RESUMEN
BACKGROUND: By alternative splicing the IGF-1 gene produces several different transcripts, including IGF-1Ec (MGF). The latter has been mainly associated with muscle regeneration processes. METHODS: We used immunohistochemistry, RT-PCR, and Western analysis to show the expression status of MGF in prostate tissue and human prostate cell lines (HPrEC, PC-3, and LNCaP) and we studied the exogenous administration of the MGF peptide E on cellular proliferation using trypan blue and MTT assays, before and after the silencing of the IGF-1 receptor and insulin receptor (siRNA methods). The MGF-induced intracellular activation was examined by Western analysis of the active forms of ERK1/2 and Akt. RESULTS: We documented that MGF is overexpressed in human prostate cancer (PCa) tissues and in human PC-3 and LNCaP cells. Notably, MGF expression was remarkably higher in PCa and prostatic intraepithelial neoplasia (PIN) than normal prostate tissues, while the normal prostate epithelial cells (HPrEC) did not express MGF. Exogenous administration of a synthetic MGF E peptide stimulated the PCa cell growth and activated ERK1/2 phosphorylation without affecting Akt phosphorylation. IGF-1R or insulin receptor (IR) silencing did not affect the mitogenic activity and intracellular signaling of the MGF E peptide in these PCa cells. CONCLUSIONS: These data suggest the possible implication of MGF E peptide in cancer biology, implying a preferential MGF expression in PCa tissues and cells. This preferential IGF-1 mRNA expression generates the MGF E peptide that possesses mitogenic activity through mechanisms independent of IGF-1R, IR, and hybrid IGF-1R/IR.
Asunto(s)
Factor I del Crecimiento Similar a la Insulina/biosíntesis , Neoplasia Intraepitelial Prostática/metabolismo , Neoplasias de la Próstata/metabolismo , Empalme Alternativo , Línea Celular Tumoral , Supervivencia Celular/fisiología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Regulación Neoplásica de la Expresión Génica , Silenciador del Gen , Humanos , Factor I del Crecimiento Similar a la Insulina/genética , Sistema de Señalización de MAP Quinasas , Masculino , Neoplasia Intraepitelial Prostática/enzimología , Neoplasias de la Próstata/enzimología , Neoplasias de la Próstata/genética , Isoformas de Proteínas , ARN Mensajero/biosíntesis , ARN Mensajero/genética , ARN Interferente Pequeño/administración & dosificación , ARN Interferente Pequeño/genética , Receptor IGF Tipo 1/biosíntesis , Receptor IGF Tipo 1/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
As the presence of tumor cells circulating in the blood is associated with systemic disease and shortened survival, the establishment of a method to detect circulating tumor cells (CTCs) is of critical importance for a more concise staging and follow-up of cancer patients. Recently, the most robust strategies for the determination of CTCs are the PCR-based methods and the CellSearch® system that exploits the immunofluorescent characterization and isolation of cancer cells. Herein, we analyzed the experimental strategies used for determining CTCs with respect to accuracy, sensitivity and reproducibility in cancers of the breast, colon, prostate and melanoma.
Asunto(s)
Neoplasias/sangre , Células Neoplásicas Circulantes/patología , Recuento de Células Sanguíneas , Células Sanguíneas/patología , Separación Celular/métodos , Femenino , Humanos , Masculino , Metástasis de la Neoplasia , Neoplasias/patología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodosRESUMEN
Disseminated malignancy is the major cause of prostate cancer-related mortality. Circulating tumor cells (CTCs) are essential for the establishment of metastasis. Various contemporary and molecular methods using prostate-specific biomarkers have been applied to detect extraprostatic disease that is undetectable by conventional imaging techniques, assessing the risk for disease recurrence after therapy of curative intent. However, the clinical relevance of CTC detection is still controversial. We review current literature regarding molecular methods used for the detection of CTCs in the peripheral blood and bone marrow biopsies of patients with prostate cancer, and we discuss the methodological pitfalls that influence the clinical significance of molecular staging.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Estadificación de Neoplasias , Células Neoplásicas Circulantes/metabolismo , Neoplasias de la Próstata , Antígenos de Neoplasias , Antígenos de Superficie/genética , Antígenos de Superficie/metabolismo , Líquidos Corporales/citología , Proteínas Ligadas a GPI , Glutamato Carboxipeptidasa II/genética , Glutamato Carboxipeptidasa II/metabolismo , Humanos , Queratina-19/genética , Queratina-19/metabolismo , Masculino , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Metástasis de la Neoplasia , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteína Relacionada con la Hormona Paratiroidea/genética , Proteína Relacionada con la Hormona Paratiroidea/metabolismo , Reacción en Cadena de la Polimerasa/métodos , Reacción en Cadena de la Polimerasa/normas , Antígeno Prostático Específico/genética , Antígeno Prostático Específico/metabolismo , Neoplasias de la Próstata/sangre , Neoplasias de la Próstata/patologíaRESUMEN
BACKGROUND/AIM: The E peptide of the IGF-1Ec transcript has been documented to stimulate the growth of different cell lines, via a type I IGF-1 receptor (IGF-1R)-independent mechanism. The aim of the present study was to determine the implication of the IGF-1Ec isoform into the posterior capsule opacification process in human lens epithelium. MATERIALS AND METHODS: The expression of the IGF-1 system was characterized in human HLE-B3 lens epithelium cells and the mitogenic activity of IGF-1 and synthetic E peptide and the effects of growth hormone (GH) and dihydrotestosterone (DHT) were examined, using qualitative real-time PCR, RT-PCR, Western blot analysis and trypan blue exclusion assays in wild-type and IGF-1R knock-out HLE-B3 cells. RESULTS: The data showed that HLE-B3 cells express only the IGF-1Ea and IGF-1R transcripts. GH increased the expression of IGF-1Ea and of the previously undetectable IGF-1Eb mRNA. Finally, IGF-1 did not present any activity in the knock-out cells. CONCLUSION: The IGF-1Ea isoform is the main source for the formation of mature IGF-1 in HLE-B3 cells. The effects of exogenous IGF-1 depend on the existence of IGF-1R. IGF-1 Ec is not expressed even in the presence of GH or DHT nor has it any effect on cell proliferation.
Asunto(s)
Células Epiteliales/metabolismo , Expresión Génica , Factor I del Crecimiento Similar a la Insulina/genética , ARN Mensajero/metabolismo , Receptor IGF Tipo 1/genética , Western Blotting , Línea Celular , Proliferación Celular , Dihidrotestosterona/farmacología , Células Epiteliales/efectos de los fármacos , Hormona del Crecimiento/farmacología , Humanos , Factor I del Crecimiento Similar a la Insulina/metabolismo , Factor I del Crecimiento Similar a la Insulina/farmacología , Cristalino/citología , Cristalino/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Interferencia de ARN , ARN Mensajero/genética , Receptor IGF Tipo 1/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
AIM: The insulin-like growth factor 1 (IGF1) gene gives rise to multiple transcripts, using an elaborate alternative splicing mechanism. The aim of this study was to shed light on the expression and role of the IGF1 system in human MG-63 osteoblast-like osteosarcoma cells. MATERIALS AND METHODS: The expression of the IGF1Ea, IGF1Eb and IGF1Ec isoforms was characterized using reverse transcription polymerase chain reaction (RT-PCR), quantitative real time-PCR (qRT-PCR) and western blot analysis. Using trypan blue exclusion assays, we also examined the mitogenic effects of IGF1 and of a synthetic peptide related to the E domain of IGF1Ec (synthetic E peptide) on MG-63 cells, as well as on MG-63 cells which had been molecularly modified to restrain the expression of type I IGF receptor (IGF1R) and of insulin receptor (INSR) by siRNA techniques (IGF1R KO or INSR KO MG-63 cells). RESULTS: MG-63 cells express only the IGF1Ea and IGF1Ec transcripts. Exogenous administration of dihydrotestosterone (DHT) significantly increased the expression of IGF1Ea and IGF1Ec mRNA and it induced the previously undetectable expression of IGF1Eb transcript. Exogenous administration of IGF1, insulin and the synthetic E peptide stimulated the growth of MG-63 cells, while only E peptide stimulated the growth of IGF1R KO and INSR KO MG-63 cells. CONCLUSION: These data suggest that the expression of all IGF1 isoforms is hormonally regulated in MG-63 cells and that the expression of IGF1Ec may be involved in osteosarcoma biology by generating the Ec peptide which acts via an IGF1R-independent and INSR-independent mechanism.
Asunto(s)
Factor I del Crecimiento Similar a la Insulina/metabolismo , Empalme Alternativo , Línea Celular Tumoral , Perfilación de la Expresión Génica/métodos , Humanos , Modelos Biológicos , Osteoblastos/metabolismo , Osteosarcoma/metabolismo , Péptidos/química , Isoformas de Proteínas , ARN Interferente Pequeño/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Receptor de Insulina/biosíntesis , TransgenesRESUMEN
BACKGROUND: The clinical relevance of positive molecular staging as defined by reverse transcriptase-polymerase chain reaction (RT-PCR) detections of both prostate-specific antigen (PSA) and prostate-specific membrane antigen (PSMA) transcripts in the peripheral blood (PB) of patients with prostate cancer is still debatable. METHODS: We analyzed the biochemical failure-free survival (bFFS) of prostate cancer patients with positive molecular staging who underwent immediate curative therapy (Group I, n=39) compared to prostate cancer patients who did convert their positive molecular staging by the administration of combined androgen blockade (CAB) for 12 months prior to curative treatment (Group II, n=15). RESULTS: The median bFFS for Group I was 9 months (95% CI 5-13 months) and was significantly lower compared to Group II (>36 months, p<0.001). In Group I, the median time for PSA values of >2.0 ng/mL was 18 months (95% CI 12-21 months, range 12-36 months). Notably, only one patient from Group II reached PSA values>2.0 ng/mL at 36 months post-curative treatment. CONCLUSIONS: In patients with clinically localized prostate cancer and positive RT-PCR detection of PSA and PSMA transcripts in PB, CAB can convert positive molecular staging status to negative and by doing so it modifies the post-curative therapy bFFS of patients with clinically localized prostate cancer.