RESUMEN
Liposomes have been extensively studied and are used in the treatment of several diseases. Liposomes improve the therapeutic efficacy by enhancing drug absorption while avoiding or minimizing rapid degradation and side effects, prolonging the biological half-life and reducing toxicity. The unique feature of liposomes is that they are biocompatible and biodegradable lipids, and are inert and non-immunogenic. Liposomes can compartmentalize and solubilize both hydrophilic and hydrophobic materials. All these properties of liposomes and their flexibility for surface modification to add targeting moieties make liposomes more attractive candidates for use as drug delivery vehicles. There are many novel liposomal formulations that are in various stages of development, to enhance therapeutic effectiveness of new and established drugs that are in preclinical and clinical trials. Recent developments in multimodality imaging to better diagnose disease and monitor treatments embarked on using liposomes as diagnostic tool. Conjugating liposomes with different labeling probes enables precise localization of these liposomal formulations using various modalities such as PET, SPECT, and MRI. In this review, we will briefly review the clinical applications of liposomal formulation and their potential imaging properties.
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Liposomas/química , Liposomas/uso terapéutico , Imagen por Resonancia Magnética/métodos , Tomografía de Emisión de Positrones/métodos , Tomografía Computarizada por Tomografía Computarizada de Emisión de Fotón Único/métodos , Animales , HumanosRESUMEN
Background. Historically, intraductal carcinoma of the prostate (IDC-P) was postulated to be a retrograde spread of high-grade invasive prostate cancer. There is evidence that IDC-P can primarily originate in the prostatic ducts. The retrograde genesis has never been experimentally or clinically confirmed before. Methods. Biopsy proven intermediate or high-risk prostate cancer was orthotopically grafted in the prostate of severe combined immunodeficiency gamma mice. Cancer growth was monitored by serum PSA levels. The animals were sacrificed and grafted areas were histological examined. Results. Twenty-one of 23 mice survived and demonstrated rising serum PSA. In 10 of 21 animals, human prostate cancer was identified orthotopically. Except for one case where the human biopsy showed a Grade Group 2 prostate cancer and mouse graft was Grade Group 5, other 9 specimens showed comparable grades. One of the specimens demonstrated a cribriform invasive prostate cancer and adjacent IDC-P. Conclusion. These experimental data offer some evidence that invasive prostate cancer can demonstrate a retrograde spread in the prostatic ducts as IDC-P. Its ability to primarily arise in the ducts has been demonstrated in other studies. However, the issue which remains unresolved is in its most common presentation of IDC-P intermixed with high-grade invasive cancer if it is the former or the latter which came first. Possibly resolving this dilemma will shed some light on the existing controversies if IDC-P should or should not be graded when invasive cancer is present.
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Carcinoma Intraductal no Infiltrante , Neoplasia Intraepitelial Prostática , Neoplasias de la Próstata , Masculino , Humanos , Animales , Ratones , Próstata/cirugía , Próstata/patología , Carcinoma Intraductal no Infiltrante/patología , Neoplasia Intraepitelial Prostática/patología , Antígeno Prostático Específico , Neoplasias de la Próstata/patologíaRESUMEN
PURPOSE: Small animal irradiation is crucial to the investigation of radiobiological mechanisms. The paradigm of clinical radiation therapy is trending toward high-precision, stereotactic treatment. However, translating this scheme to small animal irradiation is challenging owing to the lack of high-quality image guidance. To overcome this obstacle, we developed a multimodality image guided precision radiation platform. METHODS AND MATERIALS: The platform consists of 4 modules: x-ray computed tomography (CT), bioluminescence tomography (BLT), fluorescence molecular tomography (FMT), and radiation therapy. CT provides animal anatomy and material density for radiation dose calculation, as well as body contour for BLT and FMT reconstruction. BLT and FMT provide tumor localization to guide radiation beams and molecular activity to evaluate treatment outcome. Furthermore, we developed a Monte Carlo-based treatment planning system (TPS) for 3-dimensional dose calculation, calibrated it using radiochromic films sandwiched in a water-equivalent phantom, and validated it using in vivo dosimeters surgically implanted into euthanized mice (n = 4). Finally, we performed image guided irradiation on mice bearing orthotopic breast and prostate tumors and confirmed radiation delivery using γH2AX histology. RESULTS: The Monte Carlo-based TPS was successfully calibrated by benchmarking simulation dose against film measurement. For in vivo dosimetry measured in the euthanized mice, the average difference between the TPS calculated dose and measured dose was 3.86% ± 1.12%. Following the TPS-generated treatment plan, we successfully delivered 20 Gy dose to an animal bearing an orthotopic prostate tumor using 4 BLT-guided radiation beams and 5 Gy dose to an animal bearing an orthotopic breast tumor using a single FMT-guided radiation beam. γH2AX histology presented significantly more DNA damage in irradiated tumors and thus validated the dose delivery accuracy. CONCLUSIONS: Combined with Monte Carlo TPS, this multimodality CT/BLT/FMT image guided small animal radiation platform can specifically localize tumors, accurately calculate dose distribution, precisely guide radiation delivery, and molecularly evaluate treatment response. It provides an advanced toolset for radiobiology and translational cancer research.
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Neoplasias de la Mama/radioterapia , Imagen Multimodal/métodos , Neoplasias de la Próstata/radioterapia , Radioterapia Guiada por Imagen/métodos , Animales , Benchmarking , Neoplasias de la Mama/diagnóstico por imagen , Neoplasias de la Mama/patología , Calibración , Diseño de Equipo , Femenino , Mediciones Luminiscentes/métodos , Masculino , Ratones , Método de Montecarlo , Imagen Multimodal/instrumentación , Trasplante de Neoplasias , Imagen Óptica/métodos , Fantasmas de Imagen , Neoplasias de la Próstata/diagnóstico por imagen , Neoplasias de la Próstata/patología , Dosis de Radiación , Radiometría/métodos , Radioterapia Guiada por Imagen/instrumentación , Tomografía Computarizada por Rayos X/métodos , Película para Rayos XRESUMEN
Vesicular stomatitis virus (VSV) expressing IFNß induces apoptosis in multiple tumor models while maintaining an excellent safety profile. VSV-IFNß is oncoselective due to permissive replication in cells with an altered IFN pathway. The human VSV-IFNß (hIFNß) vector is currently used in clinical trials as a standalone therapy; however, we hypothesized that oncolytic virotherapy might be more effective when used in combination with radiotherapy (RT). We investigated the synergistic effects of RT and VSV-hIFNß in the subcutaneous PC3 and orthotopic LNCaP prostate xenograft models and a syngeneic RM9 prostate tumor model. VSV-IFNß combined with RT amplified tumor killing for PC3 and LNCaP xenografts, and RM9 tumors. This was attributed to the induction of proapoptotic genes leading to increased VSV-IFNß infection and replication, VSV expression, and oncolysis. In the RM9 tumors, combination therapy resulted in a robust antitumor immune response. Treated RM9 tumor-bearing mice demonstrated an increase in CD8+ and CD4+ T-cell numbers, 100% resistance to tumor rechallenge, and reduced resistance to reimplantation challenge with CD8+ knockdown. RT enhanced the activity of VSV-mediated oncolysis via attenuation of the innate antiviral response, resulting in increased VSV replication and the generation of an adaptive immune response earmarked by an increase in CD8+ lymphocyte numbers and antitumor activity. Local tumor irradiation combined with VSV-IFNß affects tumor cell death through direct and systemic activity in conjunction with pronounced antitumor immunity. IMPLICATIONS: Radiotherapy enhances VSV-mediated oncolysis and anti-tumor immunity, indicating that the ombination has promise for very high risk prostate cancer.
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Terapia Combinada/métodos , Inmunidad Innata/efectos de la radiación , Interferón beta/genética , Neoplasias de la Próstata/terapia , Vesiculovirus/fisiología , Animales , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD4-Positivos/efectos de la radiación , Linfocitos T CD8-positivos/metabolismo , Linfocitos T CD8-positivos/efectos de la radiación , Línea Celular Tumoral , Proliferación Celular , Regulación Neoplásica de la Expresión Génica/efectos de la radiación , Humanos , Masculino , Ratones , Viroterapia Oncolítica , Células PC-3 , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/inmunología , Radioterapia , Vesiculovirus/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
We have previously shown in separate studies that MDM2 knockdown via antisense MDM2 (AS-MDM2) and E2F1 overexpression via adenoviral-mediated E2F1 (Ad-E2F1) sensitized prostate cancer cells to radiation. Because E2F1 and MDM2 affect apoptosis through both common and independent pathways, we hypothesized that coupling these two treatments would result in increased killing of prostate cancer cells. In this study, the effect of Ad-E2F1 and AS-MDM2 in combination with radiation was investigated in three prostate cancer cell lines: LNCaP cells, LNCaP-Res cells [androgen insensitive with functional p53 and androgen receptor (AR)], and PC3 cells (androgen insensitive, p53(null), and AR(null)). A supra-additive radiosensitizing effect was observed in terms of clonogenic inhibition and induction of apoptosis (caspase-3 + caspase-7 activity) in response to Ad-E2F1 plus AS-MDM2 treatments in all three cell lines. In LNCaP and LNCaP-Res, these combination treatments elevated the levels of phospho-Ser(15) p53 with significant induction of p21(waf1/cip1), phospho-gammaH2AX, PUMA, and Bax levels and reduction of AR and bcl-2 expression. Similarly, AR(null) and p53(null) PC-3 cells showed elevated levels of Bax and phospho-gammaH2AX expression. These findings show that the combination of Ad-E2F1 and AS-MDM2 significantly increases cell death in prostate cancer cells exposed to radiation and that this effect occurs in the presence or absence of AR and p53.
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Andrógenos/metabolismo , Apoptosis/efectos de la radiación , ADN sin Sentido/farmacología , Factor de Transcripción E2F1/metabolismo , Neoplasias de la Próstata/radioterapia , Proteínas Proto-Oncogénicas c-mdm2/genética , Adenoviridae/genética , Caspasas/metabolismo , Línea Celular Tumoral , Factor de Transcripción E2F1/genética , Humanos , Masculino , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Receptores Androgénicos/metabolismo , Ensayo de Tumor de Célula MadreRESUMEN
PURPOSE: The image guided small animal arc radiation treatment platform has adopted onboard cone beam computed tomography and bioluminescence tomography (BLT). We used BLT to guide irradiation delivery and quantitatively assess irradiation-induced tumor response. METHODS AND MATERIALS: BLT was first validated on a tissue-simulating phantom, where the internal chemiluminescent liquid had a constant volume while its luminescence intensity gradually decayed. Then, in vivo experiments were performed on BALB/c mice orthotopically inoculated with 4T1 breast carcinoma cells expressing luciferase. Animals either received radiation treatment (radiation therapy [RT] group, n = 9) or did not (control group, n = 9). BLT was used to guide delivery of a single-fraction 5-Gy radiation dose to the tumor and to evaluate the treatment response. Terminal deoxynucleotidyl transferase deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) staining was used to evaluate irradiation-induced DNA damage and cell apoptosis. RESULTS: Phantom results showed that BLT not only recovered the constant target volume with <2% deviation but also accurately monitored the decay of the chemiluminescent molecules. For the RT group of animals, there was significant reduction in both the BLT-based tumor volume (21% ± 10%, P = .001) and bioluminescence intensity (48% ± 17%, P = .0008). For the control group, a significant increase was detected in the BLT tumor volume (35% ± 12%, P < .0001) but not the BLT bioluminescence intensity (P = .4). There was a significant difference in the BLT tumor volume between the RT and control groups 7 days after irradiation (P = .03). Regression analysis suggests a strong correlation between the BLT and cone beam computed tomography tumor volume (R2 = 0.93). Analysis using terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling staining showed a significant difference in tumor cell apoptosis between the RT and control groups (20.6% ± 2.9% and 3.2% ± 1.7%, respectively; P < .05). CONCLUSIONS: BLT onboard the image guided small animal arc radiation treatment platform can be used to accurately guide irradiation delivery and to quantitatively assess treatment response by simultaneously monitoring tumor volume and cancer cell population.
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Mediciones Luminiscentes/métodos , Neoplasias Mamarias Experimentales/radioterapia , Radioterapia Guiada por Imagen/métodos , Animales , Apoptosis/efectos de la radiación , Tomografía Computarizada de Haz Cónico , Femenino , Imagenología Tridimensional , Neoplasias Mamarias Experimentales/patología , Ratones , Ratones Endogámicos BALB C , Fantasmas de ImagenRESUMEN
Gold nanoparticles (GNP) have been intensively investigated for applications in cancer imaging and therapy. Most imaging studies focused on microscopic imaging. Their potential as optical imaging probes for whole body small animal imaging has rarely been explored. Taking advantage of their surface plasmon resonance (SPR) properties, we aim to develop a noninvasive diffuse optical imaging method to map the distribution of a special type of GNP, gold nanorods (GNR), in small animals. We developed an integrated dual-modality imaging system capable of both x-ray computed tomography (XCT) and diffuse optical tomography (DOT). XCT provides the animal anatomy and contour required for DOT; DOT maps the distribution of GNR in the animal. This SPR enhanced optical imaging (SPROI) technique was investigated using simulation, phantom and mouse experiments. The distribution of GNR at various concentrations (0.1-100 nM, or 3.5 ug/g-3.5 mg/g) was successfully reconstructed from centimeter-scaled volumes. SPROI detected GNR at 18 µg/g concentration in the mouse breast tumor, and is 3 orders more sensitive than x-ray imaging. This study demonstrated the high sensitivity of SPROI in mapping GNR distributions in small animals. It does not require additional imaging tags other than GNR themselves. SPROI can be used to detect tumors targeted by GNR via passive targeting based on enhanced permeability and retention or via active targeting using biologically conjugated ligands.
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Oro/química , Nanopartículas del Metal/química , Nanotubos/química , Fantasmas de Imagen , Tomografía Óptica/métodos , Animales , Femenino , Espectrometría de Masas , Ratones , Ratones Desnudos , Modelos Teóricos , Tomografía Computarizada por Rayos XRESUMEN
PURPOSE: Differentiating tumor from its surrounding soft tissues is challenging for x-ray computed tomography (CT). Fluorescence molecular tomography (FMT) can directly localize the internal tumors targeted with specific fluorescent probes. A FMT system was developed and integrated onto a CT-guided irradiator to improve tumor localization for image-guided radiation. METHODS: The FMT system was aligned orthogonal to the cone-beam CT onboard our previously developed image-guided small animal arc radiation treatment system (iSMAART). Through rigorous physical registration, the onboard CT provides accurate surface contour which is used to generate three-dimensional mesh for FMT reconstruction. During FMT experiments, a point laser source perpendicular to the rotating axis was used to excite the internal fluorophores. The normalized optical images from multiple projection angles were adopted for tomographic reconstruction. To investigate the accuracy of the FMT in locating the tumor and recovering its volume, in vivo experiments were conducted on two breast cancer models: MDA-MB-231 cancer xenograft on nude mice and 4T1 cancer xenograft on white mice. Both cancer cell lines overexpress the epidermal growth factor receptor (EGFR). A novel fluorescent poly(lactic-co-glycolic) acid (PLGA) nanoparticle conjugated with anti-EGFR was intravenously injected to specifically target the breast cancer cells. Another ex vivo experiment on a mouse bearing a surgically implanted Indocyanine Green-containing glass tube was conducted, to additionally validate the precision of FMT-guided radiation therapy. RESULTS: The FMT can accurately localize the single-nodule breast tumors actively targeted with fluorescent nanoparticles with localization error < 0.5 mm calculated between the centers of mass of tumors in FMT and CT. The reconstructed tumor volume in FMT was significantly correlated with that in the iodinated contrast-enhanced CT (R2 = 0.94, P < 0.001). The FMT was able to guide focal radiation delivery with submillimeter accuracy. CONCLUSION: Using the tumor-targeting fluorescent probes, the iSMAART with onboard FMT system can accurately differentiate tumors from their surrounding soft tissue, guide precise focal radiation delivery, and potentially assess tumor response in cancer research.
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Imagen Molecular , Tomografía Computarizada por Rayos X , Animales , Neoplasias de la Mama/diagnóstico por imagen , Femenino , Humanos , Ratones , Ratones Desnudos , Tomografía , Tomografía Óptica , Rayos XRESUMEN
PURPOSE: X-ray CT faces challenges in differentiating tumors from surrounding healthy tissues. A bioluminescence tomography (BLT) system which can directly reconstruct the internal luminescent tumors, was developed and integrated with CT system to accurately guide radiation dose delivery. METHODS: The BLT system, employing a lens-coupled CCD camera, was physically registered with an onboard cone beam CT system in an image-guided small animal arc radiation treatment system (iSMAART). The onboard CT provides animal anatomy and accurate surface contour used to construct the three-dimensional mesh for the BLT reconstruction. Bioluminescence projections were captured from multiple angles, once every 90° rotation. The BLT reconstruction was performed on an orthotopic prostate tumor model to evaluate its robustness and accuracy in locating and delineating bioluminescent tumors. The location and volume of the tumor identified from iodinated contrast CT was used to validate the BLT performance. Phantom experiment was also conducted to confirm the precision of BLT-guided radiation. RESULTS: The BLT was able to accurately locate the bioluminescent tumors with < 0.5 mm error. The tumor volume in BLT was significantly correlated with that in the iodinated contrast CT (R2 = 0.81, P < 0.001). Phantom experiments further validated that BLT can be used to guide radiation with submillimeter accuracy. CONCLUSION: Together with CT, BLT can provide precision radiation guidance and robust tumor volume assessment in small animal cancer research.
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Imagen Molecular , Radioterapia Guiada por Imagen , Tomografía Computarizada por Rayos X , Animales , Humanos , Masculino , Fantasmas de Imagen , Neoplasias de la Próstata/diagnóstico por imagen , Tomografía , Rayos XRESUMEN
Degradation of the extracellular matrix by proteolytic enzymes is a central aspect of physiological and pathologic tissue-remodeling processes such as trophoblastic implantation, wound healing, and tumor invasion. We have hypothesized that prostate adenocarcinoma cell invasion through the normal basal lamina is attributable in part to metalloproteinase-induced cleavage of laminin-5 (Ln-5) and enhanced motility of the cancer cells. We studied the role of membrane type-1-matrix metalloproteinase (MT1-MMP) expressed on the surface of prostate tumor cells in cleaving Ln-5 and enhancing the migration of prostate tumor cells. We also determined the nature of the MT1-MMP cleavage of human Ln-5 and how this altered Ln-5 changes the migration of prostate carcinoma cells. We found that human MT1-MMP cleaves purified human Ln-5 to an 80-kDa fragment. Mass spectrometry analyses of the 80-kDa cleaved product by trypsin and chymotrypsin gave 14 and 9 different peptide sequences, respectively, that were identical to the expected amino acid sequence of the Ln-5-beta3 chain. The recovered peptides represent 14.4% (trypsin) and 10.3% (chymotrypsin) of Ln-5-beta3 chain by amino acid count. Both trypsin and chymotrypsin digestion of MT1-MMP-cleaved product of Ln-5 did not show any other peptides that were identical to the other chains of Ln-5. Using a linear migration assay we found that the Ln-5 cleaved by MT1-MMP enhanced the migration of DU-145 prostate carcinoma cells by 2-fold compared with uncleaved Ln-5. The use of blocked antisense MT1-MMP oligonucleotides inhibited the migration of DU-145 cells on Ln-5. We also found that the prostate carcinoma cells expressing high levels of MT1-MMP, such as PC3N and PPC, demonstrated enhanced migration on human Ln-5-coated substrate, and this migration was inhibited using blocked antisense MT1-MMP oligonucleotides. In conclusion, this is a novel and important finding where we have shown that beta3-chain is cleaved by MT1-MMP, and this cleavage enhances migration of prostate cancer cells.
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Moléculas de Adhesión Celular/metabolismo , Movimiento Celular/fisiología , Metaloendopeptidasas/metabolismo , Neoplasias de la Próstata/enzimología , Neoplasias de la Próstata/patología , Secuencia de Aminoácidos , Anticuerpos Monoclonales/inmunología , Especificidad de Anticuerpos , Moléculas de Adhesión Celular/inmunología , Humanos , Masculino , Metaloproteinasas de la Matriz Asociadas a la Membrana , Metaloendopeptidasas/biosíntesis , Metaloendopeptidasas/farmacología , Datos de Secuencia Molecular , Invasividad Neoplásica , Neoplasias de la Próstata/metabolismo , Proteínas Recombinantes/farmacología , Células Tumorales Cultivadas , KalininaRESUMEN
Edelfosine is a synthetic alkyl-lysophospholipid that possesses significant antitumor activity in several human tumor models. Here, we investigated the effects of edelfosine combined with androgen deprivation (AD) in LNCaP and VCaP human prostate cancer cells. This treatment regimen greatly decreased cell proliferation compared with single agent or AD alone, resulting in higher levels of apoptosis in LNCaP compared with VCaP cells. Edelfosine caused a dose-dependent decrease in AKT activity, but did not affect the expression of total AKT in either cell line. Furthermore, edelfosine treatment inhibited the expression of androgen receptor (AR) and was associated with an increase in activating transcription factor 3 (ATF3) expression levels, a stress response gene and a negative regulator of AR transactivation. ATF3 binds to AR after edelfosine + AD and represses the transcriptional activation of AR as demonstrated by PSA promoter studies. Knockdown of ATF3 using siRNA-ATF3 reversed the inhibition of PSA promoter activity, suggesting that the growth inhibition effect of edelfosine was ATF3 dependent. Moreover, expression of AR variant 7 (ARv7) and TMPRSS2-ERG fusion gene were greatly inhibited after combined treatment with AD and edelfosine in VCaP cells. In vivo experiments using an orthotopic LNCaP model confirmed the antitumor effects of edelfosine + AD over the individual treatments. A significant decrease in tumor volume and PSA levels was observed when edelfosine and AD were combined, compared with edelfosine alone. Edelfosine shows promise in combination with AD for the treatment of prostate cancer patients. Mol Cancer Ther; 15(6); 1353-63. ©2016 AACR.
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Factor de Transcripción Activador 3/metabolismo , Antagonistas de Andrógenos/administración & dosificación , Éteres Fosfolípidos/administración & dosificación , Neoplasias de la Próstata/tratamiento farmacológico , Receptores Androgénicos/genética , Antagonistas de Andrógenos/farmacología , Animales , Apoptosis , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Sinergismo Farmacológico , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Masculino , Ratones , Trasplante de Neoplasias , Éteres Fosfolípidos/farmacología , Regiones Promotoras Genéticas/efectos de los fármacos , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptores Androgénicos/metabolismo , Transducción de Señal/efectos de los fármacos , Regulación hacia Arriba , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Small animal radiotherapy studies should be performed preferably on irradiators capable of focal tumor irradiation and healthy tissue sparing. In this study, an image guided small animal arc radiation treatment system (iSMAART) was developed which can achieve highly precise radiation targeting through the utilization of onboard cone beam computed tomography (CBCT) guidance. The iSMAART employs a unique imaging and radiation geometry where animals are positioned upright. It consists of a stationary x-ray tube, a stationary flat panel detector, and a rotatable and translational animal stage. System performance was evaluated in regards to imaging, image guidance, animal positioning, and radiation targeting using phantoms and tumor bearing animals. The onboard CBCT achieved good signal, contrast, and sub-millimeter spatial resolution. The iodine contrast CBCT accurately delineated orthotopic prostate tumors. Animal positioning was evaluated with ~0.3 mm vertical displacement along superior-inferior direction. The overall targeting precision was within 0.4 mm. Stereotactic radiation beams conformal to tumor targets can be precisely delivered from multiple angles surrounding the animal. The iSMAART allows radiobiology labs to utilize an image guided precision radiation technique that can focally irradiate tumors while sparing healthy tissues at an affordable cost.
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Neoplasias de la Próstata/veterinaria , Radiocirugia/veterinaria , Animales , Masculino , Ratones , Neoplasias de la Próstata/cirugía , Radiometría/métodos , Radiometría/veterinaria , Radiocirugia/métodosRESUMEN
The MMP, matrilysin (MMP-7), has been shown to be overexpressed in prostate cancer cells and to increase prostate cancer cell invasion. Prostate stromal fibroblasts secrete factor(s), including fibroblast growth factor-1 (FGF-1) that induces promatrilysin expression in LNCaP cells. In the present study, we investigated the signal transduction pathway involved in the FGF-1-induced expression of promatrilysin. FGF-1 treatment significantly increased the activation of extracellular signal-regulated kinases 1 and 2 (ERK1 and ERK2). This induction was time-dependent and was sustained until 24 hours after treatment. Treating the cells with MEK1/2 inhibitor (PD98059) eliminated ERK activation completely and blocked FGF-1-mediated induction of promatrilysin expression. Transient transfection studies with human matrilysin promoter resulted in a four- to five-fold increase in reporter luciferase enzyme activity that was blocked by the MEK1/2 inhibitor (PD98059). Serine phosphorylation of signal transducer and activator of transcription 3 (STAT3) was observed after FGF-1 treatment and pretreatment with 20 microM PD98059-abolished STAT3 phosphorylation. Transient transfection with dominant negative STAT3 inhibited FGF-1-induced transactivation of the matrilysin promoter indicating that STAT3 plays an important role in FGF1-induced matrilysin expression. We propose that the FGF-1-induced signaling pathway that leads to promatrilysin expression is ERK-dependent and leads to phosphorylation of Ser-727 on STAT3, phosphorylated STAT3, then binds and transactivates the matrilysin promoter. Our results demonstrate that ERK-MAP kinase and transcription factor STAT3 are important components of FGF-1-mediated signaling, which induce promatrilysin expression in LNCaP cells.
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Proteínas de Unión al ADN/metabolismo , Precursores Enzimáticos/metabolismo , Factor 1 de Crecimiento de Fibroblastos/farmacología , Metaloendopeptidasas/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Neoplasias de la Próstata/enzimología , Transactivadores/metabolismo , Western Blotting , Proteínas Quinasas Dependientes de Calcio-Calmodulina/antagonistas & inhibidores , Células Cultivadas , Cartilla de ADN/química , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Ensayo de Inmunoadsorción Enzimática , Flavonoides/farmacología , Humanos , Luciferasas/metabolismo , Masculino , Proteína Quinasa 3 Activada por Mitógenos , Reacción en Cadena de la Polimerasa , Factor de Transcripción STAT3 , Transducción de Señal , Transfección , Células Tumorales CultivadasRESUMEN
Expression of carbonic anhydrase 9 (CA9) is associated with poor prognosis and increased tumor aggressiveness and does not always correlate with HIF-1α expression. Presently, we analyzed the regulation of CA9 expression during hypoxia by HIF-1α, Notch3, and the von Hippel-Lindau (VHL) in breast carcinoma cells. Both HIF-1α and Notch3 were absolutely required for the expression of CA9 mRNA, protein, and reporter. Reciprocal co-immunoprecipitation of HIF-1α, Notch3 intracellular domain (NICD3), and pVHL demonstrated their association. The presence of common consensus prolyl hydroxylation and pVHL binding motifs (L(XY)LAP);LLPLAP(2191) suggested an oxygen-dependent regulation for NICD3. However, unlike the HIF-1α protein, NICD3 protein levels were not modulated with hypoxia or hypoxia-mimetic agents. Surprisingly, mutations of the common prolyl hydroxylation and pVHL binding domain lead to the loss of CA9 mRNA, protein, and reporter activity. Chromatin immunoprecipitation assay demonstrated the association of NICD3, HIF-1α, and pVHL at the CA9 promoter. Further, the NICD3 mutant defective in prolyl hydroxylation and subsequent pVHL binding caused a reduction in cell proliferation of breast carcinoma cells. We show here for the first time that the interaction of HIF-1α with NICD3 is important for the regulation of CA9 expression. These findings suggest that although CA9 is a hypoxia-responsive gene, its expression is modulated by the interaction of HIF-1α, Notch3, and VHL proteins. Targeting the expression of CA9 by targeting upstream regulators could be useful in cancer/stem cell therapy.
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PURPOSE: We previously showed that E2F1 overexpression radiosensitizes prostate cancer cells in vitro. Here, we demonstrate the radiosensitization efficacy of adenovirus (Ad)-E2F1 infection in growing (orthotopic) LNCaP and (subcutaneous) PC3 nude mice xenograft tumors. METHODS AND MATERIALS: Ad-E2F1 was injected intratumorally in LNCaP (3 × 10(8) plaque-forming units [PFU]) and PC3 (5 × 10(8) PFU) tumors treated with or without radiation. LNCaP tumor volumes (TV) were measured by magnetic resonance imaging, caliper were used to measure PC3 tumors, and serum prostate-specific antigen (PSA) levels were determined by enzyme-linked immunosorbent assay. Apoptosis was measured by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling, and key proteins involved in cell death signaling were analyzed by Western blotting. RESULTS: Intracellular overexpression of Ad-E2F1 had a significant effect on the regression of TV and reduction of PSA levels relative to that of adenoviral luciferase (Ad-Luc)-infected control. The in vivo regressing effect of Ad-E2F1 on LNCaP tumor growth was significant (PSA, 34 ng/ml; TV, 142 mm(3)) compared to that of Ad-Luc control (PSA, 59 ng/ml; TV, 218 mm(3); p <0.05). This effect was significantly enhanced by radiation therapy (compare: Ad-E2F1+RT/PSA, 16 ng/ml, and TV, 55 mm(3) to Ad-Luc+RT/PSA, 42 ng/ml, and TV, 174 mm(3), respectively; p <0.05). For PC3 tumors, the greatest effect was observed with Ad-E2F1 infection alone; there was little or no effect when radiotherapy (RT) was combined. However, addition of RT enhanced the level of in situ apoptosis in PC3 tumors. Molecularly, addition of Ad-E2F1 in a combination treatment abrogated radiation-induced BCL-2 protein expression and was associated with an increase in activated BAX, and together they caused a potent radiosensitizing effect, irrespective of p53 and androgen receptor functional status. CONCLUSIONS: We show here for the first time that ectopic overexpression of E2F1 in vivo, using an adenoviral vector, significantly inhibits orthotopic p53 wild-type LNCaP tumors and subcutaneous p53-null PC3 tumors in nude mice. Furthermore, we demonstrate that E2F1 strongly sensitizes LNCaP tumors to RT. These findings suggest that E2F1 overexpression can sensitize prostate tumor cells in vivo, independent of p53 or androgen receptor status.
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Adenoviridae/metabolismo , Factor de Transcripción E2F1/metabolismo , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/radioterapia , Tolerancia a Radiación/fisiología , Animales , Apoptosis , Proteínas Reguladoras de la Apoptosis/metabolismo , Factor de Transcripción E2F1/administración & dosificación , Vectores Genéticos/administración & dosificación , Humanos , Luciferasas/sangre , Masculino , Ratones , Ratones Desnudos , Antígeno Prostático Específico/sangre , Neoplasias de la Próstata/patología , Inducción de Remisión/métodos , Carga Tumoral , Ensayos Antitumor por Modelo de Xenoinjerto/métodosRESUMEN
BACKGROUND: We have shown previously that interleukin (IL) -1 beta- and IL-6-induced promatrilysin expression is mediated by an indirect pathway that requires NF kappa B-dependent synthesis of IL-6 and STAT3 signaling. We now demonstrate that IL-1 beta-induced but not IL-6-induced promatrilysin expression can be blocked by androgens in the prostate carcinoma cell line LNCaP (lymph node-derived carcinoma cells of the prostate). METHODS: By using enzyme-linked immunosorbent assay analyses, promatrilysin was measured in LNCaP cells stimulated with IL-1 beta or IL-6 LNCaP-treated cells pretreated with testosterone. In addition, promatrilysin message was measured by using Northern analyses after IL-6-treated cells pretreated with testosterone. RESULTS: In LNCaP treated with testosterone before IL-1 beta stimulation induced promatrilysin expression was completely abrogated. Furthermore, testosterone completely abrogated NF kappa B transactivation activity and induction of IL-6 protein expression and mRNA. Testosterone and 5 alpha-dihydrotestosterone did not have an inhibitory effect on IL-6-induced promatrilysin expression. Testosterone also had no effect on basal promatrilysin expression or basal NF kappa B transactivation activity. CONCLUSION: From these data, we conclude that testosterone blocks IL-1 beta-induced promatrilysin expression by inhibition of NF kappa B transactivation activity, which in turn, blocks IL-6 expression. These data suggest a mechanism in vivo by which invasive and metastatic prostatic carcinoma cell clones refractory to hormone ablation therapy may develop after chemical or surgical castration. Furthermore, these data suggest that, perhaps, upstream targets such as the cytokines IL-1 beta and IL-6 may provide alternative drug targets for inhibiting prostate cancer progression.
Asunto(s)
Carcinoma/patología , Precursores Enzimáticos/biosíntesis , Interleucina-1/farmacología , Interleucina-6/farmacología , Metaloendopeptidasas/biosíntesis , Neoplasias de la Próstata/patología , Testosterona/farmacología , Northern Blotting , Resistencia a Antineoplásicos , Ensayo de Inmunoadsorción Enzimática , Humanos , Masculino , Invasividad Neoplásica , Metástasis de la Neoplasia , Transducción de Señal , Células Tumorales CultivadasRESUMEN
Previously we have shown that the matrix metalloproteinase matrilysin (MMP-7) is overexpressed in human prostate cancers compared with normal epithelium. However, the mechanism for this overexpression is not understood. Human prostate fibroblasts have been shown to express certain fibroblast growth factors (FGFs), including FGF-1. Evidence from our laboratory and others has indicated that FGFs can regulate the expression of certain matrix metalloproteinases, including matrilysin. The goal of this study was to determine whether pharmacological inhibition of FGFR signaling would alter LNCaP tumor growth as well as expression of promatrilysin when LNCaP cells were co-injected subcutaneously with human prostate fibroblasts into athymic nude mice. For these inhibitor studies, AG1-X2 beads were coated with the pharmacological FGFR inhibitor SU5402 and were co-injected along with LNCaP and human prostate fibroblast cells (PF). Mice injected with LNCaP/PF and LNCaP/PF/beads alone demonstrated significant tumor growth, whereas mice injected with LNCaP/PF/SU5402-coated beads showed a significant decrease in tumor volume and weight. Immunohistochemical analysis showed that significant promatrilysin expression in tumors was inhibited by the FGFR inhibitor SU5402. Serum prostate-specific antigen (PSA) and promatrilysin levels were measured by enzyme-linked immunosorbent assay. The mice injected with LNCaP/PF and LNCaP/PF/beads expressed promatrilysin and serum PSA levels that were inhibited by co-injecting with SU5402. Therefore, pharmacological inhibition of FGF receptor signaling results in a decrease in the growth of LNCaP tumors generated subcutaneously by co-injecting LNCaP cells and human prostate fibroblasts. The inhibition in tumor growth was correlated with a decrease in tumor promatrilysin expression and a decrease in serum promatrilysin and PSA.