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UNLABELLED: In nonalcoholic fatty liver disease, the pathogenesis of progression from simple steatosis to steatohepatitis has not been fully clarified. Many factors, including oxidative stress and hepatic immune regulation, contribute to the inflammation in steatosis. Because regulatory T cells (Tregs) are important components of immune regulation, we have now investigated their role in the pathogenesis of nonalcoholic steatohepatitis. Wild-type C57BL/6 mice were fed a high-fat (HF) diet to induce steatosis, and the hepatic lymphocyte population was analyzed by flow cytometry. HF-induced steatosis was associated with the depletion of hepatic Tregs and led to up-regulation of the inflammatory tumor necrosis factor-alpha signaling pathway. When challenged by exogenous lipopolysaccharide, the HF-fed mice developed liver inflammation. In contrast, the adoptive transfer of Tregs decreased inflammation in HF-fed mice. In comparison with effector T cells, Tregs had a lower expression of Bcl-2 and, therefore, increased susceptibility to oxidative stress-induced apoptosis. The treatment of mice with the antioxidant Mn(III)tetrakis(4-benzoic acid)porphyrin chloride reduced Treg apoptosis, increased the number of hepatic Tregs, and decreased hepatic inflammation in HF-fed mice. CONCLUSION: Our results indicate that increased oxidative stress in a fatty liver causes the apoptosis of Tregs, reduces the number of hepatic Tregs, and leads to a lowered suppression of inflammatory responses. This scenario is likely one of the pathogenetic mechanisms that facilitate the transformation of simple steatosis into steatohepatitis when a fatty liver is exposed to second or third hits.
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Gorduras na Dieta/efeitos adversos , Fígado Gorduroso/imunologia , Hepatite/imunologia , Linfócitos T Reguladores/fisiologia , Animais , Antioxidantes/uso terapêutico , Apoptose/fisiologia , Antígenos CD4/metabolismo , Fígado Gorduroso/tratamento farmacológico , Fatores de Transcrição Forkhead/metabolismo , Hepatite/tratamento farmacológico , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Lipopolissacarídeos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Estresse Oxidativo/fisiologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Linfócitos T Reguladores/metabolismoRESUMO
The ability of cancers to evade immune surveillance and resist immunotherapy raises a fundamental question of how tumor cells survive in the presence of a competent immune system. Studies to address this question have primarily focused on mechanisms by which tumor cells avoid recognition by or induce tolerance in the immune system. However, little is known about whether cancer cells also acquire an intrinsic ability to resist killing by immune effectors. We find that cancer cells enhance their ability to withstand an attack by cytotoxic immune effector cells via acquisition of specific genetic alterations that interfere with the shared mitochondrial death signaling pathway entrained by granzyme B, IFN-gamma, and Apo2 ligand/tumor necrosis factor-related apoptosis inducing ligand (Apo2L/TRAIL), three key mediators of immunologic cell-mediated cytotoxicity. We show that the coexistence of specific mitochondrial signaling defects (either deletion of Bax, overexpression of Bcl-x(L), or deletion of Smac) with expression of X-linked inhibitor of apoptosis protein decreases the sensitivity of cancer cells to IFN-gamma/Apo2L/TRAIL- or granzyme B-induced apoptosis, lymphocyte-mediated cytotoxicity in vitro, and adoptive cellular immunotherapy in vivo. Conversely, negating X-linked inhibitor of apoptosis protein expression or function in tumor cells with defective mitochondrial signaling enables direct activation of caspase-3/-7 by granzyme B or Apo2L/TRAIL, and restores their susceptibility to immunologic cytotoxicity. These findings identify an important mechanism by which cancers evade elimination by immune effector cells and suggest that cancer immunotherapy might be improved by concurrent strategies to alleviate or circumvent the intrinsic mitochondrial death signaling defects that help cancer cells resist immunologic cytotoxicity.
Assuntos
Adenocarcinoma/imunologia , Neoplasias do Colo/imunologia , Imunoterapia Adotiva/métodos , Mitocôndrias/imunologia , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/imunologia , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/terapia , Animais , Apoptose/efeitos dos fármacos , Apoptose/imunologia , Proteínas Reguladoras de Apoptose/farmacologia , Caspases/metabolismo , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Neoplasias do Colo/terapia , Citotoxicidade Imunológica , Ativação Enzimática , Feminino , Granzimas , Células HCT116 , Humanos , Interferon gama/imunologia , Interferon gama/farmacologia , Glicoproteínas de Membrana/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Mitocôndrias/enzimologia , Proteínas Recombinantes/farmacologia , Serina Endopeptidases/farmacologia , Ligante Indutor de Apoptose Relacionado a TNF , Transfecção , Fator de Necrose Tumoral alfa/farmacologia , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/biossíntese , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
A majority of cancers fail to respond to immunotherapy with antibodies targeting immune checkpoints, such as cytotoxic T-lymphocyte antigen-4 (CTLA-4) or programmed death-1 (PD-1)/PD-1 ligand (PD-L1). Cancers frequently express transforming growth factor-ß (TGFß), which drives immune dysfunction in the tumor microenvironment by inducing regulatory T cells (Tregs) and inhibiting CD8+ and TH1 cells. To address this therapeutic challenge, we invent bifunctional antibody-ligand traps (Y-traps) comprising an antibody targeting CTLA-4 or PD-L1 fused to a TGFß receptor II ectodomain sequence that simultaneously disables autocrine/paracrine TGFß in the target cell microenvironment (a-CTLA4-TGFßRIIecd and a-PDL1-TGFßRIIecd). a-CTLA4-TGFßRIIecd is more effective in reducing tumor-infiltrating Tregs and inhibiting tumor progression compared with CTLA-4 antibody (Ipilimumab). Likewise, a-PDL1-TGFßRIIecd exhibits superior antitumor efficacy compared with PD-L1 antibodies (Atezolizumab or Avelumab). Our data demonstrate that Y-traps counteract TGFß-mediated differentiation of Tregs and immune tolerance, thereby providing a potentially more effective immunotherapeutic strategy against cancers that are resistant to current immune checkpoint inhibitors.
Assuntos
Anticorpos/uso terapêutico , Neoplasias da Mama/terapia , Imunoterapia , Melanoma/terapia , Fator de Crescimento Transformador beta/imunologia , Animais , Anticorpos/genética , Anticorpos/imunologia , Antígeno B7-H1/genética , Antígeno B7-H1/imunologia , Neoplasias da Mama/genética , Neoplasias da Mama/imunologia , Antígeno CTLA-4/genética , Antígeno CTLA-4/imunologia , Estudos de Coortes , Feminino , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/imunologia , Humanos , Linfócitos do Interstício Tumoral , Melanoma/genética , Melanoma/imunologia , Camundongos , Camundongos Endogâmicos NOD , Receptores de Fatores de Crescimento Transformadores beta/genética , Receptores de Fatores de Crescimento Transformadores beta/imunologia , Linfócitos T Reguladores/imunologia , Células Th1/imunologia , Células Th17/imunologia , Fator de Crescimento Transformador beta/genéticaRESUMO
BACKGROUND: Curcumin, a yellow polyphenol extracted from the rhizome of turmeric (Curcuma longa), has potent anti-cancer properties as demonstrated in a plethora of human cancer cell line and animal carcinogenesis models. Nevertheless, widespread clinical application of this relatively efficacious agent in cancer and other diseases has been limited due to poor aqueous solubility, and consequently, minimal systemic bioavailability. Nanoparticle-based drug delivery approaches have the potential for rendering hydrophobic agents like curcumin dispersible in aqueous media, thus circumventing the pitfalls of poor solubility. RESULTS: We have synthesized polymeric nanoparticle encapsulated formulation of curcumin - nanocurcumin - utilizing the micellar aggregates of cross-linked and random copolymers of N-isopropylacrylamide (NIPAAM), with N-vinyl-2-pyrrolidone (VP) and poly(ethyleneglycol)monoacrylate (PEG-A). Physico-chemical characterization of the polymeric nanoparticles by dynamic laser light scattering and transmission electron microscopy confirms a narrow size distribution in the 50 nm range. Nanocurcumin, unlike free curcumin, is readily dispersed in aqueous media. Nanocurcumin demonstrates comparable in vitro therapeutic efficacy to free curcumin against a panel of human pancreatic cancer cell lines, as assessed by cell viability and clonogenicity assays in soft agar. Further, nanocurcumin's mechanisms of action on pancreatic cancer cells mirror that of free curcumin, including induction of cellular apoptosis, blockade of nuclear factor kappa B (NFkappaB) activation, and downregulation of steady state levels of multiple pro-inflammatory cytokines (IL-6, IL-8, and TNFalpha). CONCLUSION: Nanocurcumin provides an opportunity to expand the clinical repertoire of this efficacious agent by enabling ready aqueous dispersion. Future studies utilizing nanocurcumin are warranted in pre-clinical in vivo models of cancer and other diseases that might benefit from the effects of curcumin.
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Purpose: We previously demonstrated an association between decreased SMAD4 expression and cetuximab resistance in head and neck squamous cell carcinoma (HNSCC). The purpose of this study was to further elucidate the clinical relevance of SMAD4 loss in HNSCC.Experimental Design: SMAD4 expression was assessed by IHC in 130 newly diagnosed and 43 patients with recurrent HNSCC. Correlative statistical analysis with clinicopathologic data was also performed. OncoFinder, a bioinformatics tool, was used to analyze molecular signaling in TCGA tumors with low or high SMAD4 mRNA levels. The role of SMAD4 was investigated by shRNA knockdown and gene reconstitution of HPV-negative HNSCC cell lines in vitro and in vivoResults: Our analysis revealed that SMAD4 loss was associated with an aggressive, HPV-negative, cetuximab-resistant phenotype. We found a signature of prosurvival and antiapoptotic pathways that were commonly dysregulated in SMAD4-low cases derived from TCGA-HNSCC dataset and an independent oral cavity squamous cell carcinoma (OSCC) cohort obtained from GEO. We show that SMAD4 depletion in an HNSCC cell line induces cetuximab resistance and results in worse survival in an orthotopic mouse model in vivo We implicate JNK and MAPK activation as mediators of cetuximab resistance and provide the foundation for the concomitant EGFR and JNK/MAPK inhibition as a potential strategy for overcoming cetuximab resistance in HNSCCs with SMAD4 loss.Conclusions: Our study demonstrates that loss of SMAD4 expression is a signature characterizing the cetuximab-resistant phenotype and suggests that SMAD4 expression may be a determinant of sensitivity/resistance to EGFR/MAPK or EGFR/JNK inhibition in HPV-negative HNSCC tumors. Clin Cancer Res; 23(17); 5162-75. ©2017 AACR.
Assuntos
Resistencia a Medicamentos Antineoplásicos/genética , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Recidiva Local de Neoplasia/tratamento farmacológico , Proteína Smad4/genética , Idoso , Animais , Anticorpos Monoclonais Humanizados/administração & dosagem , Linhagem Celular Tumoral , Cetuximab/administração & dosagem , Receptores ErbB/genética , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/patologia , Humanos , MAP Quinase Quinase 1/genética , Camundongos , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/patologia , Papillomaviridae/patogenicidade , Inibidores de Proteínas Quinases/administração & dosagem , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
A subset of patients with oral squamous cell carcinoma (OSCC), the most common subtype of head and neck squamous cell carcinoma (HNSCC), harbor dysplastic lesions (often visually identified as leukoplakia) prior to cancer diagnosis. Although evidence suggest that leukoplakia represents an initial step in the progression to cancer, signaling networks driving this progression are poorly understood. Here, we applied in silico Pathway Activation Network Decomposition Analysis (iPANDA), a new bioinformatics software suite for qualitative analysis of intracellular signaling pathway activation using transcriptomic data, to assess a network of molecular signaling in OSCC and pre-neoplastic oral lesions. In tumor samples, our analysis detected major conserved mitogenic and survival signaling pathways strongly associated with HNSCC, suggesting that some of the pathways identified by our algorithm, but not yet validated as HNSCC related, may be attractive targets for future research. While pathways activation landscape in the majority of leukoplakias was different from that seen in OSCC, a subset of pre-neoplastic lesions has demonstrated some degree of similarity to the signaling profile seen in tumors, including dysregulation of the cancer-driving pathways related to survival and apoptosis. These results suggest that dysregulation of these signaling networks may be the driving force behind the early stages of OSCC tumorigenesis. While future studies with larger leukoplakia data sets are warranted to further estimate the values of this approach for capturing signaling features that characterize relevant lesions that actually progress to cancers, our platform proposes a promising new approach for detecting cancer-promoting pathways and tailoring the right therapy to prevent tumorigenesis.
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The cornerstone of the systemic treatment of advanced colorectal cancer is 5-fluorouracil.However, 5-fluorouracil-induced apoptosis is dependent on p53, a tumor suppressor gene that is lost or inactivated in at least 85% of human colorectal cancers. Here we show that tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)/Apo2L triggers caspase-8-mediated truncation of BID, mitochondrial activation of caspase-9, and apoptosis in both p53(+/+) or p53(-/-) isogenic HCT116 colorectal cancer cells. TRAIL/Apo2L also sensitizes both p53(+/+) or p53(-/-) colorectal cancer cells to ionizing radiation. In contrast, we find that TRAIL/Apo2L fails to activate caspase-9 or induce apoptosis in isogenic HCT116 colorectal cancer cells that are deficient in BAX, a proapoptotic gene that is mutated in >50% of colorectal cancers of the microsatellite mutator phenotype. Loss of BAX also renders colorectal cancer cells resistant to TRAIL/Apo2L-mediated radiosensitization. We additionally demonstrate that TRAIL/Apo2L-induced death of p53(+/+)- or p53(-/-)- BAX-proficient but not BAX-deficient colorectal cancer cells is augmented by reducing nuclear factor-kappaB-dependent expression of Bcl-x(L) with either a peptide that disrupts the inhibitor of kappaB kinase complex or the nonsteroidal anti-inflammatory drug, sulindac sulfide. These results indicate that the combination of TRAIL/Apo2L with either irradiation or sulindac may be highly effective against both p53-proficient and p53-deficient colorectal cancers; however, BAX-deficient tumors may evade elimination by TRAIL/Apo2L-based regimens. Our findings may aid the development and genotype-specific application of TRAIL/Apo2L-based combinatorial regimens for the treatment of colorectal cancers.
Assuntos
Adenocarcinoma/patologia , Anti-Inflamatórios não Esteroides/farmacologia , Apoptose/fisiologia , Neoplasias Colorretais/patologia , Glicoproteínas de Membrana/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Proteínas Proto-Oncogênicas/fisiologia , Sulindaco/farmacologia , Fator de Necrose Tumoral alfa/farmacologia , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Sequência de Aminoácidos , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Proteínas Reguladoras de Apoptose , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Sinergismo Farmacológico , Genes p53/fisiologia , Humanos , Quinase I-kappa B , Dados de Sequência Molecular , NF-kappa B/fisiologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Proteínas Recombinantes/farmacologia , Ligante Indutor de Apoptose Relacionado a TNF , Células Tumorais Cultivadas , Proteína X Associada a bcl-2 , Proteína bcl-XRESUMO
Tumor-cell death can be triggered by engagement of specific death receptors with Apo2 ligand/tumor necrosis factor-related apoptosis-inducing ligand (Apo2L/TRAIL). Apo2L/TRAIL-induced apoptosis involves caspase-8-mediated cleavage of BID. The active truncated form of BID (tBID) triggers the mitochondrial activation of caspase-9 by inducing the activation of BAK or BAX. Although a broad spectrum of human cancer cell lines express death receptors for Apo2L/TRAIL, many remain resistant to TRAIL/Apo2L-induced death. A variety of human cancers exhibit increased activity of casein kinase II (CK2). Here we demonstrate that CK2 is at the nexus of two signaling pathways that protect tumor cells from Apo2L/TRAIL-induced apoptosis. We find that CK2 inhibits Apo2L/TRAIL-induced caspase-8-mediated cleavage of BID, thereby reducing the formation of tBID. In addition, CK2 promotes nuclear factor kappa B (NF-kappa B)-mediated expression of Bcl-x(L), which sequesters tBID and curtails its ability to activate BAX. Tumor cells with constitutive activation of CK2 exhibit a high Bcl-x(L)/tBID ratio and fail to activate caspase-9 or undergo apoptosis in response to Apo2L/TRAIL. Conversely, reduction of the Bcl-x(L)/tBID ratio by inhibition of CK2 renders such cancer cells sensitive to Apo2L/TRAIL-induced activation of caspase-9 and apoptosis. Using isogenic cancer cell lines that differ only in the presence or absence of either the p53 tumor suppressor or the BAX gene, we show that the enhancement of Apo2L/TRAIL-induced tumor-cell death by CK2 inhibitors requires BAX, but not p53. The identification of CK2 as a key survival signal that protects tumor cells from death-receptor-induced apoptosis could aid the design of Apo2L/TRAIL-based combination regimens for treatment of diverse cancers.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intracelular , Glicoproteínas de Membrana/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Fator de Necrose Tumoral alfa/farmacologia , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/enzimologia , Adenocarcinoma/patologia , Apoptose/fisiologia , Proteínas Reguladoras de Apoptose , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3 , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/enzimologia , Neoplasias da Mama/patologia , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD , Proteínas de Transporte/biossíntese , Caseína Quinase II , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/enzimologia , Neoplasias do Colo/patologia , Sinergismo Farmacológico , Humanos , NF-kappa B/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/fisiologia , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Proteínas Recombinantes/farmacologia , Ligante Indutor de Apoptose Relacionado a TNF , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/fisiologia , Proteína X Associada a bcl-2 , Proteína bcl-XRESUMO
The HER-2/neu receptor is a member of the epidermal growth factor family and is amplified in multiple cancers. It is under intense investigation both as a prognostic marker and for therapy, using monoclonal antibodies targeted against the receptor. We have developed a novel two-component gadolinium-based MR contrast agent to image the HER-2/neu receptor. Positive T1 contrast in MR images was generated by the specific binding of avidin-gadolinium complexes to tumor cells prelabeled with a biotinylated anti-HER-2/neu antibody. Significant intensity enhancement was observed in HER-2/neu-expressing cell lines and in vivo in a breast cancer model. Potential applications of this approach may include determination of the HER-2/neu status for prognosis and for selecting tumors for monoclonal antibody therapy.
Assuntos
Neoplasias Mamárias Experimentais/química , Receptor ErbB-2/análise , Animais , Avidina/metabolismo , Meios de Contraste/metabolismo , Feminino , Citometria de Fluxo , Gadolínio DTPA/metabolismo , Imageamento por Ressonância Magnética/métodos , Neoplasias Mamárias Experimentais/metabolismo , Camundongos , Camundongos SCID , Camundongos Transgênicos , Ratos , Receptor ErbB-2/metabolismo , Células Tumorais CultivadasRESUMO
The majority of colorectal cancers have lost/inactivated the p53 tumor suppressor gene. Using isogenic human colon cancer cells that differ only in their p53 status, we demonstrate that loss of p53 renders tumor cells relatively resistant to the topoisomerase I inhibitor, irinotecan. Whereas irinotecan-induced up-regulation of the proapoptotic proteins PUMA and Noxa requires p53, we find that irinotecan inhibits Janus kinase 2 (JAK2)-signal transducer and activator of transcription 3 and 5 (STAT3/5) signaling in both p53-proficient and p53-deficient tumor cells. We show that irinotecan inhibits JAK2-STAT3/5-dependent expression of survival proteins (Bcl-x(L) and XIAP) and cooperates with Apo2 ligand/tumor necrosis factor-related apoptosis-inducing ligand (Apo2L/TRAIL) to facilitate p53-independent apoptosis of colon cancer cells. Whereas xenografts of p53-deficient colon cancer cells are relatively resistant to irinotecan compared with their p53-proficient counterparts, combined treatment with irinotecan and Apo2L/TRAIL eliminates hepatic metastases of both p53-proficient and p53-deficient cancer cells in vivo and significantly improves the survival of animals relative to treatment with either agent alone. Although the synergy between chemotherapy and Apo2L/TRAIL has been ascribed to p53, our data demonstrate that irinotecan enhances Apo2L/TRAIL-induced apoptosis of tumor cells via a distinct p53-independent mechanism involving inhibition of JAK2-STAT3/5 signaling. These findings identify a novel p53-independent channel of cross-talk between topoisomerase I inhibitors and Apo2L/TRAIL and suggest that the addition of Apo2L/TRAIL can improve the therapeutic index of irinotecan against both p53-proficient and p53-deficient colorectal cancers, including those that have metastasized to the liver.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Camptotecina/análogos & derivados , Camptotecina/farmacologia , Neoplasias do Colo/tratamento farmacológico , Neoplasias Hepáticas/secundário , Glicoproteínas de Membrana/farmacologia , Fator de Necrose Tumoral alfa/farmacologia , Adenocarcinoma/patologia , Adenocarcinoma/secundário , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Proteínas Reguladoras de Apoptose , Camptotecina/administração & dosagem , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Sinergismo Farmacológico , Feminino , Células HCT116 , Humanos , Irinotecano , Janus Quinase 2 , Neoplasias Hepáticas/tratamento farmacológico , Glicoproteínas de Membrana/administração & dosagem , Camundongos , Camundongos Nus , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas/metabolismo , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Ligante Indutor de Apoptose Relacionado a TNF , Inibidores da Topoisomerase I , Transfecção , Fator de Necrose Tumoral alfa/administração & dosagem , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/fisiologia , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X , Ensaios Antitumorais Modelo de Xenoenxerto , Proteína bcl-XRESUMO
Triple negative breast cancer has an extremely poor prognosis when chemotherapy is no longer effective. To overcome drug resistance, novel drug delivery systems based on nanoparticles have had remarkable success. We produced a novel nanoparticle component 'MDC' from milk-derived colloid. In order to evaluate the anti-cancer effect of MDC, we conducted in vitro and in vivo experiments on cancer cell lines and a primary tumor derived breast xenograft. Doxorubicin (Dox) conjugated to MDC (MDC-Dox) showed higher cancer cell growth inhibition than MDC alone especially in cell lines with high EGFR expression. In a mouse melanoma model, MDC-Dox significantly suppressed tumor growth when compared with free Dox. Moreover, in a primary tumor derived breast xenograft, one of the mice treated with MDC-Dox showed partial regression, while mice treated with free Dox failed to show any suppression of tumor growth. We have shown that a novel nanoparticle compound made of simple milk-derived colloid has the capability for drug conjugation, and serves as a tumor-specific carrier of anti-cancer drugs. Further research on its safety and ability to carry various anti-cancer drugs into multiple drug-resistant primary breast models is warranted.
Assuntos
Coloides/química , Doxorrubicina/administração & dosagem , Portadores de Fármacos/química , Leite/química , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/química , Linhagem Celular Tumoral/efeitos dos fármacos , Coloides/administração & dosagem , Doxorrubicina/química , Portadores de Fármacos/administração & dosagem , Sistemas de Liberação de Medicamentos/métodos , Feminino , Humanos , Hospedeiro Imunocomprometido , Melanoma/tratamento farmacológico , Melanoma/patologia , Camundongos Endogâmicos C57BL , Nanopartículas/química , Neoplasias de Mama Triplo Negativas/patologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
INTRODUCTION: A combination of cetuximab and sorafenib in patients with recurrent and/or metastatic (R/M) head and neck squamous cell carcinoma (HNSCC) were assessed for potential benefit. MATERIAL AND METHODS: In a randomized phase II study, R/M HNSCC patients were treated with cetuximab 400mg/m(2) IV on day 1 followed by 250mg/m(2) IV weekly (Arm A), or cetuximab at the same dose/schedule plus sorafenib 400mg PO twice-a-day (Arm B). Each cycle was 21days. Tumor p16 and HPV status, and plasma immunomodulatory cytokine levels were assessed. RESULTS: Of 55 patients enrolled (Arm A-27, Arm B-28), 52 patients received assigned treatments and 43 were evaluable for response. Overall response rate was 8% for both arms. Median overall survival (OS) and progression-free survival (PFS) were 9.0 and 3.0months in Arm A, and 5.7 and 3.2months in Arm B, respectively. Forty-four patients had tumors available for p16 staining (35-negative, 9-positive). Three of nine p16-positive tumors were also HPV positive. The p16-negative patients had significantly better PFS compared to the p16-positive patients (3.7 vs. 1.6months; p-value: 0.03), regardless of study arms. Twenty-four plasma samples were tested for 12 cytokine levels and patients with higher TGFß1 levels had inferior PFS compared to lower levels (1.9 vs. 4.7months; adjusted p-value: 0.015), regardless of study arms. CONCLUSIONS: A subset of R/M patients with p16-negative tumors or lower plasma TGFß1 levels had longer PFS given the cetuximab-based therapy. However, both arms showed only modest response and sorafenib given with cetuximab did not demonstrate clinical benefit.
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Antineoplásicos/uso terapêutico , Carcinoma de Células Escamosas/tratamento farmacológico , Cetuximab/uso terapêutico , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Metástase Neoplásica , Niacinamida/análogos & derivados , Compostos de Fenilureia/uso terapêutico , Adulto , Idoso , Carcinoma de Células Escamosas/patologia , Feminino , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Niacinamida/uso terapêutico , Recidiva , SorafenibeRESUMO
Lungs resected for adenocarcinomas often harbour minute discrete foci of cytologically atypical pneumocyte proliferations designated as atypical adenomatous hyperplasia (AAH). Evidence suggests that AAH represents an initial step in the progression to adenocarcinoma in situ (AIS), minimally invasive adenocarcinoma (MIA) and fully invasive adenocarcinoma. Despite efforts to identify predictive markers of malignant transformation, alterations driving this progression are poorly understood. Here we perform targeted next-generation sequencing on multifocal AAHs and different zones of histologic progression within AISs and MIAs. Multiregion sequencing demonstrated different genetic drivers within the same tumour and reveal that clonal expansion is an early event of tumorigenesis. We find that KRAS, TP53 and EGFR mutations are indicators of malignant transition. Utilizing droplet digital PCR, we find alterations associated with early neoplasms in paired circulating DNA. This study provides insight into the heterogeneity of clonal events in the progression of early lung neoplasia and demonstrates that these events can be detected even before neoplasms have invaded and acquired malignant potential.
Assuntos
Adenocarcinoma/genética , Transformação Celular Neoplásica/genética , DNA/genética , Receptores ErbB/genética , Neoplasias Pulmonares/genética , Pulmão/patologia , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteína Supressora de Tumor p53/genética , Adenocarcinoma/patologia , Adenocarcinoma in Situ/genética , Adenocarcinoma in Situ/patologia , Adulto , Idoso , Células Clonais , Progressão da Doença , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Hiperplasia/genética , Hiperplasia/patologia , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Mutação , Reação em Cadeia da Polimerase , Lesões Pré-Cancerosas/genética , Lesões Pré-Cancerosas/patologiaRESUMO
Although specific mutations in the tyrosine kinase domain of epidermal growth factor receptor (EGFR) identify tumors that are responsive to EGFR tyrosine kinase inhibitors (TKI), these genetic alterations are present in only a minority of patients. Patients with tumors expressing wild-type EGFR lack reliable predictive markers of their clinical response to EGFR TKIs. Although epithelial-mesenchymal transition (EMT) has been inversely correlated with the response of cancers to EGFR-targeted therapy, the precise molecular mechanisms underlying this association have not been defined and no specific EMT-associated biomarker of clinical benefit has been identified. Here, we show that during transforming growth factor ß (TGFß)-mediated EMT, inhibition of the microRNAs 200 (miR200) family results in upregulated expression of the mitogen-inducible gene 6 (MIG6), a negative regulator of EGFR. The MIG6-mediated reduction of EGFR occurs concomitantly with a TGFß-induced EMT-associated kinase switch of tumor cells to an AKT-activated EGFR-independent state. In a panel of 25 cancer cell lines of different tissue origins, we find that the ratio of the expression levels of MIG6 and miR200c is highly correlated with EMT and resistance to erlotinib. Analyses of primary tumor xenografts of patient-derived lung and pancreatic cancers carrying wild-type EGFR showed that the tumor MIG6(mRNA)/miR200 ratio was inversely correlated with response to erlotinib in vivo. Our data demonstrate that the TGFß-miR200-MIG6 network orchestrates the EMT-associated kinase switch that induces resistance to EGFR inhibitors, and identify a low ratio of MIG6 to miR200 as a promising predictive biomarker of the response of tumors to EGFR TKIs.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos , Transição Epitelial-Mesenquimal , Receptores ErbB/antagonistas & inibidores , MicroRNAs/genética , Inibidores de Proteínas Quinases/farmacologia , Fator de Crescimento Transformador beta/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Resistencia a Medicamentos Antineoplásicos/genética , Ativação Enzimática/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Transição Epitelial-Mesenquimal/genética , Receptores ErbB/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Camundongos , Especificidade de Órgãos/genética , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Fenótipo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta/farmacologia , Proteínas Supressoras de Tumor/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The treatment of locoregional recurrence (LRR) of head and neck squamous cell carcinoma (HNSCC) often requires a combination of surgery, radiation therapy and/or chemotherapy. Survival outcomes are poor and the treatment outcomes are morbid. Cold atmospheric plasma (CAP) is an ionized gas produced at room temperature under laboratory conditions. We have previously demonstrated that treatment with a CAP jet device selectively targets cancer cells using in vitro melanoma and in vivo bladder cancer models. In the present study, we wished to examine CAP selectivity in HNSCC in vitro models, and to explore its potential for use as a minimally invasive surgical approach that allows for specific cancer cell or tumor tissue ablation without affecting the surrounding healthy cells and tissues. Four HNSCC cell lines (JHU-022, JHU-028, JHU-029, SCC25) and 2 normal oral cavity epithelial cell lines (OKF6 and NOKsi) were subjected to cold plasma treatment for durations of 10, 30 and 45 sec, and a helium flow of 20 l/min-1 for 10 sec was used as a positive treatment control. We showed that cold plasma selectively diminished HNSCC cell viability in a dose-response manner, as evidenced by MTT assays; the viability of the OKF6 cells was not affected by the cold plasma. The results of colony formation assays also revealed a cell-specific response to cold plasma application. Western blot analysis did not provide evidence that the cleavage of PARP occurred following cold plasma treatment. In conclusion, our results suggest that cold plasma application selectively impairs HNSCC cell lines through non-apoptotic mechanisms, while having a minimal effect on normal oral cavity epithelial cell lines.
Assuntos
Atmosfera , Carcinoma de Células Escamosas/patologia , Neoplasias de Cabeça e Pescoço/patologia , Gases em Plasma/farmacologia , Western Blotting , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Humanos , Poli(ADP-Ribose) Polimerases/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço , Ensaio Tumoral de Célula-TroncoRESUMO
Oxoglutarate dehydrogenase (OGDH) is the first and rate-limiting component of the multi-enzyme OGDH complex (OGDHC) whose malfunction is associated with neuro-degeneration. The essential role of this complex is in the degradation of glucose and glutamate and the OGDHL gene (one component of OGDHC) is down-regulated by promoter hypermethylation in many different cancer types. These properties suggest a potential growth modulating role of OGDHL in cancer; however, the molecular mechanism through which OGDHL exerts its growth modulating function has not been elucidated.Here, we report that restoration of OGDHL expression in cervical cancer cells lacking endogenous OGDHL expression suppressed cell proliferation, invasion and soft agar colony formation in vitro. Knockdown of OGDHL expression in cervical cancer cells expressing endogenous OGDHL had the opposite effect. Forced expression of OGDHL increased the production of reactive oxygen species (ROS) leading to apoptosis through caspase 3 mediated down-regulation of the AKT signaling cascade and decreased NF-κB phosphorylation. Conversely, silencing OGDHL stimulated the signaling pathway via increased AKT phosphorylation. Moreover, the addition of caspase 3 or ROS inhibitors in the presence of OGDHL increased AKT signaling and cervical cancer cell proliferation.Taken together, these data suggest that inactivation of OGDHL can contribute to cervical tumorigenesis via activation of the AKT signaling pathway and thus support it as an important anti-proliferative gene in cervical cancer.
Assuntos
Complexo Cetoglutarato Desidrogenase/metabolismo , NF-kappa B/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Apoptose/genética , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular/genética , Feminino , Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Complexo Cetoglutarato Desidrogenase/genética , Metabolismo dos Lipídeos , NF-kappa B/genética , Estresse Oxidativo , Transporte Proteico , Espécies Reativas de Oxigênio/metabolismo , Transcrição Gênica , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/metabolismoRESUMO
EGF receptor (EGFR)-targeted monoclonal antibodies (mAb), such as cetuximab, execute their antitumor effect in vivo via blockade of receptor-ligand interactions and engagement of Fcγ receptors on immune effector cells that trigger antibody-dependent cell-mediated cytotoxicity (ADCC). We show that tumors counteract the in vivo antitumor activity of anti-EGFR mAbs by increasing tumor cell-autonomous expression of TGF-ß. We show that TGF-ß suppresses the expression of key molecular effectors of immune cell-mediated cytotoxicity, including Apo2L/TRAIL, CD95L/FasL, granzyme B, and IFN-γ. In addition to exerting an extrinsic inhibition of the cytotoxic function of immune effectors, TGF-ß-mediated activation of AKT provides an intrinsic EGFR-independent survival signal that protects tumor cells from immune cell-mediated apoptosis. Treatment of mice-bearing xenografts of human head and neck squamous cell carcinoma with cetuximab resulted in emergence of resistant tumor cells that expressed relatively higher levels of TGF-ß compared with untreated tumor-bearing mice. Although treatment with cetuximab alone forced the natural selection of TGF-ß-overexpressing tumor cells in nonregressing tumors, combinatorial treatment with cetuximab and a TGF-ß-blocking antibody prevented the emergence of such resistant tumor cells and induced complete tumor regression. Therefore, elevated levels of TGF-ß in the tumor microenvironment enable tumor cells to evade ADCC and resist the antitumor activity of cetuximab in vivo. Our results show that TGF-ß is a key molecular determinant of the de novo and acquired resistance of cancers to EGFR-targeted mAbs, and provide a rationale for combinatorial targeting of TGF-ß to improve anti-EGFR-specific antibody therapy of EGFR-expressing cancers.
Assuntos
Antineoplásicos/farmacologia , Receptores ErbB/antagonistas & inibidores , Terapia de Alvo Molecular , Fator de Crescimento Transformador beta/antagonistas & inibidores , Animais , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , Anticorpos Monoclonais Humanizados , Citotoxicidade Celular Dependente de Anticorpos/efeitos dos fármacos , Carcinoma de Células Escamosas/sangue , Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/enzimologia , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cetuximab , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Receptores ErbB/metabolismo , Feminino , Neoplasias de Cabeça e Pescoço/sangue , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Neoplasias de Cabeça e Pescoço/enzimologia , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Linfócitos/efeitos dos fármacos , Linfócitos/metabolismo , Camundongos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço , Fator de Crescimento Transformador beta/sangue , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: The major established etiologic risk factor for bladder cancer is cigarette smoking and one of the major antineoplastic agents used for the treatment of advanced bladder cancer is cisplatin. A number of reports have suggested that cancer patients who smoke while receiving treatment have lower rates of response and decreased efficacy of cancer therapies. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we investigated the effect of cigarette smoke condensate (CSC) vapor on cisplatin toxicity in urothelial cell lines SV-HUC-1 and SCaBER cells. We showed that chronic exposure to CSC vapor induced cisplatin resistance in both cell lines. In addition, we found that the expression of mitochondrial-resident protein adenylate kinase-3 (AK3) is decreased by CSC vapor. We further observed that chronic CSC vapor-exposed cells displayed decreased cellular sensitivity to cisplatin, decreased mitochondrial membrane potential (ΔΨm) and increased basal cellular ROS levels compared to unexposed cells. Re-expression of AK3 in CSC vapor-exposed cells restored cellular sensitivity to cisplatin. Finally, CSC vapor increased the growth of the tumors and also curtail the response of tumor cells to cisplatin chemotherapy in vivo. CONCLUSIONS/SIGNIFICANCE: The current study provides evidence that chronic CSC vapor exposure affects AK3 expression and renders the cells resistant to cisplatin.
Assuntos
Adenilato Quinase/metabolismo , Antineoplásicos/farmacologia , Cisplatino/farmacologia , Nicotiana , Fumaça , Apoptose/efeitos dos fármacos , Linhagem Celular , Resistencia a Medicamentos AntineoplásicosRESUMO
The p53 family gene p63 plays an instrumental role in cellular stress responses including responses to DNA damage. In addition to encoding a full-length transcriptional activator, p63 also encodes several dominant inhibitory isoforms including the isoform DeltaNp63alpha, the function of which is not fully understood. DeltaNp63alpha is degraded in response to DNA damage, thereby enabling an effective cellular response to genotoxic agents. Here, we identify a key molecular mechanism underlying regulation of DeltaNp63alpha expression in response to chemotherapeutic agents or tumor necrosis factor-alpha. We found that DeltaNp63alpha interacts with IkappaB kinase (IKK), a multisubunit protein kinase that consists of two catalytic subunits, IKKalpha and IKKbeta, and a regulatory subunit, IKKgamma. The IKKbeta kinase promotes ubiquitin-mediated proteasomal degradation of DeltaNp63alpha, whereas a kinase-deficient mutant IKKbeta-K44A fails to do so. Cytokine- or chemotherapy-induced stimulation of IKKbeta caused degradation of DeltaNp63alpha and augmented transactivation of p53 family-induced genes involved in the cellular response to DNA damage. Conversely, IKKbeta inhibition attenuated cytokine- or chemotherapy-induced degradation of DeltaNp63alpha. Our findings show that IKKbeta plays an essential role in regulating DeltaNp63alpha in response to extrinsic stimuli. IKK activation represents one mechanism by which levels of DeltaNp63alpha can be reduced, thereby rendering cells susceptible to cell death in the face of cellular stress or DNA damage.
Assuntos
Quinase I-kappa B/fisiologia , Transativadores/genética , Transativadores/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Morte Celular/efeitos dos fármacos , Morte Celular/genética , Cisplatino/farmacologia , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/genética , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Quinase I-kappa B/metabolismo , NF-kappa B/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica/efeitos dos fármacos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Fatores de Transcrição , Ativação Transcricional/efeitos dos fármacos , Ativação Transcricional/fisiologia , Células Tumorais Cultivadas , Fator de Necrose Tumoral alfa/farmacologia , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Ubiquitina/metabolismoRESUMO
Curcumin or diferuloylmethane is a yellow polyphenol extracted from the rhizome of turmeric (Curcuma longa). A large volume (several hundreds) of published reports has established the anticancer and chemopreventative properties of curcumin in preclinical models of every known major cancer type. Nevertheless, the clinical translation of curcumin has been significantly hampered due to its poor systemic bioavailability, which mandates that patients consume up to 8 to 10 g of the free drug orally each day to achieve detectable levels in circulation. We have engineered a polymeric nanoparticle encapsulated curcumin formulation (NanoCurc) that shows remarkably higher systemic bioavailability in plasma and tissues compared with free curcumin upon parenteral administration. In xenograft models of human pancreatic cancer established in athymic mice, administration of parenteral NanoCurc significantly inhibits primary tumor growth in both subcutaneous and orthotopic settings. The combination of parenteral NanoCurc with gemcitabine results in enhanced tumor growth inhibition versus either single agent, suggesting an additive therapeutic influence in vivo. Furthermore, this combination completely abrogates systemic metastases in orthotopic pancreatic cancer xenograft models. Tumor growth inhibition is accompanied by significant reduction in activation of nuclear factor-kappaB, as well as significant reduction in expression of matrix metalloproteinase-9 and cyclin D1, in xenografts treated with NanoCurc and gemcitabine. NanoCurc is a promising new formulation that is able to overcome a major impediment for the clinical translation of curcumin to cancer patients by improving systemic bioavailability, and by extension, therapeutic efficacy.