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1.
Transl Res ; 229: 100-114, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33164812

RESUMO

Abundant intraperitoneal (IP) accumulation of extracellular mucus in patients with appendiceal mucinous carcinoma peritonei (MCP) causes compressive organ dysfunction and prevents delivery of chemotherapeutic drugs to cancer cells. We hypothesized that reducing extracellular mucus would decrease tumor-related symptoms and improve chemotherapeutic effect in patient-derived models of MCP. Mucolysis was achieved using a combination of bromelain (BRO) and N-acetylcysteine (NAC). Ex vivo experiments of mucolysis and chemotherapeutic drug delivery/effect were conducted with MCP and non-MCP tissue explants. In vivo experiments were performed in mouse and rat patient-derived xenograft (PDX) models of early and late (advanced) MCP. MCP tumor explants were less chemosensitive than non-MCP explants. Chronic IP administration of BRO + NAC in a mouse PDX model of early MCP and a rat PDX model of late (advanced) MCP converted solid mucinous tumors into mucinous ascites (mucolysis) that could be drained via a percutaneous catheter (rat model only), significantly reduced solid mucinous tumor growth and improved the efficacy of chemotherapeutic drugs. Combination of BRO + NAC efficiently lyses extracellular mucus in clinically relevant models of MCP. Conversion of solid mucinous tumors into mucinous ascites decreases tumor bulk and allows for minimally invasive drainage of liquified tumors. Lysis of extracellular mucus removes the protective mucinous coating surrounding cancer cells and improves chemotherapeutic drug delivery/efficacy in cancer cells. Our data provide a preclinical rationale for the clinical evaluation of BRO + NAC as a therapeutic strategy for MCP.


Assuntos
Adenocarcinoma Mucinoso/tratamento farmacológico , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Neoplasias do Apêndice/tratamento farmacológico , Muco/efeitos dos fármacos , Neoplasias Peritoneais/tratamento farmacológico , Acetilcisteína/administração & dosagem , Acetilcisteína/farmacologia , Adenocarcinoma Mucinoso/patologia , Animais , Neoplasias do Apêndice/patologia , Bromelaínas/administração & dosagem , Bromelaínas/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Humanos , Camundongos Nus , Neoplasias Peritoneais/patologia , Ratos Nus , Técnicas de Cultura de Tecidos/métodos , Ensaios Antitumorais Modelo de Xenoenxerto
2.
Orphanet J Rare Dis ; 15(1): 211, 2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32811515

RESUMO

BACKGROUND: Mucinous colon cancers (MCC) are characterized by abundant production of mucin 2 (MUC2) protein and are less sensitive to standard systemic chemotherapy. We postulated that severe/persistent endoplasmic reticulum stress (ERS) aggravation in MCC would overwhelm compensatory cytoprotective pathways and induce apoptosis. RESULTS: Basal levels of ERS markers were higher in MCC and dnTCF-LS174T cells than non-mucinous tumors and these levels were significantly increased by combinatorial treatment with ERS aggravators celecoxib + orlistat. Combination treatment inhibited cell viability and synergistically induced apoptosis. Treatment-induced cell death was ERS-dependent, apoptotic pathways were not activated following knockdown of ERS protein CHOP. Dual drug treatment significantly reduced mucinous tumor growth in vivo and induced ERS and apoptosis, consistent with in vitro experiments. CONCLUSIONS: Novel therapies are needed since MCC are more resistant to standard systemic chemotherapy. This study suggests ERS aggravation is a viable therapeutic strategy to reduce tumor growth in MCC.


Assuntos
Neoplasias do Colo , Estresse do Retículo Endoplasmático , Apoptose , Sobrevivência Celular , Neoplasias do Colo/tratamento farmacológico , Humanos
3.
Oncotarget ; 8(63): 106888-106900, 2017 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-29290997

RESUMO

Cancer cells aberrantly express mucins to enhance their survival. Relative chemoresistance of appendiceal pseudomyxoma peritonei (PMP) is attributed to abundant extracellular mucin 2 (MUC2) protein production. We hypothesized that simultaneous MUC2 inhibition and apoptosis induction would be effective against mucinous tumors. In vitro studies were conducted using LS174T cells (MUC2-secreting human colorectal cancer cells), PMP explant tissue, and epithelial organoid cultures (colonoids) derived from mucinous appendix cancers. In vivo studies were conducted using murine intraperitoneal patient-derived xenograft model of PMP. We found COX-2 over-expression in PMP explant tissue, which is known to activate G-protein coupled EP4/cAMP/PKA/CREB signaling pathway. MUC2 expression was reduced in vitro by small molecule inhibitors targeting EP4/PKA/CREB molecules and celecoxib (COX-2 inhibitor), and this was mediated by reduced CREB transcription factor binding to the MUC2 promoter. While celecoxib (5-40 µM) reduced MUC2 expression in vitro in a dose-dependent fashion, only high-dose celecoxib (≥ 20 µM) decreased cell viability and induced apoptosis. Chronic oral administration of celecoxib decreased mucinous tumor growth in our in vivo PMP model via a combination of MUC2 inhibition and induction of apoptosis. We provide a preclinical rationale for using drugs that simultaneously inhibit MUC2 production and induce apoptosis to treat patients with PMP.

4.
Transl Res ; 169: 19-30.e1, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26589109

RESUMO

Excessive accumulation of mucin 2 (MUC2; a gel-forming secreted mucin) protein in the peritoneal cavity is the major cause of morbidity and mortality in pseudomyxoma peritonei (PMP). Hypoxia (hypoxia-inducible factor-1α; HIF-1α) has been shown to regulate the expression of similar mucins (eg, MUC5AC). We hypothesized that hypoxia (HIF-1α) drives MUC2 expression in PMP and is therefore a novel target to reduce mucinous tumor growth. The regulation of MUC2 by 2% hypoxia (HIF-1α) was evaluated in MUC2-secreting LS174T cells. The effect of BAY 87-2243, an inhibitor of HIF-1α, on MUC2 expression and mucinous tumor growth was evaluated in LS174T cells, PMP explant tissue, and in a unique intraperitoneal murine xenograft model of PMP. In vitro exposure of LS174T cells to hypoxia increased MUC2 messenger RNA (mRNA) and protein expression and increased HIF-1α binding to the MUC2 promoter. Hypoxia-mediated MUC2 protein overexpression was downregulated by transfected HIF-1α small interfering RNA (siRNA) compared with scrambled siRNA in LS174T cells. BAY 87-2243 inhibited hypoxia-induced MUC2 mRNA and protein expression in LS174T cells and PMP explant tissue. In a murine xenograft model of PMP, chronic oral therapy with BAY 87-2243 inhibited mucinous tumor growth and MUC2, HIF-1α expression in the tumor tissue. Our data suggest that hypoxia (HIF-1α) induces MUC2 promoter activity to increase MUC2 expression. HIF-1α inhibition decreases MUC2 production and mucinous tumor growth, providing a preclinical rationale for the use of HIF-1α inhibitors to treat patients with PMP.


Assuntos
Hipóxia Celular , Mucina-2/biossíntese , Pseudomixoma Peritoneal/terapia , Animais , Linhagem Celular Tumoral , Xenoenxertos , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Camundongos , Pseudomixoma Peritoneal/metabolismo , RNA Interferente Pequeno/genética
5.
Transl Res ; 166(4): 344-54, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25890193

RESUMO

Excessive accumulation of mucin 2 (MUC2) protein (a gel-forming secreted mucin) within the peritoneal cavity is the major cause of morbidity and mortality in pseudomyxoma peritonei (PMP), a unique mucinous malignancy of the appendix. Mitogen-activated protein kinase (MAPK) signaling pathway is upregulated in PMP and has been shown to modulate MUC2 promoter activity. We hypothesized that targeted inhibition of the MAPK pathway would be a novel, effective, and safe therapeutic strategy to reduce MUC2 production and mucinous tumor growth. We tested RDEA119, a specific MEK1/2 (MAPK extracellular signal-regulated kinase [ERK] kinase) inhibitor, in MUC2-secreting LS174T cells, human PMP explant tissue, and in a unique intraperitoneal murine xenograft model of PMP. RDEA119 reduced ERK1/2 phosphorylation and inhibited MUC2 messenger RNA and protein expression in vitro. In the xenograft model, chronic oral therapy with RDEA119 inhibited mucinous tumor growth in an MAPK pathway-dependent manner and this translated into a significant improvement in survival. RDEA119 downregulated phosphorylated ERK1/2 and nuclear factor κB p65 protein signaling and reduced activating protein 1 (AP1) transcription factor binding to the MUC2 promoter in LS174T cells. This study provides a preclinical rationale for the use of MEK inhibitors to treat patients with PMP.


Assuntos
Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Mucina-2/biossíntese , Neoplasias Císticas, Mucinosas e Serosas/enzimologia , Neoplasias Císticas, Mucinosas e Serosas/patologia , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Difenilamina/análogos & derivados , Difenilamina/farmacologia , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos Nus , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Mucina-2/genética , NF-kappa B/metabolismo , Neoplasias Peritoneais/metabolismo , Neoplasias Peritoneais/patologia , Regiões Promotoras Genéticas/genética , Inibidores de Proteínas Quinases/farmacologia , Pseudomixoma Peritoneal/metabolismo , Pseudomixoma Peritoneal/patologia , Sulfonamidas/farmacologia , Análise de Sobrevida , Fator de Transcrição AP-1/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
6.
Exp Cell Res ; 320(2): 233-46, 2014 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-24211352

RESUMO

The Eph and Ephrin proteins, which constitute the largest family of receptor tyrosine kinases, are involved in normal tissue development and cancer progression. Here, we examined the expression and role of the B-type Eph receptor EphB2 in breast cancers. By immunohistochemistry using a progression tissue microarray of human clinical samples, we found EphB2 to be expressed in benign tissues, but strongly increased in cancers particularly in invasive and metastatic carcinomas. Subsequently, we found evidence that EphB2, whose expression varies in established cell breast lines, possesses multiple functions. First, the use of a DOX-inducible system to restore EphB2 function to low expressers resulted in decreased tumor growth in vitro and in vivo, while its siRNA-mediated silencing in high expressers increased growth. This function involves the onset of apoptotic death paralleled by caspases 3 and 9 activation. Second, EphB2 was also found to induce autophagy, as assessed by immunofluorescence and/or immunoblotting examination of the LC3, ATG5 and ATG12 markers. Third, EphB2 also has a pro-invasive function in breast cancer cells that involves the regulation of MMP2 and MMP9 metalloproteases and can be blocked by treatment with respective neutralizing antibodies. Furthermore, EphB2-induced invasion is kinase-dependent and is impeded in cells expressing a kinase-dead mutant EphB2. In summary, we identified a mechanism involving a triple role for EphB2 in breast cancer progression, whereby it regulates apoptosis, autophagy, and invasion.


Assuntos
Apoptose/genética , Autofagia/genética , Neoplasias da Mama/patologia , Receptor EphB2/fisiologia , Animais , Neoplasias da Mama/genética , Células Cultivadas , Progressão da Doença , Feminino , Humanos , Células MCF-7 , Camundongos , Camundongos Nus , Invasividade Neoplásica , Receptor EphB2/genética , Células Tumorais Cultivadas
7.
Cell Commun Signal ; 11(1): 20, 2013 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-23521888

RESUMO

BACKGROUND: Gab1 (Grb2-associated binder 1) is a key coordinator that belongs to the insulin receptor substrate-1 like family of adaptor molecules and is tyrosine phosphorylated in response to various growth factors, cytokines, and numerous other molecules. Tyrosine phosphorylated Gab1 is able to recruit a number of signaling effectors including PI3K, SHP2 and PLC-γ. In this study, we characterized the localization and regulation of tyrosine phosphorylation of Gab1 in the retina. RESULTS: Our immuno localization studies suggest that Gab1 is expressed in rod photoreceptor inner segments. We found that hydrogen peroxide activates the tyrosine phosphorylation of Gab1 ex vivo and hydrogen peroxide has been shown to inhibit the protein tyrosine phosphatase PTP1B activity. We found a stable association between the D181A substrate trap mutant of PTP1B and Gab1. Our studies suggest that PTP1B interacts with Gab1 through Tyrosine 83 and this residue may be the major PTP1B target residue on Gab1. We also found that Gab1 undergoes a light-dependent tyrosine phosphorylation and PTP1B regulates the phosphorylation state of Gab1. Consistent with these observations, we found an enhanced Gab1 tyrosine phosphorylation in PTP1B deficient mice and also in retinas treated ex vivo with a PTP1B specific allosteric inhibitor. CONCLUSIONS: Our laboratory has previously reported that retinas deficient of PTP1B are resistant to light damage compared to wild type mice. Since Gab1 is negatively regulated by PTP1B, a part of the retinal neuroprotective effect we have observed previously in PTP1B deficient mice could be contributed by Gab1 as well. In summary, our data suggest that PTP1B regulates the phosphorylation state of retinal Gab1 in vivo.

8.
Invest Ophthalmol Vis Sci ; 49(11): 4765-73, 2008 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-18566464

RESUMO

PURPOSE: The authors previously reported that physiological light induces the tyrosine phosphorylation of insulin receptors (IRs), which leads to the activation of the phosphoinositide 3-kinase (PI3K) and Akt (serine/threonine protein kinase B) survival pathway in rod photoreceptor cells. Tissue-specific deletion of IRs from photoreceptors resulted in stress-induced photoreceptor degeneration. Insulin growth factor 1 receptor (IGF-1R) is highly related in sequence and structure to the IR and shares 70% sequence identity overall and 84% identity within the tyrosine kinase domain. The role of IGF-1R in photoreceptor function is unknown. In this study the authors examined IGF-1R signaling in rod outer segment (ROS) membranes. METHODS: IGF-1R localization was examined in the plasma and disc membranes of ROS. Activation of the IGF-1R/PI3K/Akt pathway was analyzed using specific antibodies against phospho-tyrosine, IGF-1R, and phospho-Akt. PI3K activity was determined in the anti-phospho-tyrosine and anti-IGF-1R immunoprecipitates. Glutathione-S-transferase fusion proteins containing two Src homology 2 (SH2) domains of the p85 subunit of PI3K and their mutants were used to study the molecular interaction between IGF-1R and p85. In vivo IGF-1R signaling was studied in rats exposed to physiological light or to constant light. RESULTS: IGF-1R is predominately localized to plasma membranes of ROS. These studies indicate that light stress results in an increase in tyrosine phosphorylation of IGF-1R and an increase in PI3K enzyme activity in anti-phosphotyrosine and anti-IGF-1R immunoprecipitates of ROS and retinal homogenates. The authors observed that light stress induces tyrosine phosphorylation of IGF-1R in ROS membranes, which leads to the binding of p85 through N-SH2 and C-SH2 domains. Finally, the authors observed a significant activation of Akt in light-stressed retinas, indicating activation of the Akt survival pathway downstream of IGF-1R activation. CONCLUSIONS: Light stress induced the activation of PI3K through activation and binding of IGF-1R, which leads to activation of the Akt survival pathway in photoreceptors.


Assuntos
Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor IGF Tipo 1/metabolismo , Segmento Externo da Célula Bastonete/metabolismo , Segmento Externo da Célula Bastonete/patologia , Transdução de Sinais/fisiologia , Estresse Psicológico/metabolismo , Animais , Western Blotting , Sobrevivência Celular/fisiologia , Modelos Animais de Doenças , Eletroforese em Gel de Poliacrilamida , Imunoprecipitação , Estimulação Luminosa , Ratos , Ratos Sprague-Dawley , Estresse Psicológico/patologia
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