RESUMO
Renal medullary carcinoma (RMC) is an aggressive kidney cancer that almost exclusively develops in individuals with sickle cell trait (SCT) and is always characterized by loss of the tumor suppressor SMARCB1. Because renal ischemia induced by red blood cell sickling exacerbates chronic renal medullary hypoxia in vivo, we investigated whether the loss of SMARCB1 confers a survival advantage under the setting of SCT. Hypoxic stress, which naturally occurs within the renal medulla, is elevated under the setting of SCT. Our findings showed that hypoxia-induced SMARCB1 degradation protected renal cells from hypoxic stress. SMARCB1 wild-type renal tumors exhibited lower levels of SMARCB1 and more aggressive growth in mice harboring the SCT mutation in human hemoglobin A (HbA) than in control mice harboring wild-type human HbA. Consistent with established clinical observations, SMARCB1-null renal tumors were refractory to hypoxia-inducing therapeutic inhibition of angiogenesis. Further, reconstitution of SMARCB1 restored renal tumor sensitivity to hypoxic stress in vitro and in vivo. Together, our results demonstrate a physiological role for SMARCB1 degradation in response to hypoxic stress, connect the renal medullary hypoxia induced by SCT with an increased risk of SMARCB1-negative RMC, and shed light into the mechanisms mediating the resistance of SMARCB1-null renal tumors against angiogenesis inhibition therapies.
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Carcinoma de Células Renais , Neoplasias Renais , Traço Falciforme , Animais , Humanos , Camundongos , Carcinoma de Células Renais/patologia , Hipóxia/genética , Hipóxia/metabolismo , Rim/metabolismo , Neoplasias Renais/patologia , Traço Falciforme/genética , Traço Falciforme/metabolismo , Proteína SMARCB1/genética , Proteína SMARCB1/metabolismoRESUMO
Hepatocellular carcinoma (HCC) is one of the most lethal and chemo-refractory cancers, clearly, alternative treatment strategies are needed. We utilized 10nm gold nanoparticles as a scaffold to synthesize nanoconjugates bearing a targeting antibody (cetuximab, C225) and gemcitabine. Loading efficiency of gemcitabine on the gold nanoconjugates was 30%. Targeted gold nanoconjugates in combination with RF were selectively cytotoxic to EGFR expressing Hep3B and SNU449 cells when compared to isotype particles with/without RF (P<0.05). In animal experiments, targeted gold nanoconjugates halted the growth of subcutaneous Hep3B xenografts in combination with RF exposure (P<0.05). These xenografts also demonstrated increased apoptosis, necrosis and decreased proliferation compared to controls. Normal tissues were unharmed. We have demonstrated that non-invasive RF-induced hyperthermia when combined with targeted delivery of gemcitabine is more effective and safe at dosages ~275-fold lower than the current clinically-delivered systemic dose of gemcitabine. FROM THE CLINICAL EDITOR: In a model of hepatocellular carcinoma, the authors demonstrate that non-invasive RF-induced hyperthermia applied with cetuximab targeted delivery of Au NP-gemcitabine conjugate is more effective and safe at dosages ~ 275-fold lower than the current clinically-used systemic dose of gemcitabine.
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Anticorpos Monoclonais Humanizados/uso terapêutico , Carcinoma Hepatocelular/terapia , Desoxicitidina/análogos & derivados , Ouro/uso terapêutico , Neoplasias Hepáticas/terapia , Nanoconjugados/uso terapêutico , Animais , Anticorpos Monoclonais Humanizados/química , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Cetuximab , Desoxicitidina/química , Desoxicitidina/uso terapêutico , Sistemas de Liberação de Medicamentos , Ouro/química , Humanos , Hipertermia Induzida , Fígado/efeitos dos fármacos , Fígado/patologia , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/patologia , Nanopartículas Metálicas/química , Nanopartículas Metálicas/uso terapêutico , Camundongos Endogâmicos BALB C , Nanoconjugados/química , GencitabinaRESUMO
Mesenchymal plasticity has been extensively described in advanced and metastatic epithelial cancers; however, its functional role in malignant progression, metastatic dissemination and therapy response is controversial. More importantly, the role of epithelial mesenchymal transition (EMT) and cell plasticity in tumor heterogeneity, clonal selection and clonal evolution is poorly understood. Functionally, our work clarifies the contribution of EMT to malignant progression and metastasis in pancreatic cancer. We leveraged ad hoc somatic mosaic genome engineering, lineage tracing and ablation technologies and dynamic genetic reporters to trace and ablate tumor-specific lineages along the phenotypic spectrum of epithelial to mesenchymal plasticity. The experimental evidences clarify the essential contribution of mesenchymal lineages to pancreatic cancer evolution and metastatic dissemination. Spatial genomic analysis combined with single cell transcriptomic and epigenomic profiling of epithelial and mesenchymal lineages reveals that EMT promotes with the emergence of chromosomal instability (CIN). Specifically tumor lineages with mesenchymal features display highly conserved patterns of genomic evolution including complex structural genomic rearrangements and chromotriptic events. Genetic ablation of mesenchymal lineages robustly abolished these mutational processes and evolutionary patterns, as confirmed by cross species analysis of pancreatic and other human epithelial cancers. Mechanistically, we discovered that malignant cells with mesenchymal features display increased chromatin accessibility, particularly in the pericentromeric and centromeric regions, which in turn results in delayed mitosis and catastrophic cell division. Therefore, EMT favors the emergence of high-fitness tumor cells, strongly supporting the concept of a cell-state, lineage-restricted patterns of evolution, where cancer cell sub-clonal speciation is propagated to progenies only through restricted functional compartments. Restraining those evolutionary routes through genetic ablation of clones capable of mesenchymal plasticity and extinction of the derived lineages completely abrogates the malignant potential of one of the most aggressive form of human cancer.
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Molecular routes to metastatic dissemination are critical determinants of aggressive cancers. Through in vivo CRISPR-Cas9 genome editing, we generated somatic mosaic genetically engineered models that faithfully recapitulate metastatic renal tumors. Disruption of 9p21 locus is an evolutionary driver to systemic disease through the rapid acquisition of complex karyotypes in cancer cells. Cross-species analysis revealed that recurrent patterns of copy number variations, including 21q loss and dysregulation of the interferon pathway, are major drivers of metastatic potential. In vitro and in vivo genomic engineering, leveraging loss-of-function studies, along with a model of partial trisomy of chromosome 21q, demonstrated a dosage-dependent effect of the interferon receptor genes cluster as an adaptive mechanism to deleterious chromosomal instability in metastatic progression. This work provides critical knowledge on drivers of renal cell carcinoma progression and defines the primary role of interferon signaling in constraining the propagation of aneuploid clones in cancer evolution.
Assuntos
Carcinoma de Células Renais , Neoplasias Renais , Humanos , Carcinoma de Células Renais/genética , Variações do Número de Cópias de DNA/genética , Instabilidade Cromossômica/genética , Aneuploidia , Neoplasias Renais/genéticaRESUMO
BACKGROUND: Several studies have reported targeted hyperthermia at the cellular level using remote activation of nanoparticles by radiofrequency waves. To date, methods to quantify intracellular thermal dose have not been reported. In this report we study the relationship between radio wave exposure and luciferase denaturation with and without intracellular nanoparticles. The findings are used to devise a strategy to quantify targeted thermal dose in a primary human liver cancer cell line. METHODS: Water bath or non-invasive external Kanzius RF generator (600 W, 13.56 MHz) was used for hyperthermia exposures. Luciferase activity was measured using a bioluminescence assay and viability was assessed using Annexin V-FITC and propidium iodide staining. Heat shock proteins were analysed using western blot analysis. RESULTS: Duration-dependent luciferase denaturation was observed in SNU449 cells exposed to RF field that preceded measurable loss in viability. Loss of luciferase activity was higher in cetuximab-conjugated gold nanoparticle (C225-AuNP) treated cells. Using a standard curve from water bath experiments, the intracellular thermal dose was calculated. Cells treated with C225-AuNP accumulated 6.07 times higher intracellular thermal dose than the untreated controls over initial 4 min of RF exposure. CONCLUSION: Cancer cells when exposed to an external RF field exhibit dose-dependent protein denaturation. Luciferase denaturation assay can be used to quantify thermal dose delivered after RF exposures to cancer cells with and without nanoparticles.
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Anticorpos Monoclonais/administração & dosagem , Hipertermia Induzida/métodos , Luciferases/metabolismo , Desnaturação Proteica , Anticorpos Monoclonais Humanizados , Linhagem Celular Tumoral , Cetuximab , Ouro/uso terapêutico , Humanos , Medições Luminescentes , Nanopartículas Metálicas/uso terapêutico , Terapia por Radiofrequência , TermômetrosRESUMO
The use of noninvasive radiofrequency (RF) electric fields as an energy source for thermal activation of nanoparticles within cancer cells could be a valuable addition to the emerging field of nano-mediated cancer therapies. Based on investigations of cell death through hyperthermia, and offering the ability for total-body penetration by RF fields, this technique is thought to complement and possibly outperform existing nano-heat treatments that utilize alternative heat production via optical or magnetic stimuli. However, it remains a challenge to understand fully the complex RF-nanoparticle-intracellular interactions before full system optimization can be engineered. Herein we have shown that liver cancer cells can selectively internalize antibody-conjugated gold nanoparticles (AuNPs) through receptor-mediated endocytosis, with the nanoparticles predominantly accumulating and aggregating within cytoplasmic endolysosomes. After exposure to an external RF field, nonaggregated AuNPs absorbed and dissipated energy as heat, causing thermal damage to the targeted cancer cells. We also observed that RF absorption and heat dissipation is dependent on solubility of AuNPs in the colloid, which is pH dependent. Furthermore, by modulating endolysosomal pH it is possible to prevent intracellular AuNP aggregation and enhance thermal cytotoxicity in hepatocellular cancer cells. FROM THE CLINICAL EDITOR: Gold nanoparticles absorb energy from RF fields and can exert hyperthermic effects leading to cell death. Combining this known effect with antibody-based targeting of the nanoparticles, selective cancer specific hyperthermia induced cell death therapies can be designed, as demonstrated in this article.
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Ouro/uso terapêutico , Hipertermia Induzida/métodos , Imunoconjugados/uso terapêutico , Neoplasias Hepáticas/terapia , Nanopartículas/uso terapêutico , Terapia por Radiofrequência , Anticorpos/química , Anticorpos/uso terapêutico , Linhagem Celular Tumoral , Ouro/química , Humanos , Concentração de Íons de Hidrogênio , Imunoconjugados/química , Fígado/metabolismo , Fígado/patologia , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Lisossomos/metabolismo , Lisossomos/patologia , Nanopartículas/química , SolubilidadeRESUMO
Pancreatic ductal adenocarcinoma (PDAC) is almost universally lethal. A critical unmet need exists to explore essential susceptibilities in PDAC and to identify druggable targets to improve PDAC treatment. KRAS mutations dominate the genetic landscape of PDAC and lead to activation of multiple downstream pathways and cellular processes. Here, we investigated the requirement of these pathways for tumor maintenance using an inducible KrasG12D -driven PDAC mouse model (iKras model), identifying that RAF-MEK-MAPK signaling is the major effector for oncogenic KRAS-mediated tumor maintenance. However, consistent with previous studies, MEK inhibition had minimal therapeutic effect as a single agent for PDAC in vitro and in vivo. Although MEK inhibition partially downregulated transcription of glycolysis genes, it failed to suppress glycolytic flux in PDAC cells, which is a major metabolic effector of oncogenic KRAS. Accordingly, an in vivo genetic screen identified multiple glycolysis genes as potential targets that may sensitize tumor cells to MEK inhibition. Inhibition of glucose metabolism with low-dose 2-deoxyglucose in combination with a MEK inhibitor induced apoptosis in KrasG12D -driven PDAC cells in vitro. The combination also inhibited xenograft PDAC tumor growth and prolonged overall survival in a genetically engineered PDAC mouse model. Molecular and metabolic analyses indicated that co-targeting glycolysis and MAPK signaling results in apoptosis via induction of lethal endoplasmic reticulum stress. Together, our work suggests that combined inhibition of glycolysis and the MAPK pathway may serve as an effective approach to target KRAS-driven PDAC. SIGNIFICANCE: This study demonstrates the critical role of glucose metabolism in resistance to MAPK inhibition in KRAS-driven pancreatic cancer, uncovering a potential therapeutic approach for treating this aggressive disease.
Assuntos
Adenocarcinoma/tratamento farmacológico , Carcinoma Ductal Pancreático/tratamento farmacológico , Glucose/metabolismo , Inibidores de Proteínas Quinases/uso terapêutico , Animais , Humanos , Camundongos , Inibidores de Proteínas Quinases/farmacologiaRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
Transcriptomic profiling classifies pancreatic ductal adenocarcinoma (PDAC) into several molecular subtypes with distinctive histological and clinical characteristics. However, little is known about the molecular mechanisms that define each subtype and their correlation with clinical outcome. Mutant KRAS is the most prominent driver in PDAC, present in over 90% of tumors, but the dependence of tumors on oncogenic KRAS signaling varies between subtypes. In particular, the squamous subtype is relatively independent of oncogenic KRAS signaling and typically displays much more aggressive clinical behavior versus the progenitor subtype. Here, we identified that yes-associated protein 1 (YAP1) activation is enriched in the squamous subtype and associated with poor prognosis. Activation of YAP1 in progenitor subtype cancer cells profoundly enhanced malignant phenotypes and transformed progenitor subtype cells into squamous subtype. Conversely, depletion of YAP1 specifically suppressed tumorigenicity of squamous subtype PDAC cells. Mechanistically, we uncovered a significant positive correlation between WNT5A expression and YAP1 activity in human PDAC and demonstrated that WNT5A overexpression led to YAP1 activation and recapitulated a YAP1-dependent but Kras-independent phenotype of tumor progression and maintenance. Thus, our study identifies YAP1 oncogene as a major driver of squamous subtype PDAC and uncovers the role of WNT5A in driving PDAC malignancy through activation of the YAP pathway.
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Proteínas Adaptadoras de Transdução de Sinal/genética , Carcinoma Ductal Pancreático/genética , Oncogenes , Neoplasias Pancreáticas/genética , Fatores de Transcrição/genética , Carcinoma Ductal Pancreático/patologia , Humanos , Neoplasias Pancreáticas/patologia , Proteína Wnt-5a/genética , Proteínas de Sinalização YAPRESUMO
Triple-negative breast cancer (TNBC), which lacks estrogen receptor α (ERα), progesterone receptor, and human epidermal growth factor receptor 2 (HER2) expression, is closely related to basal-like breast cancer. Previously, we and others report that cyclin E/cyclin-dependent kinase 2 (CDK2) phosphorylates enhancer of zeste homolog 2 (EZH2) at T416 (pT416-EZH2). Here, we show that transgenic expression of phospho-mimicking EZH2 mutant EZH2T416D in mammary glands leads to tumors with TNBC phenotype. Coexpression of EZH2T416D in mammary epithelia of HER2/Neu transgenic mice reprograms HER2-driven luminal tumors into basal-like tumors. Pharmacological inhibition of CDK2 or EZH2 allows re-expression of ERα and converts TNBC to luminal ERα-positive, rendering TNBC cells targetable by tamoxifen. Furthermore, the combination of either CDK2 or EZH2 inhibitor with tamoxifen effectively suppresses tumor growth and markedly improves the survival of the mice bearing TNBC tumors, suggesting that the mechanism-based combination therapy may be an alternative approach to treat TNBC.
Assuntos
Quinase 2 Dependente de Ciclina/metabolismo , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Receptor alfa de Estrogênio/efeitos dos fármacos , Neoplasias Mamárias Experimentais/genética , Neoplasias de Mama Triplo Negativas/genética , Animais , Benzamidas/farmacologia , Compostos de Bifenilo , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Óxidos N-Cíclicos , Quinase 2 Dependente de Ciclina/antagonistas & inibidores , Proteína Potenciadora do Homólogo 2 de Zeste/antagonistas & inibidores , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Feminino , Humanos , Indolizinas , Glândulas Mamárias Humanas/efeitos dos fármacos , Glândulas Mamárias Humanas/metabolismo , Neoplasias Mamárias Experimentais/metabolismo , Camundongos , Camundongos Transgênicos , Morfolinas , Fosforilação , Compostos de Piridínio/farmacologia , Piridonas/farmacologia , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Receptores de Progesterona/metabolismo , Neoplasias de Mama Triplo Negativas/metabolismoRESUMO
Pancreatic ribonuclease (RNase) is a secreted enzyme critical for host defense. We discover an intrinsic RNase function, serving as a ligand for epidermal growth factor receptor (EGFR), a member of receptor tyrosine kinase (RTK), in pancreatic ductal adenocarcinoma (PDAC). The closely related bovine RNase A and human RNase 5 (angiogenin [ANG]) can trigger oncogenic transformation independently of their catalytic activities via direct association with EGFR. Notably, high plasma ANG level in PDAC patients is positively associated with response to EGFR inhibitor erlotinib treatment. These results identify a role of ANG as a serum biomarker that may be used to stratify patients for EGFR-targeted therapies, and offer insights into the ligand-receptor relationship between RNase and RTK families.
Assuntos
Carcinoma Ductal Pancreático/patologia , Cloridrato de Erlotinib/farmacologia , Neoplasias Pancreáticas/patologia , Ribonuclease Pancreático/sangue , Ribonuclease Pancreático/metabolismo , Animais , Sítios de Ligação , Biomarcadores/sangue , Carcinoma Ductal Pancreático/sangue , Carcinoma Ductal Pancreático/tratamento farmacológico , Bovinos , Linhagem Celular Tumoral , Receptores ErbB/química , Receptores ErbB/metabolismo , Cloridrato de Erlotinib/uso terapêutico , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HeLa , Humanos , Camundongos , Transplante de Neoplasias , Neoplasias Pancreáticas/sangue , Neoplasias Pancreáticas/tratamento farmacológico , Ribonuclease Pancreático/química , Transdução de SinaisRESUMO
BACKGROUND: Gemcitabine is a potent nucleoside analogue against solid tumors, but development of drug resistance is a substantial problem. Removal of gemcitabine incorporated into DNA by repair mechanisms may contribute to resistance in chemo-refractory solid tumors. Human hepatocellular carcinoma (HCC) is usually very chemoresistant to gemcitabine. METHODS: We treated HCC in vitro and in vivo (orthotopic murine model with human Hep3B or HepG2 xenografts, 7-10 CB17SCID mice per group) with gemcitabine. The role of homologous recombination repair proteins in repairing stalled replication forks was evaluated with hyperthermia exposure and cell-cycle analysis. The Student t-test was used for two-sample comparisons. Multiple group data were analyzed using one-way analysis of variance. All statistical tests were two-sided. RESULTS: We demonstrated that Mre11-mediated homologous recombination repair of gemcitabine-stalled replication forks is crucial to survival of HCC cells. Furthermore, we demonstrated inhibition of Mre11 by an exonuclease inhibitor or concomitant hyperthermia. In orthotopic murine models of chemoresistant HCC, the Hep3B tumor mass with radiofrequency plus gemcitabine treatment (mean ± SD, 180±91mg) was statistically significantly smaller compared with gemcitabine alone (661±419mg, P = .0063). CONCLUSIONS: This study provides mechanistic understanding of homologous recombination inhibiting-strategies, such as noninvasive radiofrequency field-induced hyperthermia, to overcome resistance to gemcitabine in refractory human solid tumors.
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Antimetabólitos Antineoplásicos/farmacologia , Carcinoma Hepatocelular/terapia , Replicação do DNA/efeitos dos fármacos , DNA de Neoplasias/efeitos dos fármacos , Proteínas de Ligação a DNA/antagonistas & inibidores , Desoxicitidina/análogos & derivados , Hipertermia Induzida/métodos , Neoplasias Hepáticas/terapia , Ondas de Rádio , Reparo de DNA por Recombinação/efeitos dos fármacos , Animais , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/genética , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Replicação do DNA/genética , DNA de Neoplasias/genética , Desoxicitidina/farmacologia , Modelos Animais de Doenças , Resistencia a Medicamentos Antineoplásicos , Exonucleases/antagonistas & inibidores , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/genética , Proteína Homóloga a MRE11 , Camundongos , Camundongos SCID , Neoplasias Experimentais/terapia , Terapia por Radiofrequência , GencitabinaRESUMO
AIM: Although thioredoxin 1 (TXN) has pleiotropic cellular functions as a redox-sensitive protein, very little is known about its role in tumor survival and growth under hypoxia. MHCC97H hepatocellular carcinoma cells have a high metastatic potential and high thioredoxin expression levels compared with their parent cell line, MHCC97. Thus, we used this cell line to explore the functional connections between TXN and hypoxia. METHODS: MHCC97H cells were cultured under normoxia and hypoxia for specific periods after nucleofection with TXN siRNA or control siRNA. We assessed the ß-phenylethyl isothiocyanate (PEITC) sensitivity of the cells, cell proliferation, cell cycle and senescence, and DNA damage response by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays, colony formation assays, flow cytometry, ß-galactosidase staining, western blotting, and immunohistochemistry. RESULTS: ß-phenylethyl isothiocyanate treatment shifted reduced TXN to oxidized TXN in MHCC97H cells. Although silencing of TXN via siRNA had no effect on the PEITC sensitivity of the cells, it suppressed cell proliferation and colony formation under both normoxia and hypoxia. Under hypoxia, silencing TXN did not induce apoptosis but induced DNA damage response and cellular senescence. CONCLUSIONS: High TXN levels in MHCC97H cells protect them from DNA damage and cellular senescence under hypoxia. Targeting TXN might enhance the chemotherapeutic effects of some DNA-damaging agents against hepatocellular carcinoma.
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Hepatocellular carcinoma (HCC) and pancreatic carcinoma (PC) cells often have inherent urea cycle defects rendering them auxotrophic for the amino acid l-arginine (l-arg). Most HCC and PC require extracellular sources of l-arg and undergo cell cycle arrest and apoptosis when l-arg is restricted. Systemic, enzyme-mediated depletion of l-arg has been investigated in mouse models and human trials. Non-human enzymes elicit neutralizing antibodies, whereas human arginases display poor pharmacological properties in serum. Co(2+) substitution of the Mn(2+) metal cofactor in human arginase I (Co-hArgI) was shown to confer more than 10-fold higher catalytic activity (k(cat)/K(m)) and 5-fold greater stability. We hypothesized that the Co-hArgI enzyme would decrease tumor burden by systemic elimination of l-arg in a murine model. Co-hArgI was conjugated to 5-kDa PEG (Co-hArgI-PEG) to enhance circulation persistence. It was used as monotherapy for HCC and PC in vitro and in vivo murine xenografts. The mechanism of cell death was also investigated. Weekly treatment of 8 mg/kg Co-hArgI-PEG effectively controlled human HepG2 (HCC) and Panc-1 (PC) tumor xenografts (P = .001 and P = .03, respectively). Both cell lines underwent apoptosis in vitro with significant increased expression of activated caspase-3 (P < .001). Furthermore, there was evidence of autophagy in vitro and in vivo. We have demonstrated that Co-hArgI-PEG is effective at controlling two types of l-arg-dependent carcinomas. Being a nonessential amino acid, arginine deprivation therapy through Co-hArgI-PEG holds promise as a new therapy in the treatment of HCC and PC.
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There is a paucity of data regarding the safety of administering solid gold nanoparticles (AuNPs) in large animal tumor models. We assessed the acute toxicity and biodistribution of 5 nm and 25 nm solid AuNPs in New Zealand White rabbits (n = 6 in each) with implanted liver Vx2 tumors 24 h after intravenous injection. Gold concentration was determined by inductively coupled plasma atomic emission spectrometry (ICP) and imaged with transmission electron microscopy (TEM). There was no clinico-pathologic evidence of renal, hepatic, pulmonary, or other organ dysfunction. After 25 nm AuNP administration, the concentration of white blood cells increased after treatment (p = 0.001). Most other blood studies were unchanged. AuNPs were distributed to the spleen, liver, and Vx2 tumors, but not to other tissues. The urinary excretion of AuNPs was bimodal as measured by ICP. 25 nm AuNPs were more evenly distributed throughout tissues and may be better tools for medical therapy.
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Ouro/farmacocinética , Ouro/toxicidade , Neoplasias Hepáticas Experimentais/metabolismo , Nanopartículas Metálicas/toxicidade , Animais , Contagem de Células Sanguíneas , Ouro/administração & dosagem , Ouro/urina , Histocitoquímica , Injeções Intravenosas , Neoplasias Hepáticas Experimentais/química , Neoplasias Hepáticas Experimentais/patologia , Espectrometria de Massas , Nanopartículas Metálicas/administração & dosagem , Microscopia Eletrônica de Transmissão , Tamanho da Partícula , Coelhos , Distribuição TecidualRESUMO
PURPOSE: The resistance of tumors to antiangiogenic therapies is becoming increasingly relevant. There are currently no validated predictive biomarkers for selecting which cancer patients will benefit from antiangiogenic therapy. Also lacking are resistance biomarkers that can identify which escape pathways should be targeted after tumors develop resistance to VEGF treatment. Recent studies showed that anti-VEGF treatment can make tumor cells more aggressive and metastatic. However, the mechanisms and mediators of this are unidentified. Therefore, we aimed this study at directly identifying the tumor cell-initiated mechanisms responsible for the resistance of pancreatic cancer to anti-VEGF treatment. EXPERIMENTAL DESIGN: We established and validated two murine models of human pancreatic cancer resistant to the VEGF-specific antibody bevacizumab in vivo. We used a genome-wide analysis to directly identify which tumor-secreted factors were overexpressed by pancreatic cancer cells that were resistant to anti-VEGF treatment. RESULTS: Rather than direct proangiogenic factors, we identified several proinflammatory factors that were expressed at higher levels in cells resistant to anti-VEGF treatment than in treatment-sensitive control cells. These proinflammatory factors acted in a paracrine manner to stimulate the recruitment of CD11b(+) proangiogenic myeloid cells. Also, we found that secreted factors overexpressed by anti-VEGF treatment-resistant pancreatic cancer cells acted in an autocrine manner to induce epithelial-to-mesenchymal transition (EMT) and were thus responsible for increased aggressiveness of bevacizumab-resistant pancreatic tumors. CONCLUSIONS: Our results identified proinflammatory factors and EMT markers as potential biomarkers for selecting patients with pancreatic cancer for antiangiogenic therapy.
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Inibidores da Angiogênese/uso terapêutico , Anticorpos Monoclonais Humanizados/uso terapêutico , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/fisiopatologia , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Animais , Bevacizumab , Biomarcadores Tumorais/análise , Antígeno CD11b/biossíntese , Linhagem Celular Tumoral , Quimiocinas/biossíntese , Citocinas/biossíntese , Progressão da Doença , Resistencia a Medicamentos Antineoplásicos , Feminino , Estudo de Associação Genômica Ampla , Humanos , Camundongos , Camundongos Nus , Análise de Sequência com Séries de Oligonucleotídeos , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Transplante Heterólogo , Evasão Tumoral/genéticaRESUMO
BACKGROUND: Gold and carbon nanoparticles absorb nonionizing radio frequency (RF) energy and release heat. Solid gold nanoparticles are delivered to cancer cells via conjugation with targeting antibodies. Here, 20-nm gold particles were conjugated to cetuximab, which is an epidermal growth factor receptor-1 (EGFR-1) antibody. METHODS: A pancreatic carcinoma cell line that highly expresses EGFR-1, Panc-1, and Cama-1, which is a breast carcinoma cell line that minimally expresses EGFR-1, were treated with 100-nmol/L cetuximab-conjugated gold nanoparticles for 3 h (n = 4). Thirty-six hours later, the dishes were placed in an RF field with a generator power of 200 W for 5 min. After another 36 h, cell injury and death were evaluated with flow cytometry. RESULTS: The targeted cell line Panc-1 had a viability of 46% +/- 12%, whereas the Cama-1 cell had a viability of 92% +/- 2% after RF field exposure (P < .008). Transmission electron microscopy showed gold nanoparticle uptake in Panc-1 cells but negligible uptake by Cama-1 cells. Nontargeted cells do not internalize a sufficient amount of antibody-conjugated gold nanoparticles to induce injury in a noninvasive RF field. CONCLUSION: This technique could be useful in cancer treatment if a cancer-specific antibody is used to localize gold nanoparticles to malignant cells.
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Nanopartículas Metálicas/uso terapêutico , Neoplasias Pancreáticas/terapia , Terapia por Radiofrequência , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/uso terapêutico , Anticorpos Monoclonais Humanizados , Linhagem Celular Tumoral , Sobrevivência Celular , Cetuximab , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/metabolismo , Ouro/administração & dosagem , Ouro/uso terapêutico , Humanos , Hipertermia Induzida , Nanopartículas Metálicas/administração & dosagem , Microscopia Eletrônica de Transmissão , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologiaRESUMO
PURPOSE: Pancreatic carcinoma is one of the deadliest cancers with few effective treatments. Gold nanoparticles (AuNP) are potentially therapeutic because of the safety demonstrated thus far and their physiochemical characteristics. We used the astounding heating rates of AuNPs in nonionizing radiofrequency (RF) radiation to investigate human pancreatic xenograft destruction in a murine model. EXPERIMENTAL DESIGN: Weekly, Panc-1 and Capan-1 human pancreatic carcinoma xenografts in immunocompromised mice were exposed to an RF field 36 hours after treatment (intraperitoneal) with cetuximab- or PAM4 antibody-conjugated AuNPs, respectively. Tumor sizes were measured weekly, whereas necrosis and cleaved caspase-3 were investigated with hematoxylin-eosin staining and immunofluorescence, respectively. In addition, AuNP internalization and cytotoxicity were investigated in vitro with confocal microscopy and flow cytometry, respectively. RESULTS: Panc-1 cells demonstrated increased apoptosis with decreased viability after treatment with cetuximab-conjugated AuNPs and RF field exposure (P = 0.00005). Differences in xenograft volumes were observed within 2 weeks of initiating therapy. Cetuximab- and PAM4-conjugated AuNPs demonstrated RF field-induced destruction of Panc-1 and Capan-1 pancreatic carcinoma xenografts after 6 weeks of weekly treatment (P = 0.004 and P = 0.035, respectively). There was no evidence of injury to murine organs. Cleaved caspase-3 and necrosis were both increased in treated tumors. CONCLUSIONS: This study demonstrates a potentially novel cancer therapy by noninvasively inducing intracellular hyperthermia with targeted AuNPs in an RF field. While the therapy is dependent on the specificity of the targeting antibody, normal tissues were without toxicity despite systemic therapy and whole-body RF field exposure.
Assuntos
Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/radioterapia , Anticorpos Monoclonais/administração & dosagem , Ouro/uso terapêutico , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/radioterapia , Terapia por Radiofrequência , Adenocarcinoma/patologia , Animais , Anticorpos Monoclonais Humanizados , Antineoplásicos/administração & dosagem , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Linhagem Celular Tumoral , Cetuximab , Terapia Combinada , Sistemas de Liberação de Medicamentos/métodos , Ouro/administração & dosagem , Ouro/efeitos adversos , Humanos , Nanopartículas Metálicas/administração & dosagem , Nanopartículas Metálicas/efeitos adversos , Nanopartículas Metálicas/uso terapêutico , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias Pancreáticas/patologia , Ondas de Rádio/efeitos adversos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Hepatocellular and pancreatic carcinomas are often auxotrophic for L-arginine, a semi-essential amino acid. The purpose of this study was to investigate cancer cell death using a significantly more active, cobalt-substituted bioengineered arginase. METHODS: Panc-1, a human pancreatic carcinoma cell line, and Hep 3B, a human hepatocellular carcinoma cell line, were exposed to L-arginase. Flow cytometry was used to measure expression of Ki-67, caspase-3, and argininosuccinate synthetase-1 (ASS-1) 4 days after treatment. An MTT assay measured proliferation. The Student t test determined statistical significance. RESULTS: Viability decreased by 31% +/- 2% for Panc-1 cells (P < .0001) and 34% +/- 1% (P < .0001) for Hep 3B cells after treatment. Both cell lines demonstrated a 4-fold increase activated caspase-3 expression after high dose treatment (P < .01), and 5-fold increase in ASS-1 expression (P < .002). Ki-67 expression did not vary in Hep 3B cells but decreased for Panc-1 cells (P < .015). The 50% inhibitory concentration was 8-fold higher for Panc-1 cells than for Hep 3B cells (P < .03). CONCLUSION: Increased ASS-1 expression by these cells, in order to increase L-arginine concentration, is inadequate, suggesting a mechanism by which arginine depletion can be used in multimodality therapy for arginine-dependent cancers.
Assuntos
Arginase/farmacologia , Argininossuccinato Sintase/biossíntese , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/enzimologia , Caspase 3/metabolismo , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/enzimologia , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/enzimologia , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Arginase/administração & dosagem , Arginina/metabolismo , Carcinoma Hepatocelular/patologia , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Indução Enzimática/efeitos dos fármacos , Humanos , Antígeno Ki-67/metabolismo , Neoplasias Hepáticas/patologia , Modelos Biológicos , Neoplasias Pancreáticas/patologia , Engenharia de Proteínas , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/farmacologiaRESUMO
PURPOSE: Development of novel agents and drug combinations are urgently needed for treatment of pancreatic cancer. Oxaliplatin belongs to an important class of DNA-damaging organoplatinum agents, useful in pancreatic cancer therapy. However, increased ability of cancer cells to recognize and repair DNA damage enables resistance to these agents. Poly (ADP ribose) polymerase-1 is a sensor of DNA damage with key roles in DNA repair. Here, we report the therapeutic activity of the poly (ADP ribose) polymerase-1 inhibitor BSI-401, as a single agent and in combination with oxaliplatin in orthotopic nude mouse models of pancreatic cancer, and its effect on oxaliplatin-induced acute neurotoxicity. EXPERIMENTAL DESIGN: We determined in vitro the effect of BSI-401 and its synergism with oxaliplatin on the growth of pancreatic cancer cells. Activity of different dosages of parenteral and oral BSI-401, alone and in combination with oxaliplatin, was evaluated in orthotopic nude mouse models with luciferase-expressing pancreatic cancer cells. The effect of BSI-401 in preventing oxaliplatin-induced acute cold allodynia was measured in rats using a temperature-controlled plate. RESULTS: BSI-401 alone and in synergism with oxaliplatin significantly inhibited the growth of pancreatic cancer cells in vitro. In nude mice, i.p. [200 mg/kg once a week (QW) x 4] and oral [400 mg/kg days 1-5 of each week (QD5 + R2) x 4] administration of BSI-401 significantly reduced tumor burden and prolonged survival (46 versus 144 days, P = 0.0018; 73 versus 194 days, P = 0.0017) compared with no treatment. BSI-401 combined with oxaliplatin had potent synergistic antitumor activity (46 versus 132 days, P = 0.0063), and significantly (P = 0.0148) prevented acute oxaliplatin-induced neurotoxicity. CONCLUSIONS: BSI-401, alone or in combination with oxaliplatin, is a promising new therapeutic agent that warrants further evaluation for treatment of pancreatic cancer.