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1.
Cancers (Basel) ; 16(1)2024 Jan 02.
Artículo en Inglés | MEDLINE | ID: mdl-38201638

RESUMEN

The tumor microenvironment (TME) is a critical determinant of tumor progression, metastasis, and therapeutic outcomes [...].

2.
PLoS One ; 16(9): e0257984, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-34570813

RESUMEN

Prostate cancer is the second leading cause of cancer related death in American men. Several therapies have been developed to treat advanced prostate cancer, but these therapies often have severe side effects. To improve the outcome with fewer side effects we focused on the furanocoumarin bergamottin, a natural product found in grapefruit juice and a potent CYP3A inhibitor. Our recent studies have shown that CYP3A5 inhibition can block androgen receptor (AR) signaling, critical for prostate cancer growth. We observed that bergamottin reduces prostate cancer (PC) cell growth by decreasing both total and nuclear AR (AR activation) reducing downstream AR signaling. Bergamottin's role in reducing AR activation was confirmed by confocal microscopy studies and reduction in prostate specific antigen (PSA) levels, which is a marker for prostate cancer. Further studies revealed that bergamottin promotes cell cycle block and accumulates G0/G1 cells. The cell cycle block was accompanied with reduction in cyclin D, cyclin B, CDK4, P-cdc2 (Y15) and P-wee1 (S642). We also observed that bergamottin triggers apoptosis in prostate cancer cell lines as evident by TUNEL staining and PARP cleavage. Our data suggests that bergamottin may suppress prostate cancer growth, especially in African American (AA) patients carrying wild type CYP3A5 often presenting aggressive disease.


Asunto(s)
Antagonistas de Receptores Androgénicos/uso terapéutico , Antineoplásicos/uso terapéutico , Apoptosis/efectos de los fármacos , Inhibidores del Citocromo P-450 CYP3A/uso terapéutico , Furocumarinas/uso terapéutico , Fase G1/efectos de los fármacos , Neoplasias de la Próstata/tratamiento farmacológico , Fase de Descanso del Ciclo Celular/efectos de los fármacos , Western Blotting , Fraccionamiento Celular , Línea Celular Tumoral , Citrus paradisi/química , Regulación hacia Abajo , Jugos de Frutas y Vegetales/análisis , Humanos , Masculino , Microscopía Confocal , Receptores Androgénicos/efectos de los fármacos
3.
Cancers (Basel) ; 12(4)2020 Apr 17.
Artículo en Inglés | MEDLINE | ID: mdl-32316460

RESUMEN

Androgen receptor signaling is crucial for prostate cancer growth and is positively regulated in part by intratumoral CYP3A5. As African American (AA) men often carry the wild type CYP3A5 and express high levels of CYP3A5 protein, we blocked the wild type CYP3A5 in AA origin prostate cancer cells and tested its effect on androgen receptor signaling. q-PCR based profiler assay identified several AR regulated genes known to regulate AR nuclear translocation, cell cycle progression, and cell growth. CYP3A5 processes several commonly prescribed drugs and many of these are CYP3A5 inducers or inhibitors. In this study, we test the effect of these commonly prescribed CYP3A5 inducers/inhibitors on AR signaling. The results show that the CYP3A5 inducers promoted AR nuclear translocation, downstream signaling, and cell growth, whereas CYP3A5 inhibitors abrogated them. The observed changes in AR activity is specific to alterations in CYP3A5 activity as the effects are reduced in the CYP3A5 knockout background. Both the inducers tested demonstrated increased cell growth of prostate cancer cells, whereas the inhibitors showed reduced cell growth. Further, characterization and utilization of the observation that CYP3A5 inducers and inhibitors alter AR signaling may provide guidance to physicians prescribing CYP3A5 modulating drugs to treat comorbidities in elderly patients undergoing ADT, particularly AA.

4.
Cancer Epidemiol Biomarkers Prev ; 28(6): 1003-1009, 2019 06.
Artículo en Inglés | MEDLINE | ID: mdl-30787054

RESUMEN

BACKGROUND: Given the scarcity of cell lines from underrepresented populations, it is imperative that genetic ancestry for these cell lines is characterized. Consequences of cell line mischaracterization include squandered resources and publication retractions. METHODS: We calculated genetic ancestry proportions for 15 cell lines to assess the accuracy of previous race/ethnicity classification and determine previously unknown estimates. DNA was extracted from cell lines and genotyped for ancestry informative markers representing West African (WA), Native American (NA), and European (EUR) ancestry. RESULTS: Of the cell lines tested, all previously classified as White/Caucasian were accurately described with mean EUR ancestry proportions of 97%. Cell lines previously classified as Black/African American were not always accurately described. For instance, the 22Rv1 prostate cancer cell line was recently found to carry mixed genetic ancestry using a much smaller panel of markers. However, our more comprehensive analysis determined the 22Rv1 cell line carries 99% EUR ancestry. Most notably, the E006AA-hT prostate cancer cell line, classified as African American, was found to carry 92% EUR ancestry. We also determined the MDA-MB-468 breast cancer cell line carries 23% NA ancestry, suggesting possible Afro-Hispanic/Latina ancestry. CONCLUSIONS: Our results suggest predominantly EUR ancestry for the White/Caucasian-designated cell lines, yet high variance in ancestry for the Black/African American-designated cell lines. In addition, we revealed an extreme misclassification of the E006AA-hT cell line. IMPACT: Genetic ancestry estimates offer more sophisticated characterization leading to better contextualization of findings. Ancestry estimates should be provided for all cell lines to avoid erroneous conclusions in disparities literature.


Asunto(s)
Población Negra/genética , Neoplasias de la Mama/clasificación , Neoplasias de la Mama/genética , Línea Celular Tumoral/clasificación , Neoplasias de la Próstata/clasificación , Neoplasias de la Próstata/genética , Población Blanca/genética , Anciano , Biomarcadores de Tumor/genética , Neoplasias de la Mama/diagnóstico , Línea Celular Tumoral/patología , Femenino , Pruebas Genéticas/métodos , Células HeLa , Humanos , Masculino , Persona de Mediana Edad , Polimorfismo de Nucleótido Simple , Neoplasias de la Próstata/diagnóstico
5.
BMC Cancer ; 17(1): 631, 2017 Sep 06.
Artículo en Inglés | MEDLINE | ID: mdl-28877685

RESUMEN

BACKGROUND: Neoplastic cells proliferate rapidly and obtain requisite building blocks by reprogramming metabolic pathways that favor growth. Previously, we observed that prostate cancer cells uptake and store lipids in the form of lipid droplets, providing building blocks for membrane synthesis, to facilitate proliferation and growth. Mechanisms of lipid uptake, lipid droplet dynamics and their contribution to cancer growth have yet to be defined. This work is focused on elucidating the prostate cancer-specific modifications in lipid storage pathways so that these modified gene products can be identified and therapeutically targeted. METHODS: To identify genes that promote lipid droplet formation and storage, the expression profiles of candidate genes were assessed and compared between peripheral blood mononuclear cells and prostate cancer cells. Subsequently, differentially expressed genes were inhibited and growth assays performed to elucidate their role in the growth of the cancer cells. Cell cycle, apoptosis and autophagy assays were performed to ascertain the mechanism of growth inhibition. RESULTS: Our results indicate that DGAT1, ABHD5, ACAT1 and ATGL are overexpressed in prostate cancer cells compared to PBMCs and of these overexpressed genes, DGAT1 and ABHD5 aid in the growth of the prostate cancer cells. Blocking the expression of both DGAT1 and ABHD5 results in inhibition of growth, cell cycle block and cell death. DGAT1 siRNA treatment inhibits lipid droplet formation and leads to autophagy where as ABHD5 siRNA treatment promotes accumulation of lipid droplets and leads to apoptosis. Both the siRNA treatments reduce AMPK phosphorylation, a key regulator of lipid metabolism. While DGAT1 siRNA reduces phosphorylation of ACC, the rate limiting enzyme in de novo fat synthesis and triggers phosphorylation of raptor and ULK-1 inducing autophagy and cell death, ABHD5 siRNA decreases P70S6 phosphorylation, leading to PARP cleavage, apoptosis and cell death. Interestingly, DGAT-1 is involved in the synthesis of triacylglycerol where as ABHD5 is a hydrolase and participates in the fatty acid oxidation process, yet inhibition of both enzymes similarly promotes prostate cancer cell death. CONCLUSION: Inhibition of either DGAT1 or ABHD5 leads to prostate cancer cell death. Both DGAT1 and ABHD5 can be selectively targeted to block prostate cancer cell growth.


Asunto(s)
1-Acilglicerol-3-Fosfato O-Aciltransferasa/metabolismo , Diacilglicerol O-Acetiltransferasa/metabolismo , Gotas Lipídicas/metabolismo , Neoplasias de la Próstata/metabolismo , 1-Acilglicerol-3-Fosfato O-Aciltransferasa/genética , Apoptosis/genética , Autofagia , Ciclo Celular/genética , Línea Celular Tumoral , Diacilglicerol O-Acetiltransferasa/genética , Regulación Neoplásica de la Expresión Génica , Humanos , Metabolismo de los Lípidos , Masculino , Modelos Biológicos , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Interferencia de ARN , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo
6.
Drug Deliv Transl Res ; 6(6): 735-754, 2016 12.
Artículo en Inglés | MEDLINE | ID: mdl-27798766

RESUMEN

The eye is a distinctive organ with protective anatomy and physiology. Several pharmacokinetics compartment models of ocular drug delivery have been developed for describing the absorption, distribution, and elimination of ocular drugs in the eye. Determining pharmacokinetics parameters in ocular tissues is a major challenge because of the complex anatomy and dynamic physiological barrier of the eye. In this review, pharmacokinetics of these compartments exploring different drugs, delivery systems, and routes of administration is discussed including factors affecting intraocular bioavailability. Factors such as precorneal fluid drainage, drug binding to tear proteins, systemic drug absorption, corneal factors, melanin binding, and drug metabolism render ocular delivery challenging and are elaborated in this manuscript. Several compartment models are discussed; these are developed in ocular drug delivery to study the pharmacokinetics parameters. There are several transporters present in both anterior and posterior segments of the eye which play a significant role in ocular pharmacokinetics and are summarized briefly. Moreover, several ocular pharmacokinetics animal models and relevant studies are reviewed and discussed in addition to the pharmacokinetics of various ocular formulations.


Asunto(s)
Ojo/metabolismo , Soluciones Oftálmicas/farmacocinética , Administración Oftálmica , Animales , Ojo/anatomía & histología , Humanos , Proteínas de Transporte de Membrana/metabolismo
7.
Prostate ; 75(5): 527-38, 2015 Apr 01.
Artículo en Inglés | MEDLINE | ID: mdl-25586052

RESUMEN

BACKGROUND: The central role of androgen receptor (AR) signaling is established in prostate cancer growth and progression. We propose CYP3A5 is part of a feedback loop that modulates the sensitivity of AR to androgen exposure. The purpose of this study is to elucidate the mechanism of regulation of AR expression by CYP3A5. METHODS: To identify the role of CYP3A5 in regulating AR signaling, CYP3A5 protein expression was inhibited using CYP3A5 siRNA and azamulin. Both cell fractionation and immunocytochemical approaches in combination with dihydrotestosterone (DHT) and R1881 treatment were used to evaluate changes in AR nuclear translocation. RESULTS: CYP3A5 siRNA blocked growth of LNCaP and C4-2 cells by 30-60% (P ≤ 0.005). Azamulin, a CYP3A pharmacologic inhibitor, reduced the growth of LNCaP, C4-2 and 22RV1 lines by ∼ 40% (P ≤ 0.005). CYP3A5 siRNA inhibited growth in response to DHT and R1881 treatment in LNCaP and C4-2 by decreasing nuclear AR localization and resulting in diminished PSA and TMPRSS2 expression. Decreased AR nuclear localization resulting from CYP3A5 inhibition resulted in growth inhibition comparable to IC60 and IC40 of bicalutamide in LNCaP and C4-2 cell lines. Conversely, the CYP3A inducer rifampicin enhanced AR nuclear localization. CONCLUSION: As CYP3A5 regulates the nuclear translocation of AR; co-targeting CYP3A5 may provide a novel strategy for enhancing the efficacy of androgen deprivation therapy. Consequentially, these data suggest that concomitant medications may impact androgen deprivation therapy's efficacy.


Asunto(s)
Citocromo P-450 CYP3A/fisiología , Neoplasias de la Próstata/enzimología , Receptores Androgénicos/metabolismo , Transducción de Señal/fisiología , Western Blotting , Hidrocarburos Aromáticos con Puentes/farmacología , Línea Celular Tumoral , Inhibidores del Citocromo P-450 CYP3A/farmacología , Dihidrotestosterona/farmacología , Técnica del Anticuerpo Fluorescente , Humanos , Masculino , Metribolona/farmacología , Neoplasias de la Próstata/patología , ARN Interferente Pequeño/farmacología , Reacción en Cadena en Tiempo Real de la Polimerasa , Triazoles/farmacología
8.
BMC Cancer ; 14: 91, 2014 Feb 15.
Artículo en Inglés | MEDLINE | ID: mdl-24528787

RESUMEN

BACKGROUND: Reprogramming of energy metabolism of malignant cancer cells confers competitive advantage in growth environments with limited resources. However, not every process of cancer development is associated with competition for resources. During hematogenous transport, cancer cells are exposed to high levels of oxygen and nutrients. Does energy metabolism of cancer cells change as a function of exposure to the bloodstream? Could such changes be exploited to improve the detection of circulating tumor cells (CTC)? These questions have clinical significance, but have not yet been sufficiently examined. METHODS: The energy metabolism was examined as a function of incubation in nutrient-rich plasma in prostate metastatic cancer cells LNCaP and non-transformed prostate epithelial cells RWPE1. Uptake kinetics of a fluorescent glucose analog (2-NBD) and lipophilic dyes (DiD & Bodipy) were measured in both cell lines, as well as in peripheral blood mononuclear cells (PBMC). RESULTS: LNCaP cells exhibited hyper-acetylation of low molecular weight proteins compared to RWPE1 cells. Following plasma incubation, protein lysine acetylation profile was unchanged for LNCaP cells while significantly altered for RWPE1 cells. O-linked glycosylated protein profiles were different between LNCaP and RWPE1 cells and varied in both cell lines with plasma incubation. Maximal respiration or glycolytic capacities was unchanged in LNCaP cells and impaired in RWPE1 cells following plasma incubation. However, the uptake rates of 2-NBD and DiD were insufficient for discrimination of LNCaP, or RWPE1 cells from PBMC. On the other hand, both RWPE1 and LNCaP cells exhibited intracellular lipid bodies following plasma incubation; whereas, PBMC did not. The presence of lipid bodies in LNCaP cells permitted retention of Bodipy dye and allowed discrimination of LNCaP cells from PBMC with flow cytometry. CONCLUSIONS: Despite clear differences in energy metabolism, metastatic prostate cancer cells could not be efficiently distinguished from non-transformed prostate epithelial cells using fluorescent glucose or lipid uptake kinetics. However, metastatic prostate cancer cells in plasma could be clearly distinguished from blood nucleated cells due to the presence of intracellular lipid bodies. Fluorescent labeling of lipid bodies permitted a simple and sensitive means for high throughput detection of metastatic prostate cancer cells in human plasma.


Asunto(s)
Transformación Celular Neoplásica/patología , Lípidos de la Membrana/sangre , Células Neoplásicas Circulantes/patología , Neoplasias de la Próstata/patología , Coloración y Etiquetado/métodos , Transformación Celular Neoplásica/química , Colorantes Fluorescentes , Humanos , Leucocitos Mononucleares/química , Leucocitos Mononucleares/patología , Masculino , Lípidos de la Membrana/química , Células Neoplásicas Circulantes/química , Neoplasias de la Próstata/sangre , Neoplasias de la Próstata/química
9.
Prostate ; 73(7): 706-14, 2013 May.
Artículo en Inglés | MEDLINE | ID: mdl-23138928

RESUMEN

BACKGROUND: Neutral endopeptidase (NEP) is a transmembrane cell surface peptidase present on prostatic epithelial cells that catalytically inactivates small peptide substrates. Neutral endopeptidase loss is associated with prostate cancer growth, progression, and increased angiogenesis. We examined whether NEP expression is regulated by hypoxia, frequently encountered in the tumor microenvironment. METHODS: NEP expression was compared in prostate cancer cell lines cultured in normoxic and hypoxic conditions. The NEP activity, protein levels, and mRNA levels were determined using enzyme assay, Western blotting and q-PCR analysis, respectively. Electrophoretic mobility shift assay and chromatin immunoprecipitation assay (ChIP) was used to confirm the negative regulation of NEP at the transcriptional level by hypoxia responsive elements (HREs). RESULTS: The results indicate that NEP expression was inhibited under hypoxic conditions in the NEP positive LNCaP, C4-2, and 22RV1 cells and human umbilical vascular endothelial (HUVEC) cells. NEP regulation appeared to be predominantly at the transcriptional level as NEP mRNA expression significantly reduced with hypoxia, concordant with the kinetics of protein levels, and NEP enzyme activity. A search of the NEP gene sequence revealed three putative HREs upstream of the NEP promoter. Two of the HREs demonstrated a specific reduction of shift in the presence of cobalt chloride; specificity of the binding sites was confirmed by ChIP. CONCLUSIONS: Our data indicate a novel mechanism where hypoxia negatively regulates the tumor suppressor function of NEP in prostate cancer. The negative regulation of NEP is mediated by binding of the HIF1-α protein binding to the HREs present upstream of the NEP promoter.


Asunto(s)
Neprilisina/metabolismo , Próstata/enzimología , Neoplasias de la Próstata/enzimología , ARN Mensajero/metabolismo , Western Blotting , Hipoxia de la Célula , Inmunoprecipitación de Cromatina , Ensayo de Inmunoadsorción Enzimática , Humanos , Masculino , Neprilisina/genética , Próstata/patología , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Reacción en Cadena en Tiempo Real de la Polimerasa , Células Tumorales Cultivadas
10.
BMC Cancer ; 12: 540, 2012 Nov 21.
Artículo en Inglés | MEDLINE | ID: mdl-23171028

RESUMEN

BACKGROUND: Circulating tumour cells (CTC) are an important indicator of metastasis and associated with a poor prognosis. Detection sensitivity and specificity of CTC in the peripheral blood of metastatic cancer patient remain a technical challenge. METHODS: Coherent anti-Stokes Raman scattering (CARS) microscopy was employed to examine the lipid content of CTC isolated from the peripheral blood of metastatic prostate cancer patients. CARS microscopy was also employed to evaluate lipid uptake and mobilization kinetics of a metastatic human prostate cancer cell line. RESULTS: One hundred CTC from eight metastatic prostate cancer patients exhibited strong CARS signal which arose from intracellular lipid. In contrast, leukocytes exhibited weak CARS signal which arose mostly from cellular membrane. On average, CARS signal intensity of prostate CTC was 7-fold higher than that of leukocytes (P<0.0000001). When incubated with human plasma, C4-2 metastatic human prostate cancer cells exhibited rapid lipid uptake kinetics and slow lipid mobilization kinetics. Higher expression of lipid transport proteins in C4-2 cells compared to non-transformed RWPE-1 and non-malignant BPH-1 prostate epithelial cells further indicated strong affinity for lipid of metastatic prostate cancer cells. CONCLUSIONS: Intracellular lipid could serve as a biomarker for prostate CTC which could be sensitively detected with CARS microscopy in a label-free manner. Strong affinity for lipid by metastatic prostate cancer cells could be used to improve detection sensitivity and therapeutic targeting of prostate CTC.


Asunto(s)
Metabolismo de los Lípidos/fisiología , Células Neoplásicas Circulantes/metabolismo , Células Neoplásicas Circulantes/patología , Neoplasias de la Próstata/sangre , Neoplasias de la Próstata/patología , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Línea Celular , Línea Celular Tumoral , Membrana Celular/genética , Membrana Celular/metabolismo , Membrana Celular/patología , Células Epiteliales/metabolismo , Células Epiteliales/patología , Humanos , Leucocitos/metabolismo , Leucocitos/patología , Masculino , Microscopía/métodos , Neoplasias de la Próstata/diagnóstico , Neoplasias de la Próstata/genética , Sensibilidad y Especificidad , Espectrometría Raman/métodos
11.
J Biol Chem ; 286(20): 17543-59, 2011 May 20.
Artículo en Inglés | MEDLINE | ID: mdl-21402692

RESUMEN

CYP3A4 expression in breast cancer correlates with decreased overall survival, but the mechanisms are unknown. Cytochrome P450 gene profiling by RNAi silencing demonstrates that CYP3A or 2C8 gene expression is specifically required for growth of the breast cancer lines MCF7, T47D, and MDA-MB-231. CYP3A4 silencing blocks the cell cycle at the G(2)/M checkpoint and induces apoptosis in the MCF7 line, thereby inhibiting anchorage-dependent growth and survival. CYP3A4 was profiled for NADPH-dependent arachidonic acid (AA) metabolism and synthesized AA epoxygenase products (±)-8,9-, (±)-11,12-, and (±)-14,15-epoxyeicosatrienoic acid (EET) (total turnover of ∼2 pmol/pmol CYP3A4/min) but not hydroxylase products (±)-15-, (±)-19-, or 20-hydroxyeicosatetraenoic acid. Furthermore, eicosanoid profiling revealed that MCF7 cells synthesize EETs in a CYP3A4-dependent manner. The (±)-14,15-EET regioisomer selectively rescues breast cancer cells from CYP3A4 silencing in a concentration-dependent fashion and promotes mitogenesis and anchorage-dependent cloning. Stat3 (Tyr-705) phosphorylation was inhibited by CYP3A4 silencing, providing a potential mechanism for CYP3A4 involvement in breast cancer cell growth. Silencing Stat3 blocks breast cancer cell growth and abrogates (±)-14,15-EET-induced proliferation, indicating a Stat3 requirement for (±)-14,15-EET-mediated cell growth. Although silencing of CYP3A4 reduces nuclear Tyr(P)-705-Stat3, (±)-14,15-EET restores this signaling process and promotes Tyr(P)-705-Stat3 translocation to the nucleus, suggesting that (±)-14,15-EET may be involved in an autocrine/paracrine pathway driving cell growth. These studies indicate that CYP3A4 is a highly active AA epoxygenase that promotes Stat3-mediated breast cancer cell growth in part through (±)-14,15-EET biosynthesis. Furthermore, these studies indicate an essential role for Stat3 as a mediator of epoxygenase activity in breast cancer.


Asunto(s)
Ácido 8,11,14-Eicosatrienoico/análogos & derivados , Neoplasias de la Mama/metabolismo , División Celular , Citocromo P-450 CYP3A/metabolismo , Fase G2 , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Factor de Transcripción STAT3/metabolismo , Ácido 8,11,14-Eicosatrienoico/genética , Ácido 8,11,14-Eicosatrienoico/metabolismo , Transporte Activo de Núcleo Celular/genética , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Línea Celular Tumoral , Citocromo P-450 CYP3A/genética , Femenino , Silenciador del Gen , Humanos , Fosforilación/genética , Factor de Transcripción STAT3/genética , Transducción de Señal/genética
12.
J Thorac Oncol ; 6(4): 661-70, 2011 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-21270666

RESUMEN

INTRODUCTION: Ritonavir is a potential therapeutic agent in lung cancer, but its targets in lung adenocarcinoma are unknown, as are candidate biomarkers for its activity. METHODS: RNAi was used to identify genes whose expression affects ritonavir sensitivity. Synergy between ritonavir, gemcitabine, and cisplatin was tested by isobologram analysis. RESULTS: Ritonavir inhibits growth of K-ras mutant lung adenocarcinoma lines A549, H522, H23, and K-ras wild-type line H838. Ritonavir causes G0/G1 arrest and apoptosis. Associated with G0/G1 arrest, ritonavir down-regulates cyclin-dependent kinases, cyclin D1, and retinoblastoma protein phosphorylation. Associated with induction of apoptosis, ritonavir reduces survivin messenger RNA and protein levels more than twofold. Ritonavir inhibits phosphorylation of c-Src and signal transducer and activator of transcription protein 3, which are important events for survivin gene expression and cell growth, and induces cleavage of PARP1. Although knock down of survivin, c-Src, or signal transducer and activator of transcription protein 3 inhibits cell growth, only survivin knock down enhances ritonavir inhibition of growth and survivin overexpression promotes ritonavir resistance. Ritonavir was tested in combination with gemcitabine or cisplatin, exhibiting synergistic and additive effects, respectively. The combination of ritonavir/gemcitabine/cisplatin is synergistic in the A549 line and additive in the H522 line, at clinically feasible ritonavir concentrations (<10 µM). CONCLUSIONS: Ritonavir is of interest for lung adenocarcinoma therapeutics, and survivin is an important target and potential biomarker for its sensitivity. Ritonavir cooperation with gemcitabine/cisplatin might be explained by involvement of PARP1 in repair of cisplatin-mediated DNA damage and survivin in repair of gemcitabine-mediated double-stranded DNA breaks.


Asunto(s)
Adenocarcinoma/tratamiento farmacológico , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Inhibidores de la Proteasa del VIH/farmacología , Proteínas Inhibidoras de la Apoptosis/antagonistas & inhibidores , Neoplasias Pulmonares/tratamiento farmacológico , Ritonavir/farmacología , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Apoptosis/efectos de los fármacos , Western Blotting , Ciclo Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Cisplatino/administración & dosificación , Ciclina D1/genética , Ciclina D1/metabolismo , Roturas del ADN de Doble Cadena/efectos de los fármacos , Desoxicitidina/administración & dosificación , Desoxicitidina/análogos & derivados , Quimioterapia Combinada , Humanos , Proteínas Inhibidoras de la Apoptosis/genética , Proteínas Inhibidoras de la Apoptosis/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Poli(ADP-Ribosa) Polimerasa-1 , Poli(ADP-Ribosa) Polimerasas/genética , Poli(ADP-Ribosa) Polimerasas/metabolismo , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Survivin , Células Tumorales Cultivadas , Gemcitabina
13.
Clin Cancer Res ; 17(9): 2934-46, 2011 May 01.
Artículo en Inglés | MEDLINE | ID: mdl-21242119

RESUMEN

PURPOSE: This study was performed to discover prognostic genomic markers associated with postoperative outcome of stage I to III non-small cell lung cancer (NSCLC) that are reproducible between geographically distant and demographically distinct patient populations. EXPERIMENTAL DESIGN: American patients (n = 27) were stratified on the basis of recurrence and microarray profiling of their tumors was performed to derive a training set of 44 genes. A larger Korean patient validation cohort (n = 138) was also stratified by recurrence and screened for these genes. Four reproducible genes were identified and used to construct genomic and clinicogenomic Cox models for both cohorts. RESULTS: Four genomic markers, DBN1 (drebrin 1), CACNB3 (calcium channel beta 3), FLAD1 (PP591; flavin adenine dinucleotide synthetase), and CCND2 (cyclin D2), exhibited highly significant differential expression in recurrent tumors in the training set (P < 0.001). In the validation set, DBN1, FLAD1 (PP591), and CACNB3 were significant by Cox univariate analysis (P ≤ 0.035), whereas only DBN1 was significant by multivariate analysis. Genomic and clinicogenomic models for recurrence-free survival (RFS) were equally effective for risk stratification of stage I to II or I to III patients (all models P < 0.0001). For stage I to II or I to III patients, 5-year RFS of the low- and high-risk patients was approximately 70% versus 30% for both models. The genomic model for overall survival of stage I to III patients was improved by addition of pT and pN stage (P < 0.0013 vs. 0.010). CONCLUSION: A 4-gene prognostic model incorporating the multivariate marker DBN1 exhibits potential clinical utility for risk stratification of stage I to III NSCLC patients.


Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/diagnóstico , Carcinoma de Pulmón de Células no Pequeñas/cirugía , Perfilación de la Expresión Génica , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/cirugía , Adulto , Anciano , Anciano de 80 o más Años , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/patología , Supervivencia sin Enfermedad , Femenino , Perfilación de la Expresión Génica/normas , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Masculino , Análisis por Micromatrices , Persona de Mediana Edad , Modelos Genéticos , Técnicas de Diagnóstico Molecular/normas , Estadificación de Neoplasias , Periodo Posoperatorio , Pronóstico , Recurrencia , Estándares de Referencia , Estudios de Validación como Asunto
14.
Clin Cancer Res ; 12(6): 1883-96, 2006 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-16551874

RESUMEN

PURPOSE: These studies were designed to determine whether ritonavir inhibits breast cancer in vitro and in vivo and, if so, how. EXPERIMENTAL DESIGN: Ritonavir effects on breast cancer cell growth were studied in the estrogen receptor (ER)-positive lines MCF7 and T47D and in the ER-negative lines MDA-MB-436 and MDA-MB-231. Effects of ritonavir on Rb-regulated and Akt-mediated cell proliferation were studied. Ritonavir was tested for inhibition of a mammary carcinoma xenograft. RESULTS: ER-positive estradiol-dependent lines (IC50, 12-24 micromol/L) and ER-negative (IC50, 45 micromol/L) lines exhibit ritonavir sensitivity. Ritonavir depletes ER-alpha levels notably in ER-positive lines. Ritonavir causes G1 arrest, depletes cyclin-dependent kinases 2, 4, and 6 and cyclin D1 but not cyclin E, and depletes phosphorylated Rb and Ser473 Akt. Ritonavir induces apoptosis independent of G1 arrest, inhibiting growth of cells that have passed the G1 checkpoint. Myristoyl-Akt, but not activated K-Ras, rescues ritonavir inhibition. Ritonavir inhibited a MDA-MB-231 xenograft and intratumoral Akt activity at a clinically attainable serum Cmax of 22 +/- 8 micromol/L. Because heat shock protein 90 (Hsp90) substrates are depleted by ritonavir, ritonavir effects on Hsp90 were tested. Ritonavir binds Hsp90 (K(D), 7.8 micromol/L) and partially inhibits its chaperone function. Ritonavir blocks association of Hsp90 with Akt and, with sustained exposure, notably depletes Hsp90. Stably expressed Hsp90alpha short hairpin RNA also depletes Hsp90, inhibiting proliferation and sensitizing breast cancer cells to low ritonavir concentrations. CONCLUSIONS: Ritonavir inhibits breast cancer growth in part by inhibiting Hsp90 substrates, including Akt. Ritonavir may be of interest for breast cancer therapeutics and its efficacy may be increased by sustained exposure or Hsp90 RNA interference.


Asunto(s)
Proliferación Celular/efectos de los fármacos , Inhibidores de la Proteasa del VIH/farmacología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ritonavir/farmacología , Animales , Western Blotting , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Ciclina D1/metabolismo , Quinasas Ciclina-Dependientes/metabolismo , Relación Dosis-Respuesta a Droga , Femenino , Proteínas HSP90 de Choque Térmico/metabolismo , Humanos , Inmunohistoquímica , Inmunoprecipitación , Neoplasias Mamarias Experimentales/tratamiento farmacológico , Neoplasias Mamarias Experimentales/metabolismo , Neoplasias Mamarias Experimentales/patología , Ratones , Ratones Desnudos , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , Ritonavir/sangre , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto
15.
Appl Environ Microbiol ; 71(7): 3716-24, 2005 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-16000781

RESUMEN

Nitrogen fixation in Azotobacter vinelandii is regulated by the nifLA operon. NifA activates the transcription of nif genes, while NifL antagonizes the transcriptional activator NifA in response to fixed nitrogen and molecular oxygen levels. However, transcriptional regulation of the nifLA operon of A. vinelandii itself is not fully understood. Using the S1 nuclease assay, we mapped the transcription start site of the nifLA operon, showing it to be similar to the sigma54-dependent promoters. We also identified a positive cis-acting regulatory element (+134 to +790) of the nifLA operon within the coding region of the nifL gene of A. vinelandii. Deletion of this element results in complete loss of promoter activity. Several protein factors bind to this region, and the specific binding sites have been mapped by DNase I foot printing. Two of these sites, namely dR1 (+134 to +204) and dR2 (+745 to +765), are involved in regulating the nifLA promoter activity. The absence of NtrC-like binding sites in the upstream region of the nifLA operon in A. vinelandii makes the identification of these downstream elements a highly significant finding. The interaction of the promoter with the proteins binding to the dR2 region spanning +745 to +765 appears to be dependent on the face of the helix as introduction of 4 bases just before this region completely disrupts promoter activity. Thus, the positive regulatory element present within the BglII-BglII fragment may play, in part; an important role in nifLA regulation in A. vinelandii.


Asunto(s)
Azotobacter vinelandii/genética , Proteínas Bacterianas/genética , Elementos de Facilitación Genéticos , Regulación Bacteriana de la Expresión Génica , Operón , Factores de Transcripción/genética , Azotobacter vinelandii/metabolismo , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Sitios de Unión , Huella de ADN , Eliminación de Gen , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Factores de Transcripción/metabolismo , Transcripción Genética
16.
J Pharm Sci ; 91(4): 1135-46, 2002 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-11948552

RESUMEN

The purpose of this work was to elucidate the transport pathways of zinc insulin across the Calu-3 cell monolayer, an in vitro model of the human airway epithelium. Calu-3 cells grown in liquid-covered conditions formed a confluent monolayer with a high transepithelial electrical resistance value of 1000 +/- 150 Omega small middle dot cm(2). The cell monolayer was characterized by a low mannitol permeability of 4.7 +/- 0.5 10(-7)cm/s. Transport of zinc insulin (donor concentration 1 U/mL) in Dulbecco's modified phosphate buffer saline at 37 degrees C was found to be higher in the basolateral (BL) to apical (AP) (P(app) = 3.0 +/- 0.2 10(-8) cm/s), than in the AP to BL direction (P(app) = 0.41 +/- 0.02 10(-8) cm/s). P-glycoprotein efflux or specific enzymatic degradation did not appear to contribute toward this asymmetric transport. Insulin receptors, though apparently more abundant on the BL side than on the AP side of Calu-3 cells, did not mediate the direction-dependent transport of insulin. However, transport of a monomeric human insulin analog, Asp(B10)des(B28-30), across the Calu-3 cell monolayer was similar in both directions (BL to AP and AP to BL). The corresponding permeability, P(app) = 2.9 +/- 0.2 10(-8) cm/s, was not significantly different from the permeability of zinc insulin in the BL to AP direction. The paracellular pathway seems to play a major role in the insulin transport across the Calu-3 cell monolayers. We hypothesize that the transport of zinc insulin oligomers is restricted at the AP surface by the presence of the tight junctional complexes. From the BL side, oligomers may undergo dissociation in the intercellular space and diffuse readily as monomers to the AP surface of the membrane.


Asunto(s)
Bronquios/citología , Bronquios/metabolismo , Insulina/análogos & derivados , Insulina/farmacocinética , Mucosa Respiratoria/citología , Mucosa Respiratoria/metabolismo , Animales , Sitios de Unión , Células Cultivadas , Humanos , Hipoglucemiantes/química , Hipoglucemiantes/farmacocinética , Pulmón/citología , Pulmón/metabolismo , Transporte de Proteínas , Receptor de Insulina/metabolismo , Porcinos , Células Tumorales Cultivadas
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