genética , Ftalazinas/farmacologia , Piperazinas/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Sequenciamento do Exoma , Ensaios Antitumorais Modelo de Xenoenxerto
10.
Curr Cancer Drug Targets ; 18(9): 905-911, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-28786349

RESUMO

BACKGROUND: Recent epidemiological and experimental studies have shown that obesity is a major risk factor for Colorectal Cancer (CRC). Regular intake of high fat-containing diet can promote obesity and metabolic syndrome by increasing the insulin resistance and inflammatory response which contribute to carcinogenesis. Previously, we have shown that inhibition of polyol pathway enzyme aldose reductase (AR) prevents carcinogens- and inflammatory growth factorsinduced CRC. However, the effect of AR inhibition on a high-fat diet (HFD)-induced formation of intestinal polyps in Apc-deficient Min (multiple intestinal neoplasia; ApcMin/+) mice is not known. METHODS: We examined the effect of AR inhibitor, fidarestat on the HFD-induced formation of preneoplastic intestinal polyps in ApcMin/+ mice which is an excellent model of colon cancer. RESULTS: APCMin/+ mice fed for 12 weeks of HFD caused a significant increase in the formation of polyps in the small and large intestines and fidarestat given along with the HFD prevented the number of intestinal polyps. Fidarestat also decreased the size of the polyps in the intestines of HFDtreated APC Min mice. Further, the expression levels of beta-catenin, PCNA, PKC-ß2, P-AKT, Pp65, COX-2, and iNOS in the small and large intestines of HFD-treated mice significantly increased, and AR inhibitor prevented it. CONCLUSION: Our results thus suggest that fidarestat could be used as a potential chemopreventive drug for intestinal cancers due to APC gene mutations.


Assuntos
Proteína da Polipose Adenomatosa do Colo/fisiologia , Aldeído Redutase/antagonistas & inibidores , Neoplasias do Colo/prevenção & controle , Dieta Hiperlipídica/efeitos adversos , Inibidores Enzimáticos/farmacologia , Imidazolidinas/farmacologia , Pólipos Intestinais/prevenção & controle , Animais , Neoplasias do Colo/enzimologia , Neoplasias do Colo/etiologia , Feminino , Pólipos Intestinais/enzimologia , Pólipos Intestinais/etiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL
11.
Mol Cancer Ther ; 17(10): 2176-2186, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30065100

RESUMO

Prostate-specific membrane antigen (PSMA) is a membrane-bound glutamate carboxypeptidase that is highly expressed in nearly all prostate cancers with the highest expression in metastatic castration-resistant prostate cancer (mCRPC). The prevalence of increased surface expression and constitutive internalization of PSMA make it an attractive target for an antibody-drug conjugate (ADC) approach to treating patients with mCRPC. MEDI3726 (previously known as ADCT-401) is an ADC consisting of an engineered version of the anti-PSMA antibody J591 site specifically conjugated to the pyrrolobenzodiazepine (PBD) dimer tesirine. MEDI3726 specifically binds the extracellular domain of PSMA and, once internalized, releases the PBD dimer to crosslink DNA and trigger cell death. In vitro, MEDI3726 demonstrated potent and specific cytotoxicity in a panel of PSMA-positive prostate cancer cell lines, consistent with internalization and DNA interstrand crosslinking. In vivo, MEDI3726 showed robust antitumor activity against the LNCaP and the castration-resistant CWR22Rv1 prostate cancer cell line xenografts. MEDI3726 also demonstrated durable antitumor activity in the PSMA-positive human prostate cancer patient-derived xenograft (PDX) LuCaP models. This activity correlated with increased phosphorylated Histone H2AX in tumor xenografts treated with MEDI3726. MEDI3726 is being evaluated in a phase I clinical trial as a treatment for patients with metastatic castrate-resistant prostate cancer (NCT02991911). Mol Cancer Ther; 17(10); 2176-86. ©2018 AACR.


Assuntos
Antineoplásicos Imunológicos/farmacologia , Biomarcadores Tumorais/antagonistas & inibidores , Glutamato Carboxipeptidase II/antagonistas & inibidores , Imunoconjugados/farmacologia , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/imunologia , Animais , Antígenos de Superfície/genética , Antígenos de Superfície/metabolismo , Linhagem Celular Tumoral , Reações Cruzadas/imunologia , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Expressão Gênica , Glutamato Carboxipeptidase II/genética , Glutamato Carboxipeptidase II/metabolismo , Humanos , Imuno-Histoquímica , Macaca fascicularis , Masculino , Camundongos , Neoplasias da Próstata/patologia , Ensaios Antitumorais Modelo de Xenoenxerto
12.
Endocrinology ; 148(9): 4371-84, 2007 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-17584970

RESUMO

Diabetes is associated with increased generation of cytokines and tissue inflammation, but it is unclear how increased cytokine synthesis is causally related to the development of diabetic complications. Here, we report that exposure to high (25 mm) glucose, but not iso-osmotic concentrations of mannitol or 3-methyl glucose, increased TNF-alpha secretion by rat and human aortic smooth muscle cells in culture. The increase in TNF-alpha production was prevented by actinomycin D and cycloheximide, indicating transcriptional activation of TNF-alpha gene. High glucose (HG)-induced TNF-alpha release was specifically inhibited by protein kinase C (PKC)-delta inhibitor (Rottlerin; EMD Biosciences, San Diego, CA), but not PKC-beta2 inhibitor (CGP53353; Tocris Cookson Inc., Ellisville, MO), indicating the possible involvement of PKC-delta in HG signaling. TNF-alpha secretion was also prevented by pretreating cells with aldose reductase (AR) inhibitors, sorbinil or tolrestat and in cells treated with antisense AR mRNA. Inhibition of AR also prevented the increase in TNF-alpha mRNA. Addition of anti-TNF-alpha antibodies or soluble TNF-alpha receptors 1 and 2 to the medium or RNA interference ablation of TNF-alpha attenuated nuclear factor-kappaB activation and prevented HG-stimulated cell growth. These data indicate that AR is required for HG-induced TNF-alpha synthesis and release. In vivo, the release of TNF-alpha by HG leading to autocrine stimulation of TNF-alpha synthesis may be a critical step in the development of the cardiovascular complications of diabetes. Interruption of the autocrine effects of TNF-alpha may be a useful strategy for treating diabetic vasculopathies.


Assuntos
Aldeído Redutase/metabolismo , Músculo Liso Vascular/citologia , Fator de Necrose Tumoral alfa/biossíntese , Fator de Necrose Tumoral alfa/genética , Animais , Aorta , Divisão Celular/efeitos dos fármacos , Glucose/farmacologia , Humanos , Músculo Liso Vascular/efeitos dos fármacos , NF-kappa B/fisiologia , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa
13.
Diabetes ; 55(4): 901-10, 2006 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-16567509

RESUMO

The objective of this study was to determine whether the polyol pathway enzyme aldose reductase mediates diabetes abnormalities in vascular smooth muscle cell (SMC) growth. Aldose reductase inhibitors (tolrestat or sorbinil) or antisense aldose reductase mRNA prevented hyperproliferation of cultured rat aortic SMCs induced by high glucose. Cell cycle progression in the presence of high glucose was blocked by tolrestat, which induced a G0-G1 phase growth arrest. In situ, diabetes increased SMC growth and intimal hyperplasia in balloon-injured carotid arteries of streptozotocin-treated rats, when examined 7 or 14 days after injury. Treatment with tolrestat (15 mg x kg(-1) x day(-1)) diminished intimal hyperplasia and decreased SMC content of the lesion by 25%. Although tolrestat treatment increased immunoreactivity of the lesion with antibodies raised against protein adducts of the lipid peroxidation product 4-hydroxy trans-2-nonenal, no compensatory increase in lesion fibrosis was observed. Collectively, these results suggest that inhibition of aldose reductase prevents glucose-induced stimulation of SMC growth in culture and in situ. Even though inhibition of aldose reductase increases vascular oxidative stress, this approach may be useful in preventing abnormal SMC growth in vessels of diabetic patients.


Assuntos
Aldeído Redutase/metabolismo , Diabetes Mellitus Experimental/patologia , Glucose/farmacologia , Músculo Liso Vascular/citologia , Aldeído Redutase/antagonistas & inibidores , Aldeído Redutase/genética , Animais , Aorta , Ciclo Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Inibidores Enzimáticos/farmacologia , Hiperplasia/prevenção & controle , Imidazolidinas/farmacologia , Masculino , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/patologia , Naftalenos/farmacologia , Ratos , Ratos Sprague-Dawley , Valores de Referência , Túnica Íntima/citologia , Túnica Íntima/efeitos dos fármacos , Túnica Íntima/patologia
14.
Free Radic Biol Med ; 42(8): 1290-302, 2007 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-17382209

RESUMO

Aldose reductase (AR) is a ubiquitously expressed protein with pleiotrophic roles as an efficient catalyst for the reduction of toxic lipid aldehydes and mediator of hyperglycemia, cytokine, and growth factor-induced redox-sensitive signals that cause secondary diabetic complications. Although AR inhibition has been shown to be protective against oxidative stress signals, the role of AR in regulating nitric oxide (NO) synthesis and NO-mediated apoptosis has not been elucidated to date. We therefore investigated the role of AR in regulating lipopolysaccharide (LPS)-induced NO synthesis and apoptosis in RAW 264.7 macrophages. Inhibition or RNA interference ablation of AR suppressed LPS-stimulated production of NO and overexpression of iNOS mRNA. Inhibition or ablation of AR also prevented the LPS-induced apoptosis, cell cycle arrest, activation of caspase-3, p38-MAPK, JNK, NF-kappaB, and AP1. In addition, AR inhibition prevented the LPS-induced down-regulation of Bcl-xl and up-regulation of Bax and Bak in macrophages. L-Arginine increased and L-NAME decreased the severity of cell death caused by LPS and AR inhibitors prevented it. Furthermore, inhibition of AR prevents cell death caused by HNE and GS-HNE, but not GS-DHN. Our findings for the first time suggest that AR-catalyzed lipid aldehyde-glutathione conjugates regulate the LPS-induced production of inflammatory marker NO and cytotoxicity in RAW 264.7 cells. Inhibition or ablation of AR activity may be a potential therapeutic target in endotoximia and other inflammatory diseases.


Assuntos
Aldeído Redutase/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Endotoxinas/toxicidade , Macrófagos/fisiologia , Óxido Nítrico/biossíntese , Aldeído Redutase/antagonistas & inibidores , Aldeídos/metabolismo , Animais , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Células Cultivadas , Glutationa/metabolismo , Lipopolissacarídeos/toxicidade , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Camundongos , Óxido Nítrico/toxicidade
15.
Cancer Lett ; 252(2): 299-306, 2007 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-17300864

RESUMO

Pro-inflammatory cytokines such as TNF-alpha play an important role in the pathophysiology of diseases such as Crohn's and ulcerative colitis which cause increased risk of colorectal cancer. However, the mechanisms underlying colon carcinogenesis are not well understood. Herein we report that inhibition/antisense abolition of polyol pathway enzyme, aldose reductase (AR) inhibited the TNF-alpha-induced synthesis of prostaglandin E2 and the activity of cyclooxygenase (Cox) in human colon cancer cells, Caco-2. Inhibition of AR prevented TNF-alpha-induced activation of PKC and NF-kappaB which resulted in the abrogation of Cox-2 mRNA and protein expression. These results suggest that inhibition of AR could be a novel chemopreventive approach to colon cancer.


Assuntos
Aldeído Redutase/metabolismo , Neoplasias do Colo/metabolismo , Dinoprostona/sangue , Fator de Necrose Tumoral alfa/fisiologia , Aldeído Redutase/antagonistas & inibidores , Sequência de Bases , Células CACO-2 , Neoplasias do Colo/enzimologia , Neoplasias do Colo/patologia , Primers do DNA , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Humanos , NF-kappa B/metabolismo , Proteína Quinase C/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa
16.
Diabetes ; 54(3): 818-29, 2005 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-15734861

RESUMO

Activation of protein kinase C (PKC) has been linked to the development of secondary diabetes complications. However, the underlying molecular mechanisms remain unclear. We examined the contribution of aldose reductase, which catalyzes the first, and the rate-limiting, step of the polyol pathway of glucose metabolism, to PKC activation in vascular smooth muscle cells (VSMCs) isolated from rat aorta and exposed to high glucose in culture. Exposure of VSMCs to high glucose (25 mmol/l), but not iso-osmotic mannitol, led to an increase in total membrane-associated PKC activity, which was prevented by the aldose reductase inhibitors tolrestat or sorbinil or by the ablation of aldose reductase by small interfering RNA (siRNA). The VSMCs were found to express low levels of sorbitol dehydrogenase, and treatment with the sorbitol dehydrogenase inhibitor CP-166572 did not prevent high-glucose-induced PKC activation. Stimulation with high glucose caused membrane translocation of conventional (alpha, beta1, beta2, and gamma) and novel (delta and epsilon) isoforms of PKC. Inhibition of aldose reductase prevented membrane translocation of PKC-beta2 and -delta and delayed the activation of PKC-beta1 and -epsilon, whereas membrane translocation of PKC-alpha and -gamma was not affected. Treatment with tolrestat prevented phosphorylation of PKC-beta2 and -delta. High glucose increased the formation of diacylglycerol (DAG) and enhanced phosphorylation of phospholipase C-gamma1 (PLC-gamma1). Inhibition of aldose reductase prevented high glucose-induced DAG formation and phosphorylation of PLC-gamma1 and PLC-beta2 and -delta. Inhibition of phospholipid hydrolysis by D609, but not by the synthetic alkyl-1-lysophospholipid 1-O-octadecyl-2-O-methyl-rac-glycerophosphocholine, or edelfosine, prevented DAG formation. Treatment with sorbinil decreased the levels of reactive oxygen species in high-glucose-stimulated VSMCs. Hence, inhibition of aldose reductase, independent of sorbitol dehydrogenase, appears to be effective in diminishing oxidative stress and hyperglycemic changes in signaling events upstream to the activation of multiple PKC isoforms and PLC-gamma1 and may represent a useful approach for preventing the development of secondary vascular complications of diabetes.


Assuntos
Aldeído Redutase/metabolismo , Diglicerídeos/biossíntese , Hiperglicemia/metabolismo , Músculo Liso Vascular/enzimologia , Proteína Quinase C/metabolismo , Aldeído Redutase/antagonistas & inibidores , Animais , Células Cultivadas , Ativação Enzimática , Expressão Gênica , L-Iditol 2-Desidrogenase/metabolismo , Fosfolipase C gama , Fosforilação , RNA Interferente Pequeno , Ratos , Espécies Reativas de Oxigênio , Fatores de Tempo , Fosfolipases Tipo C/metabolismo
17.
Invest Ophthalmol Vis Sci ; 47(12): 5395-403, 2006 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-17122129

RESUMO

PURPOSE: Bacterial infections are one of the major causes of human eye disease. Because the bacterial endotoxin lipopolysaccharide (LPS) is known to cause cytotoxicity through oxidative stress and an earlier study has shown that aldose reductase (AR) mediates oxidative stress signals, the purpose of this study was to investigate the anti-inflammatory effects of AR inhibition on LPS-induced activation of NF-kappaB-dependent signals in human lens epithelial cells (HLECs). METHODS: Growth-arrested HLECs were cultured without or with AR inhibitors or transfected with an AR small interfering (si)RNA. Subsequently, the cells were stimulated with LPS (1-10 mug/mL) for 24 hours. The cell viability was assessed by cell counts and MTT assay, and apoptosis was measured by nucleosomal degradation. Electrophoretic mobility gel shift assays were performed to determine the activation of NF-kappaB and AP1. The levels of nitric oxide, MMP-2, MMP-9, Cox-2, and TNF-alpha were measured by using specific ELISA kits. Western blot analysis was performed to determine the cleavage of poly(ADP-ribose) polymerase (PARP) and the activation of PKC and mitogen-activated protein kinase (MAPK). RESULTS: Bacterial LPS caused apoptosis of HLECs. Inhibition of AR by two structurally unrelated inhibitors, sorbinil and tolrestat, or ablation by AR siRNA prevented the LPS-induced apoptosis, activation of caspase-3 and cleavage of PARP protein. Inhibition of AR in HLECs also prevented the LPS-induced activation of redox-sensitive transcription factors such as NF-kappaB and AP1 and their downstream signals that lead to expression of Cox-2, MMP-2, MMP-9, and TNF-alpha proteins. In addition, inhibition of AR prevented LPS-induced activation of protein kinases upstream to NF-kappaB activation such as PKC and MAPK in HLECs. CONCLUSIONS: The results indicate that AR mediates the bacterial endotoxin signaling that could damage HLECs by regulating the signals that activate the redox-sensitive transcription factor NF-kappaB and cause inflammation. Thus, inhibition of AR could be a therapeutic target for Gram-negative bacterial infection-induced visual complications.


Assuntos
Aldeído Redutase/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Cristalino/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Western Blotting , Caspase 3/metabolismo , Colágeno Tipo XI/metabolismo , Ciclo-Oxigenase 2/metabolismo , Dinoprostona/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Ensaio de Imunoadsorção Enzimática , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Humanos , Imidazolidinas/farmacologia , Lactente , Cristalino/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Naftalenos/farmacologia , Óxido Nítrico/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
18.
Mol Cancer Ther ; 15(4): 689-701, 2016 04.
Artigo em Inglês | MEDLINE | ID: mdl-26880266

RESUMO

HER3/ERBB3 is a kinase-deficient member of the EGFR family receptor tyrosine kinases (RTK) that is broadly expressed and activated in human cancers. HER3 is a compelling cancer target due to its important role in activation of the oncogenic PI3K/AKT pathway. It has also been demonstrated to confer tumor resistance to a variety of cancer therapies, especially targeted drugs against EGFR and HER2. HER3 can be activated by its ligand (heregulin/HRG), which induces HER3 heterodimerization with EGFR, HER2, or other RTKs. Alternatively, HER3 can be activated in a ligand-independent manner through heterodimerization with HER2 in HER2-amplified cells. We developed a fully human mAb against HER3 (KTN3379) that efficiently suppressed HER3 activity in both ligand-dependent and independent settings. Correspondingly, KTN3379 inhibited tumor growth in divergent tumor models driven by either ligand-dependent or independent mechanisms in vitro and in vivo Most intriguingly, while investigating the mechanistic underpinnings of tumor response to KTN3379, we discovered an interesting dichotomy in that PTEN loss, a frequently occurring oncogenic lesion in a broad range of cancer types, substantially blunted the tumor response in HER2-amplified cancer, but not in the ligand-driven cancer. To our knowledge, this represents the first study ascertaining the impact of PTEN loss on the antitumor efficacy of a HER3 mAb. KTN3379 is currently undergoing a phase Ib clinical trial in patients with advanced solid tumors. Our current study may help us optimize patient selection schemes for KTN3379 to maximize its clinical benefits. Mol Cancer Ther; 15(4); 689-701. ©2016 AACR.


Assuntos
Anticorpos Monoclonais/farmacologia , Antineoplásicos/farmacologia , Neoplasias/metabolismo , Receptor ErbB-3/antagonistas & inibidores , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Expressão Gênica , Humanos , Ligantes , Camundongos , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/patologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Fosforilação , Multimerização Proteica/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Interferência de RNA , Receptor ErbB-2/química , Receptor ErbB-2/metabolismo , Receptor ErbB-3/química , Receptor ErbB-3/metabolismo , Transdução de Sinais/efeitos dos fármacos , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
19.
Mol Cancer Ther ; 14(7): 1637-49, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25948294

RESUMO

ADAM17 is the primary sheddase for HER pathway ligands. We report the discovery of a potent and specific ADAM17 inhibitory antibody, MEDI3622, which induces tumor regression or stasis in many EGFR-dependent tumor models. The inhibitory activity of MEDI3622 correlated with EGFR activity both in a series of tumor models across several indications as well in as a focused set of head and neck patient-derived xenograft models. The antitumor activity of MEDI3622 was superior to that of EGFR/HER pathway inhibitors in the OE21 esophageal model and the COLO205 colorectal model suggesting additional activity outside of the EGFR pathway. Combination of MEDI3622 and cetuximab in the OE21 model was additive and eradicated tumors. Proteomics analysis revealed novel ADAM17 substrates that function outside of the HER pathways and may contribute toward the antitumor activity of the monoclonal antibody.


Assuntos
Proteínas ADAM/antagonistas & inibidores , Anticorpos Monoclonais/farmacologia , Receptores ErbB/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto , Proteínas ADAM/imunologia , Proteínas ADAM/metabolismo , Proteína ADAM17 , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/farmacocinética , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Western Blotting , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cetuximab/administração & dosagem , Cetuximab/farmacologia , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Receptores ErbB/metabolismo , Feminino , Células HCT116 , Células HT29 , Humanos , Camundongos Endogâmicos DBA , Camundongos Nus , Neoplasias/imunologia , Neoplasias/metabolismo , Resultado do Tratamento
20.
Cancer Lett ; 355(1): 141-7, 2014 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-25218594

RESUMO

Type-2 diabetes and obesity-related metabolic abnormalities are major risk factors for the development of colon cancer. In the present study, we examined the effects of polyol pathway enzyme aldose reductase (AR) inhibitor, fidarestat, on the development of azoxymethane (AOM)-induced colonic premalignant lesions in C57BL/KsJ-db/db obese mice. Our results indicate that fidarestat given in the drinking water caused a significant reduction in the total number of colonic premalignant lesions in the AOM treated obese mice. Further, the expression levels of PKC-ß2, AKT, COX-2 and iNOS in the colonic mucosa of AOM-treated mice were significantly decreased by fidarestat. The serum levels of IL-1α, IP-10, MIG, TNF-α and VEGF are significantly suppressed in AOM + fidarestat treated obese mice. Fidarestat also decreased the expression of COX-2, iNOS, XIAP, survivin, ß-catenin and NF-κB in high glucose-treated HT29 colon cancer cells. In conclusion, our results indicate that fidarestat inhibits the development of colonic premalignant lesions in an obesity-related colon cancer and is chemopreventive to colorectal carcinogenesis in obese individuals.


Assuntos
Aldeído Redutase/antagonistas & inibidores , Anticarcinógenos/farmacologia , Azoximetano , Colo/efeitos dos fármacos , Neoplasias do Colo/prevenção & controle , Diabetes Mellitus Tipo 2/complicações , Inibidores Enzimáticos/farmacologia , Imidazolidinas/farmacologia , Mucosa Intestinal/efeitos dos fármacos , Obesidade/complicações , Lesões Pré-Cancerosas/prevenção & controle , Aldeído Redutase/metabolismo , Animais , Colo/enzimologia , Colo/patologia , Neoplasias do Colo/enzimologia , Neoplasias do Colo/patologia , Diabetes Mellitus Tipo 2/enzimologia , Diabetes Mellitus Tipo 2/genética , Modelos Animais de Doenças , Células HT29 , Humanos , Mediadores da Inflamação/metabolismo , Mucosa Intestinal/enzimologia , Mucosa Intestinal/patologia , Masculino , Camundongos Endogâmicos C57BL , Obesidade/enzimologia , Obesidade/genética , Estresse Oxidativo/efeitos dos fármacos , Lesões Pré-Cancerosas/enzimologia , Lesões Pré-Cancerosas/patologia , Transdução de Sinais/efeitos dos fármacos
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