RESUMEN
The application of endoscopic imaging in the biopsy of malignant laryngeal lesions is one of the current research hotspots in the medical field. Based on the narrowband imaging resolution enhancement technology, a model for postoperative examination of laryngeal malignant tumor was constructed in this paper. The article calculated the biopsy detection rate of malignant lesions and the correct biopsy detection rate of the two groups and made a statistical comparison. In the NBI mode group, the mucosal morphology and superficial mucosal microvascular morphology of the same lesion under two different modes of white light and NBI were compared, which solved the problem of data processing of cases. During the case comparison process, patients who needed biopsies to be sent for pathology were selected for inclusion in the study and were randomly divided into two groups. The coincidence rate of EUS combined with NBI diagnosis was significantly higher than that of ordinary white light gastroscopy (47.92%), and the difference was statistically significant (P=0.000 < 0.05). The experimental results compared the accuracy of the normal white light mode and the NBI mode to diagnose the nature of the lesions: according to the Kudo classification criteria, 23 cases of tumor lesions were to be diagnosed in the normal white light mode, with an accuracy rate of 69.70%, and the NBI mode was to be used to diagnose tumors. There were 81 cases of sexual lesions, with an accuracy rate of 93.94%. The NBI mode was more accurate in diagnosing the nature of the lesions under the Kudo classification standard (P < 0.05). In 64 cases, the accuracy rate was 63.63%. Under the NBI mode, 29 cases of tumor lesions were proposed to be diagnosed, and the accuracy rate was 87.88% to promote the application of NBI endoscopy in the biopsy of malignant laryngeal lesions.
Asunto(s)
Refuerzo Biomédico , Neoplasias Laríngeas , Humanos , Neoplasias Laríngeas/diagnóstico por imagen , Neoplasias Laríngeas/cirugía , Luz , Imagen de Banda Estrecha , Sensibilidad y EspecificidadRESUMEN
Aim: The paper aims to combine mathematical statistics to assess the effect of nutritional intervention in the population of nasopharyngeal cancer patients. Methodology. After following the inclusion and exclusion criteria, a total of 120 patients with nasopharyngeal carcinoma were selected. All patients are treated with intensity-modulated radiotherapy (IMRT). The nurse collects relevant clinical treatment data during the radiotherapy of the patient. After the patient's radiotherapy, the nurse remeasures the patient's nutritional status indicators. Three months after the completion of radiotherapy, the patient will be reexamined by MRI, and the radiotherapist will assess the patient's radiosensitivity based on the results of the MRI examination. All the blood biochemical indicators and body measurement indicators were also assessed and coordinated with nasopharyngeal carcinoma patients. This study performs multiple linear regression analysis on treatment-related factors that affect nutritional status during radiotherapy. Results: The experimental results showed that the side effects of radiotherapy are independent influencing factors of nutritional status. Radiotherapy damages the DNA of cells, so that cells cannot continue to divide and grow, and all cells in the treatment area were affected by radiation. The standard radiotherapy treatment is quite long, and the oral cavity, throat, and parotid gland, are all within the irradiation range. In addition to killing the tumor cells, the radiation can also cause certain damage to the surrounding tissues of the tumor. This article takes radiosensitivity as the dependent variable (insensitivity = 0; sensitivity = 1) and takes the nutritional index NI, age, gender, education level, marriage, smoking, chronic disease history, TNM staging, whether the chemotherapy steps are the same or not, GTVnx prescription dose, and the number of radiotherapies as independent variables. AMC, albumin, hemoglobin, serum prealbumin, and transferrin are all correlated with radiosensitivity, which is consistent with the results of most studies. The results of multivariate logistic regression analysis showed that nutritional index (NI) was correlated with the radiosensitivity of nasopharyngeal carcinoma. Conclusion: Finally, this paper concludes that nutritional intervention has a certain effect on the treatment of patients with nasopharyngeal carcinoma.
Asunto(s)
Carcinoma , Neoplasias Nasofaríngeas , Radioterapia de Intensidad Modulada , Carcinoma/tratamiento farmacológico , Carcinoma/patología , Carcinoma/radioterapia , Humanos , Carcinoma Nasofaríngeo/etiología , Carcinoma Nasofaríngeo/radioterapia , Neoplasias Nasofaríngeas/tratamiento farmacológico , Neoplasias Nasofaríngeas/patología , Neoplasias Nasofaríngeas/radioterapia , Radioterapia de Intensidad Modulada/efectos adversos , Radioterapia de Intensidad Modulada/métodos , Estudios RetrospectivosRESUMEN
The new and rapid advancement in the complexity of biologics drug discovery has been driven by a deeper understanding of biological systems combined with innovative new therapeutic modalities, paving the way to breakthrough therapies for previously intractable diseases. These exciting times in biomedical innovation require the development of novel technologies to facilitate the sophisticated, multifaceted, high-paced workflows necessary to support modern large molecule drug discovery. A high-level aspiration is a true integration of "lab-on-a-chip" methods that vastly miniaturize cellulmical experiments could transform the speed, cost, and success of multiple workstreams in biologics development. Several microscale bioprocess technologies have been established that incrementally address these needs, yet each is inflexibly designed for a very specific process thus limiting an integrated holistic application. A more fully integrated nanoscale approach that incorporates manipulation, culture, analytics, and traceable digital record keeping of thousands of single cells in a relevant nanoenvironment would be a transformative technology capable of keeping pace with today's rapid and complex drug discovery demands. The recent advent of optical manipulation of cells using light-induced electrokinetics with micro- and nanoscale cell culture is poised to revolutionize both fundamental and applied biological research. In this review, we summarize the current state of the art for optical manipulation techniques and discuss emerging biological applications of this technology. In particular, we focus on promising prospects for drug discovery workflows, including antibody discovery, bioassay development, antibody engineering, and cell line development, which are enabled by the automation and industrialization of an integrated optoelectronic single-cell manipulation and culture platform. Continued development of such platforms will be well positioned to overcome many of the challenges currently associated with fragmented, low-throughput bioprocess workflows in biopharma and life science research.
Asunto(s)
Automatización , Productos Biológicos , Descubrimiento de Drogas , Dispositivos Laboratorio en un Chip , HumanosRESUMEN
The treatment of castration-resistant (CR) prostate cancer (PCa) is limited. A sub-population of CR PCa tumors can synthesize androgens for intracrine androgen receptor (AR) activation, thus targeting androgen biosynthesis could be an effective therapeutic option for these patients. We determined that androgen biosynthesis inhibitors simvastatin, atorvastatin, and ketoconazole directly inhibit growth, migration, and colony formation of LNCaP C-81 cells, which exhibit de novo androgen biosynthesis, with simvastatin being the most effective. Importantly, in combination treatments, statins specifically enhanced growth suppression with added effects by anti-androgen abiraterone acetate on the CR PCa cells. Thus, statins can be used in conjunction with abiraterone acetate to enhance anti-androgen therapy for CR PCa.
RESUMEN
While androgen deprivation therapy (ADT) reduces tumor burden, autocrine growth factor loops such as human epidermal growth factor receptor 2 (HER2/ErbB-2/neu) have been proposed to contribute to prostate cancer (PCa) survival and relapse. However, the role of ErbB-2 in regulating androgen-sensitive (AS) and castration-resistant (CR) cell proliferation remains unclear. Here, we determined the role of ErbB-2 in PCa progression and survival under steroid-reduced conditions using two independent PCa cell progression models. In AR-positive androgen-independent (AI) PCa cells that exhibit the CR phenotype, ErbB-2 was constitutively activated, compared to corresponding AS PCa cells. In AS LNCaP C-33 cells, androgen-induced ErbB-2 activation through ERK1/2 mediates PCa cell proliferation. Further, the ErbB-2-specific but not EGFR-specific inhibitor suppresses basal and androgen-stimulated cell proliferation and also blocks ERK1/2 activation. ErbB-2 ectopic expression and cPAcP siRNA transfection of LNCaP C-33 cells each increases ErbB-2 tyrosine phosphorylation, correlating with increased AI PSA secretion and cell proliferation. Conversely, trapping ErbB-2 by transfected endoplasmic reticulum-targeting ScFv5R expression vector abolished DHT-induced LNCaP C-33 cell growth. Moreover, inhibition of ErbB-2 but not EGFR in AI LNCaP C-81 and MDA PCa2b-AI PCa cells significantly abolished AI cell growth. In contrast to androgens via ErbB-2/ERK1/2 signaling in AS PCa cells, the inhibition of ErbB-2 abrogated AI cell proliferation by inhibiting the cell survival protein Akt in those AI cells. These results suggest that ErbB-2 is a prominent player in mediating the ligand-dependent and -independent activation of AR in AS and AI/CR PCa cells respectively for PCa progression and survival.
Asunto(s)
Antagonistas de Andrógenos/uso terapéutico , Receptores ErbB/metabolismo , Neoplasias de la Próstata Resistentes a la Castración/patología , Receptor ErbB-2/metabolismo , Receptores Androgénicos/metabolismo , Fosfatasa Ácida/genética , Línea Celular Tumoral , Proliferación Celular/genética , Supervivencia Celular/genética , Retículo Endoplásmico , Receptores ErbB/antagonistas & inhibidores , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Calicreínas/metabolismo , Masculino , Fosforilación/genética , Antígeno Prostático Específico/metabolismo , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Interferencia de ARN , ARN Interferente Pequeño/genética , Receptor ErbB-2/antagonistas & inhibidores , Receptor ErbB-2/genética , Transducción de SeñalRESUMEN
BACKGROUND: Prostate cancer (PCa) is the most commonly diagnosed solid tumor and the second leading cancer death in the United States, and also one of the major cancer-related deaths in Chinese. Androgen deprivation therapy (ADT) is the first line treatment for metastatic PCa. PCa ultimately relapses with subsequent ADT treatment failure and becomes castrate-resistant (CR). It is important to develop effective therapies with a surrogate marker towards CR PCa. METHOD: Histone deacetylase (HDAC) inhibitors were examined to determine their effects in androgen receptor (AR)/cellular prostatic acid phosphatase (cPAcP)-positive PCa cells, including LNCaP C-33, C-81, C4-2 and C4-2B and MDA PCa2b androgen-sensitive and androgen-independent cells, and AR/cPAcP-negative PCa cells, including PC-3 and DU 145 cells. Cell growth was determined by cell number counting. Western blot analyses were carried out to determine AR, cPAcP and PSA protein levels. RESULTS: cPAcP protein level was increased by HDAC inhibitor treatment. Valproic acid, a HDAC inhibitor, suppressed the growth of AR/cPAcP-positive PCa cells by over 50% in steroid-reduced conditions, higher than on AR/cPAcP-negative PCa cells. Further, HDAC inhibitor pretreatments increased androgen responsiveness as demonstrated by PSA protein level quantitation. CONCLUSION: Our results clearly demonstrate that HDAC inhibitors can induce cPAcP protein level, increase androgen responsiveness, and exhibit higher inhibitory activities on AR/cPAcP-positive PCa cells than on AR/cPAcP-negative PCa cells. Upon HDAC inhibitor pretreatment, PSA level was greatly elevated by androgens. This data indicates the potential clinical importance of cPAcP serving as a useful biomarker in the identification of PCa patient sub-population suitable for HDAC inhibitor treatment.
RESUMEN
Many efforts are underway to develop selective inhibitors of the voltage-gated sodium channel NaV1.7 as new analgesics. Thus far, however, in vitro selectivity has proved difficult for small molecules, and peptides generally lack appropriate pharmacokinetic properties. We previously identified the NaV1.7 inhibitory peptide GpTx-1 from tarantula venom and optimized its potency and selectivity via structure-guided analoging. To further understand GpTx-1 binding to NaV1.7, we have mapped the binding site to transmembrane segments 1-4 of the second pseudosubunit internal repeat (commonly referred to as Site 4) using NaV1.5/NaV1.7 chimeric protein constructs. We also report that select GpTx-1 amino acid residues apparently not contacting NaV1.7 can be derivatized with a hydrophilic polymer without adversely affecting peptide potency. Homodimerization of GpTx-1 with a bifunctional polyethylene glycol (PEG) linker resulted in a compound with increased potency and a significantly reduced off-rate, demonstrating the ability to modulate the function and properties of GpTx-1 by linking to additional molecules.
Asunto(s)
Canal de Sodio Activado por Voltaje NAV1.7/metabolismo , Péptidos/química , Péptidos/farmacología , Ingeniería de Proteínas , Bloqueadores del Canal de Sodio Activado por Voltaje/farmacología , Dimerización , Relación Dosis-Respuesta a Droga , Humanos , Conformación Molecular , Unión Proteica , Estructura Terciaria de Proteína , Relación Estructura-Actividad , Bloqueadores del Canal de Sodio Activado por Voltaje/químicaRESUMEN
ORAI1 is the pore-forming component of calcium release-activated calcium (CRAC) channels. CRAC channels are the primary route for calcium ion (Ca(2+)) entry into T-cells following antigen stimulation. This Ca(2+) entry induces proliferation and cytokine production through activation of calcineurin and the nuclear factor of activated T-cells (NFAT) transcription factor along with subsequent cytokine-related genes. It was hypothesized that the in vivo inhibition of T-cell function by blocking ORAI1 or calcineurin would lead to similar functional consequences. To test this hypothesis the activity of 2C1.1, a fully human anti-ORAI1 monoclonal antibody, and cyclosporin A (CsA) were tested in vivo for their suppressive effect on T-cell-derived cytokine production and a T-cell-dependent antibody response (TDAR) using sheep red blood cells (SRBC) in cynomolgus monkeys. Despite showing similar inhibition of ex vivo interleukin (IL)-2 production by stimulated T-cells, both molecules exhibited different pharmacologic effects on the SRBC antibody response. CsA blocked the development of SRBC-specific antibodies, while 2C1.1 failed to inhibit the antigen-specific antibody response. These surprising observations suggest that full inhibition of the CRAC channel is required to inhibit a functional immune response, consistent with findings from human patients with loss of function mutations in ORAI1.
Asunto(s)
Anticuerpos Monoclonales/administración & dosificación , Canales de Calcio/metabolismo , Ciclosporina/administración & dosificación , Macaca fascicularis , Linfocitos T/inmunología , Animales , Formación de Anticuerpos , Calcineurina/metabolismo , Canales de Calcio/inmunología , Bovinos , Células Cultivadas , Eritrocitos/inmunología , Humanos , Interleucina-2/metabolismo , Activación de Linfocitos , Masculino , Factores de Transcripción NFATC/metabolismo , Proteína ORAI1 , OvinosRESUMEN
p66Shc functions as a longevity protein in murine and exhibits oxidase activity in regulating diverse biological activities. In this study, we investigated the role of p66Shc protein in regulating ovarian cancer (OCa) cell proliferation. Among three cell lines examined, the slowest growing OVCAR-3 cells have the lowest level of p66Shc protein. Transient transfection with p66Shc cDNA expression vector in OVCAR-3 cells increases cell proliferation. Conversely, knock-down of p66Shc by shRNA in rapidly growing SKOV-3 cells results in decreased cell growth. In estrogen (E2)-treated CaOV-3 cells, elevated p66Shc protein level correlates with ROS level, ErbB-2 and ERK/MAPK activation, and cell proliferation. Further, the E2-stimulated proliferation of CaOV-3 cells was blocked by antioxidants and ErbB-2 inhibitor. Additionally, in E2-stimulated cells, the tartrate-sensitive, but not the tartrate-resistant, phosphatase activity decreases; concurrently, the tyrosine phosphorylation of ErbB-2 increases. Conversely, inhibition of phosphatase activity by L(+)-tartrate treatment increases p66Shc protein level, ErbB-2 tyrosine phosphorylation, ERK/MAPK activation, and cell growth. Further, inhibition of the ERK/MAPK pathway by PD98059 blocks E2-induced ERK/MAPK activation and cell proliferation in CaOV-3 cells. Moreover, immunohistochemical analyses showed that the p66Shc protein level was significantly higher in cancerous cells than in noncancerous cells in archival OCa tissues (n = 76; P = 0.00037). These data collectively indicate that p66Shc protein plays a critical role in up-regulating OCa progression.
Asunto(s)
Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología , Receptor ErbB-2/metabolismo , Proteínas Adaptadoras de la Señalización Shc/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Estrógenos/farmacología , Femenino , Flavonoides/farmacología , Humanos , Neoplasias Ováricas/genética , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Adaptadoras de la Señalización Shc/genética , Transducción de Señal/efectos de los fármacos , Proteína Transformadora 1 que Contiene Dominios de Homología 2 de Src , Regulación hacia ArribaRESUMEN
We determined the expression of ORAI1 protein in rodent and non-rodent tissues using a monoclonal antibody directed against an extracellular loop of the protein. Previous reports using antibodies directed at the C-terminus of ORAI1 have not detected central nervous system (CNS) expression. Our results demonstrate broad tissue expression that includes the CNS using a unique monoclonal antibody specific to an extracellular loop of ORAI1. In addition, we present in situ hybridization (ISH) results using a probe within the middle of the mouse coding region showing CNS expression of Orai1 RNA. We contrast the patterns of rodent and human tissue expression and conclude that rodents have similar expression of ORAI1 in most tissue types when compared to primates, with an important exception being the male reproductive system, where human-specific expression is observed.
Asunto(s)
Canales de Calcio/análisis , Inmunohistoquímica/métodos , Animales , Anticuerpos Monoclonales/análisis , Canales de Calcio/genética , Línea Celular , Sistema Nervioso Central/química , Sistema Nervioso Central/metabolismo , Sistema Nervioso Central/ultraestructura , Femenino , Humanos , Hibridación in Situ/métodos , Macaca fascicularis , Masculino , Ratones , Ratones Endogámicos C57BL , Proteína ORAI1 , ARN Mensajero/análisis , ARN Mensajero/genética , Ratas , Ratas Sprague-Dawley , Análisis de Matrices Tisulares/métodosRESUMEN
Calcium entry into T cells following antigen stimulation is crucial for nuclear factor of activated T cells (NFAT)-mediated T cell activation. The movement of calcium is mediated by calcium release-activated calcium (CRAC) channels. There are two key components of this channel: Orai1 is the pore-forming subunit located in the plasma membrane, and stromal interaction molecule 1 (STIM1) functions as a Ca(2+) sensor in the endoplasmic reticulum. A subset of human patients carry mutations in either STIM1 or Orai1 that affect protein function or expression, resulting in defective store-operated Ca(2+) influx and CRAC channel function, and impaired T cell activation. These patients suffer from a hereditary form of severe combined immune deficiency syndrome, highlighting the importance of the CRAC channel for T lymphocyte function in humans. Since autoreactive T cells play an important role in the development of autoimmune diseases such as rheumatoid arthritis, multiple sclerosis, and organ transplantation, Orai1 becomes an attractive therapeutic target for ameliorating autoimmune disease. We developed a novel approach to inhibiting CRAC function by generating high-affinity fully human monoclonal antibodies to human Orai1. These antibodies inhibited ICRAC current, store-operated Ca(2+) influx, NFAT transcription, and cytokine release. These fully human antibodies to human Orai1 may represent a novel therapeutic approach for the treatment of autoimmunity.
Asunto(s)
Anticuerpos Bloqueadores/uso terapéutico , Anticuerpos Monoclonales/uso terapéutico , Enfermedades Autoinmunes/tratamiento farmacológico , Canales de Calcio/efectos de los fármacos , Canales de Calcio/inmunología , Aequorina/farmacología , Secuencia de Aminoácidos , Animales , Anticuerpos Bloqueadores/biosíntesis , Anticuerpos Monoclonales/biosíntesis , Western Blotting , Quimera , Citocinas/sangre , Mapeo Epitopo , Epítopos/efectos de los fármacos , Citometría de Flujo , Genes Reporteros , Células HEK293 , Humanos , Células Jurkat , Cinética , Luciferasas/genética , Ratones , Datos de Secuencia Molecular , Factores de Transcripción NFATC/biosíntesis , Factores de Transcripción NFATC/genética , Proteína ORAI1 , Técnicas de Placa-Clamp , Polimorfismo de Nucleótido Simple , RatasRESUMEN
Steroid hormones exhibit diverse biological activities. Despite intensive studies on steroid function at the genomic level, their nongenomic actions remain an enigma. In this study, we investigated the role of reactive oxygen species (ROS) in androgen-stimulated prostate cancer (PCa) cell proliferation. In androgen-treated PCa cells, increased cell growth and ROS production correlated with elevated p66Shc protein, an authentic oxidase. This growth stimulation was blocked by antioxidants. Further, elevated expression of p66Shc protein by cDNA transfection encoding wild-type protein, but not a redox-deficient (W134F) mutant, was associated with increased PCa cell proliferation. Conversely, knockdown of p66Shc expression by shRNA resulted in diminished cell growth. Increased p66Shc expression in PCa cells enhanced their tumorigenicity in xenograft animals. Importantly, p66Shc protein level is higher in clinical prostate adenocarcinomas than in adjacent noncancerous cells. Expression of redox-deficient p66Shc mutant protein abolished androgen-stimulated cell growth. In androgen-treated, H(2)O(2)-treated, and p66Shc cDNA-transfected PCa cells, cellular prostatic acid phosphatase, an authentic tyrosine phosphatase, was inactivated by reversible oxidation; subsequently, ErbB-2 was activated by phosphorylation at tyrosine-1221/1222. These results together support the notion that androgens induce ROS production through the elevation of p66Shc protein, which inactivates tyrosine phosphatase activity for the activation of interacting tyrosine kinase, leading to increased cell proliferation and enhanced tumorigenicity. Our results thus suggest that p66Shc protein functions at the critical junction point between androgens and tyrosine phosphorylation signaling in human PCa cells.
Asunto(s)
Andrógenos/farmacología , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Especies Reactivas de Oxígeno/metabolismo , Proteínas Adaptadoras de la Señalización Shc/metabolismo , Tirosina/metabolismo , Animales , Western Blotting , Proliferación Celular , Humanos , Peróxido de Hidrógeno/farmacología , Técnicas para Inmunoenzimas , Inmunoprecipitación , Longevidad , Masculino , Ratones , Oxidantes/farmacología , Oxidación-Reducción , Fosforilación , Proteínas Tirosina Fosfatasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Transducción de Señal , Proteína Transformadora 1 que Contiene Dominios de Homología 2 de Src , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
We identified the molecular target by histone deacetylase (HDAC) inhibitors for exploring their potential prostate cancer (PCa) therapy. Upon HDAC inhibitors-treatment, LNCaP cell growth was suppressed, correlating with increased cellular prostatic acid phosphatase (cPAcP) expression, an authentic protein tyrosine phosphatase. In those cells, ErbB-2 was dephosphorylated, histone H3/H4 acetylation and methylation increased and cyclin proteins decreased. In PAcP shRNA-transfected C-81 cells, valproic acid (VPA) efficacy of growth suppression was diminished. Further, VPA pre-treatment enhanced androgen responsiveness of C-81, C4-2 and MDA PCa2b-AI cells. Thus, cPAcP expression is involved in growth suppression by HDAC inhibitors in PCa cells, and VPA pre-treatments increase androgen responsiveness.
Asunto(s)
Inhibidores de Histona Desacetilasas/farmacología , Neoplasias de la Próstata/metabolismo , Proteínas Tirosina Fosfatasas/biosíntesis , Ácido Valproico/farmacología , Fosfatasa Ácida , Antineoplásicos/farmacología , Northern Blotting , Western Blotting , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Separación Celular , Citometría de Flujo , Humanos , Masculino , Fosforilación , Receptor ErbB-2/metabolismoRESUMEN
BACKGROUND: p66Shc, an isoform of Shc adaptor proteins, mediates diverse signals, including cellular stress and mouse longevity. p66Shc protein level is elevated in several carcinomas and steroid-treated human cancer cells. Several lines of evidence indicate that p66Shc plays a critical role in steroid-related carcinogenesis, and steroids play a role in its elevated levels in those cells without known mechanism. METHODS AND FINDINGS: In this study, we investigated the molecular mechanism by which steroid hormones up-regulate p66Shc protein level. In steroid-treated human prostate and ovarian cancer cells, p66Shc protein levels were elevated, correlating with increased cell proliferation. These steroid effects on p66Shc protein and cell growth were competed out by the respective antagonist. Further, actinomycin D and cyclohexamide could only partially block the elevated p66Shc protein level by steroids. Treatment with proteasomal inhibitors, but not lysosomal protease inhibitor, resulted in elevated p66Shc protein levels, even higher than that by steroids. Using prostate cancer cells as a model, immunoprecipitation revealed that androgens and proteasomal inhibitors reduce the ubiquitinated p66Shc proteins. CONCLUSIONS: The data collectively indicate that functional steroid receptors are required in steroid up-regulation of p66Shc protein levels in prostate and ovarian cancer cells, correlating with cell proliferation. In these steroid-treated cells, elevated p66Shc protein level is apparently in part due to inhibiting its ubiquitination. The results may lead to an impact on advanced cancer therapy via the regulation of p66Shc protein by up-regulating its ubiquitination pathway.
Asunto(s)
Proliferación Celular/efectos de los fármacos , Neoplasias Ováricas/metabolismo , Neoplasias de la Próstata/metabolismo , Proteínas Adaptadoras de la Señalización Shc/genética , Esteroides/farmacología , Ubiquitinación/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacos , Línea Celular Tumoral , Femenino , Humanos , Masculino , Neoplasias Ováricas/química , Neoplasias Ováricas/patología , Neoplasias de la Próstata/química , Neoplasias de la Próstata/patología , Receptores de Esteroides/fisiología , Proteínas Adaptadoras de la Señalización Shc/metabolismo , Proteína Transformadora 1 que Contiene Dominios de Homología 2 de SrcRESUMEN
Cellular prostatic acid phosphatase (cPAcP), an authentic tyrosine phosphatase, is proposed to function as a negative growth regulator of prostate cancer (PCa) cells in part through its dephosphorylation of ErbB-2. Nevertheless, the direct interaction between cPAcP and ErbB-2 has not been shown nor the specific dephosphorylation site of ErbB-2 by cPAcP. In this report, our data show that the phosphorylation level of ErbB-2 primarily at Tyr(1221/2) correlates with the growth rate of both LNCaP and MDA PCa2b human PCa cells. Further, cPAcP reciprocally co-immunoprecipitated with ErbB-2 in a non-permissive growth condition. Expression of wild type cPAcP, but not inactive mutant, by cDNA in cPAcP-null LNCaP C-81 cells results in decreased tyrosine phosphorylation of ErbB-2 including Tyr(1221/2). Concurrently, Tyr(317) phosphorylation of p52(Shc), proliferating cell nuclear antigen expression, and cell growth are decreased in these cells. Conversely, decreased cPAcP expression by short hairpin RNA in LNCaP C-33 cells was associated with elevated phosphorylation of ErbB-2 initially at Tyr(1221/2). Its downstream p52(Shc), ERK1/2, Akt, Src, STAT-3, and STAT-5 were activated, and cell proliferation, proliferating cell nuclear antigen, and cyclin D1 expression were increased. Stable subclones of C-33 cells by small interfering PAcP had elevated Tyr(1221/2) phosphorylation of ErbB-2 and exhibited androgen-independent growth and increased tumorigenicity in xenograft female animals. In summary, our data together indicate that in prostate epithelia, cPAcP interacts with and dephosphorylates ErbB-2 primarily at Tyr(1221/2) and hence blocks downstream signaling, leading to reduced cell growth. In PCa cells, decreased cPAcP expression is associated with androgen-independent cell proliferation and tumorigenicity as seen in advanced hormone-refractory prostate carcinomas.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Neoplasias de la Próstata/metabolismo , Proteínas Tirosina Fosfatasas/fisiología , Receptor ErbB-2/metabolismo , Fosfatasa Ácida , Animales , Línea Celular Tumoral , Proliferación Celular , Femenino , Humanos , Masculino , Ratones , Trasplante de Neoplasias , Fosforilación , Proteínas Tirosina Fosfatasas/metabolismo , Fracciones Subcelulares , Tirosina/químicaRESUMEN
We examined the efficacy of combination treatments utilizing cytotoxic drugs plus inhibitors to members of the ErbB-ERK signal pathway in human prostate cancer (PCa) LNCaP C-81 cells. Under an androgen-reduced condition, 50nM gemcitabine caused about 40% growth suppression on C-81 cells. Simultaneous treatment of gemcitabine plus 10microM AG825 produced 60% suppression (p<0.03); while, 85% growth inhibition (p<0.02) was seen if AG825 was added to gemcitabine-treated cells after a 24h-interval. Our data thus showed that in androgen-reduced conditions, inhibition of ErbB-2 increases the cytotoxic efficacy of gemcitabine in PCa cells. This finding has significant implications in the choice of drugs for combination therapy as well as the order of administration for treating cancer patients.
Asunto(s)
Andrógenos/metabolismo , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Neoplasias de la Próstata/patología , Receptor ErbB-2/antagonistas & inhibidores , Andrógenos/deficiencia , Antimetabolitos Antineoplásicos/farmacología , Benzotiazoles/farmacología , Línea Celular Tumoral , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacología , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo , Quinasas MAP Reguladas por Señal Extracelular/antagonistas & inhibidores , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Flavonoides/farmacología , Humanos , Masculino , Neoplasias de la Próstata/enzimología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Quinazolinas , Receptor ErbB-2/metabolismo , Factores de Tiempo , Tirfostinos/farmacología , Proteína X Asociada a bcl-2/metabolismo , Proteína bcl-X/metabolismo , GemcitabinaRESUMEN
The CB2 receptor is an attractive therapeutic target for analgesic and anti-inflammatory agents. Herein we describe the discovery of a novel class of oxadiazole derivatives from which potent and selective CB2 agonist leads were developed. Initial hit 7 was identified from a cannabinoid target-biased library generated by virtual screening of sample collections using a pharmacophore model in combination with a series of physicochemical filters. 7 was demonstrated to be a selective CB2 agonist (CB2 EC50 = 93 nM, Emax = 98%, CB1 EC50 > 10 microM). However, this compound exhibited poor solubility and relatively high clearance in rat, resulting in low oral bioavailability. In this paper, we report detailed SAR studies on 7 en route toward improving potency, physicochemical properties, and solubility. This effort resulted in identification of 63 that is a potent and selective agonist at CB2 (EC50 = 2 nM, Emax = 110%) with excellent pharmacokinetic properties.
Asunto(s)
Aminoquinolinas/síntesis química , Oxadiazoles/síntesis química , Receptor Cannabinoide CB2/agonistas , Administración Oral , Aminoquinolinas/administración & dosificación , Aminoquinolinas/farmacocinética , Animales , Disponibilidad Biológica , Células CHO , Cricetinae , Cricetulus , Evaluación Preclínica de Medicamentos , Humanos , Modelos Moleculares , Oxadiazoles/administración & dosificación , Oxadiazoles/farmacocinética , Ratas , Relación Estructura-ActividadRESUMEN
BACKGROUND: The expression of prostate-derived factor (PDF) is significantly elevated in human prostate tumors. We investigate the functional role and signaling of PDF in androgen receptor (AR)-positive human prostate cancer cells. METHODS: Transient or stable expression of PDF by cDNA transfection, antisense-mediated gene silencing, media conditioned by PDF-elevated cells, and antibody (Ab) neutralization were employed. RESULTS: Elevated endogenous and exogenous expression of PDF and treatment of PDF-enriched media were associated with increased proliferation and clonogenic growth of the cells. On the contrary, knockdown of PDF or addition of PDF neutralizing Ab resulted in diminished proliferation and reduced anchorage-independent growth. Further, ERK1/2 and p90RSK, but not Smad2/3, were activated in PDF-elevated cells as well as in cells treated with PDF-enriched media, while inhibition of ERK1/2 decreased the growth of those cells. CONCLUSION: PDF promotes AR-positive prostate tumor progression through upregulating cell proliferation via ERK1/2 signal pathway.
Asunto(s)
Proteínas Morfogenéticas Óseas/biosíntesis , Neoplasias de la Próstata/metabolismo , Receptores Androgénicos/biosíntesis , Western Blotting , Proteínas Morfogenéticas Óseas/genética , Proteínas Morfogenéticas Óseas/metabolismo , Adhesión Celular/fisiología , Procesos de Crecimiento Celular/fisiología , Línea Celular Tumoral , Silenciador del Gen , Factor 15 de Diferenciación de Crecimiento , Humanos , Masculino , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Proteínas Quinasas S6 Ribosómicas 90-kDa/metabolismo , Transducción de Señal , Transfección , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
PURPOSE: To determine whether specific dietary and synthetic flavonoids can protect human retinal pigment epithelial (RPE) cells from oxidative-stress-induced death. METHODS: The efficacy and potency were determined of a variety of dietary and synthetic flavonoids on the survival of human ARPE-19 cells and primary human RPE cells treated with either hydrogen peroxide (H2O2) or t-butyl hydroperoxide (t-BOOH). We determined the effective concentrations (EC50s) and the toxicities (LD50s) of the flavonoids after 24 hours, by using the MTT assay. The efficacy of vitamins E and C on RPE cell survival were compared under identical conditions. The ability of specific flavonoids to protect RPE cells from cell death was determined at various time intervals after the cells were exposed to oxidative stress. The ability of flavonoids to block the accumulation of intracellular reactive oxygen species was examined with dichlorofluorescein (DCF) fluorescence. Finally, the ability of flavonoids to induce phase-2 detoxifying enzymes was tested by immunoblot analysis for the transcription factor Nrf2 and the phase-2 gene product heme-oxygenase 1. RESULTS: Specific flavonoids protected human RPE cells from oxidative-stress-induced death with efficacies between 80% and 100% and potencies in the high-nanomolar and low-micromolar range. The toxicities of most of the effective flavonoids were low. The effective flavonoids included the dietary flavonoids fisetin, luteolin, quercetin, eriodictyol, baicalein, galangin and EGCG, and the synthetic flavonoids, 3,6-dihydroxy flavonol and 3,7 dihydroxy flavonol. Several flavonoids can protect RPE cells even when they are added after the cells have been exposed to oxidative stress. The flavonoids acted through an intracellular route to block the accumulation of reactive oxygen species. Many of these flavonoids induced the expression of Nrf2 and the phase-2 gene product heme-oxygenase 1 in human RPE cells. CONCLUSIONS: The results identify a select group of flavonoids that protect RPE cells from oxidative-stress-induced death with a high degree of potency and low toxicity. Many of these flavonoids also induce the expression of phase-2 detoxification proteins which could function to provide additional protection against oxidative stress. This select group of flavonoids and the foods that contain high levels of these compounds may have some clinical benefit for patients with retinal diseases associated with oxidative stress.
Asunto(s)
Citoprotección/efectos de los fármacos , Flavonoides/farmacología , Estrés Oxidativo/efectos de los fármacos , Epitelio Pigmentado Ocular/efectos de los fármacos , Western Blotting , Recuento de Células , Técnicas de Cultivo de Célula , Diferenciación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Dieta , Relación Dosis-Respuesta a Droga , Hemo-Oxigenasa 1/metabolismo , Humanos , Peróxido de Hidrógeno/toxicidad , Factor 2 Relacionado con NF-E2/metabolismo , Epitelio Pigmentado Ocular/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sales de Tetrazolio , Tiazoles , terc-Butilhidroperóxido/toxicidadRESUMEN
Neuroendocrine (NE) cells are the minor cell populations in normal prostate epithelial compartments. During prostate carcinogenesis, the number of NE cells in malignant lesions increases, correlating with its tumorigenicity and hormone-refractory growth. It is thus proposed that cancerous NE cells promote prostate cancer (PCa) cell progression and its androgen-independent proliferation, although the origin of the cancerous NE cells is not clear. To investigate the role of cancerous NE cells in prostate carcinogenesis, we characterized three NE subclone cell lines-NE-1.3, NE-1.8 and NE-1.9, which were transdifferentiated from androgen-sensitive human PCa LNCaP cells by culturing in an androgen-depleted environment, resembling clinical androgen-ablation therapy. These subclone cells acquire many features of NE cells seen in clinical prostate carcinomas, for example exhibiting a neuronal morphology and expressing multiple NE markers, including neuron-specific enolase, chromogranin B, neurotensin, parathyroid hormone-related peptide, and to a lesser degree for chromogranin A, while lacking androgen receptor (AR) or prostate specific antigen (PSA) expression. These cells represent terminally differentiated stable cells because after 3 months of re-culturing in a medium containing androgenic activity, they still retained the NE phenotype and expressed NE markers. Despite these NE cells having a slow growth rate, they readily developed xenograft tumors. Furthermore, media conditioned by these NE cells exhibited a stimulatory effect on proliferation and PSA secretion by LNCaP cells in androgen-deprived conditions. Additionally, we found that receptor protein tyrosine phosphatase alpha plays a role in upregulating multiple NE markers and acquiring the NE phenotype. These NE cells thus represent cancerous NE cells and could serve as a useful cell model system for investigating the role of cancerous NE cells in hormone-refractory proliferation of PCa cells.