RESUMEN
AIM: To evaluate and compare the expression profiles of CXCL12 (SDF-1), CCL19 (MIP-3beta), CCL20 (MIP-3alpha) and CCL21 (6Ckine, Exodus2) and their receptors on RNA and protein levels in hepatocellular carcinoma (HCC) versus colorectal liver metastases (CRLM) and to elucidate their impact on the carcinogenesis and progression of malignant liver diseases. METHODS: Chemokine expression was analyzed by RT-PCR and ELISA in 11 cases of HCC specimens and in 23 cases of CRLM and corresponding adjacent non-tumorous liver tissues, respectively. Expressions of their receptors CXCR4, CCR6 and CCR7 were analyzed by RT-PCR and Western blot analysis in the same cases of HCC and CRLM. RESULTS: Significant up-regulation for CCL20/CCR6 was detected in both cancer types. Moreover, CCL20 demonstrated significant overexpression in CRLM in relation to the HCC tissues. Being significantly up-regulated only in CRLM, CXCR4 displayed an aberrant expression pattern with respect to the HCC tissues. CONCLUSION: Correlation of CXCR4 expression with CRLM suggests CXCR4 as a potential predictive factor for CRLM. High level expression of CCL20 and its receptor CCR6 in HCC and CRLM with marked up-regulation of CCL20 in CRLM in relation to HCC tissues indicates involvement of the CCL20/CCR6 ligand-receptor pair in the carcinogenesis and progression of hepatic malignancies.
Asunto(s)
Carcinoma Hepatocelular/metabolismo , Quimiocinas/metabolismo , Neoplasias Colorrectales/metabolismo , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/secundario , Adulto , Anciano , Anciano de 80 o más Años , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Quimiocina CCL20 , Quimiocina CXCL12 , Quimiocinas/genética , Quimiocinas CC/genética , Quimiocinas CC/metabolismo , Quimiocinas CXC/genética , Quimiocinas CXC/metabolismo , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Femenino , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Neoplasias Hepáticas/genética , Proteínas Inflamatorias de Macrófagos/genética , Proteínas Inflamatorias de Macrófagos/metabolismo , Masculino , Persona de Mediana Edad , Metástasis de la Neoplasia/genética , Metástasis de la Neoplasia/patología , Valor Predictivo de las Pruebas , Receptores CCR6 , Receptores CXCR4/genética , Receptores CXCR4/metabolismo , Receptores de Quimiocina/genética , Receptores de Quimiocina/metabolismo , Regulación hacia ArribaRESUMEN
Various chemokine receptors, namely CXCR4, CCR6 and CCR7, have recently been shown to be involved in the regulation of metastasis in malignant tumors. However, little is known about the role of these receptors in promoting tumor metastasis of colorectal cancer (CRC) to the primary site of CRC metastasis in the liver. To investigate this issue, we analyzed the expression of the chemokine receptors CXCR4, CCR6 and CCR7 in colorectal tumors and colorectal liver metastases. In the present study, 30 human cancer samples from colorectal tissue, 30 human samples from colorectal liver metastases and the adjacent nontumorous liver tissues were screened using quantitative real-time PCR, Western blot analysis, histochemistry, microdissection and the enzyme-linked immunosorbent assay (ELISA). While an overexpression of all the chemokine receptors was found in CRC, in colorectal liver metastases only the chemokine receptors CXCR4 and CCR6 were significantly upregulated. Consequently, we investigated the expression of the corresponding ligands CXCL12/SDF1alpha, CCL20/MIP3alpha, CCL19/MIP3beta and CCL21/6Ckine in various organs, such as the stomach, esophagus, pancreas, colon and rectum, in comparison with their expression in the liver as the primary site of metastatic spread in CRC. We found that only CCL20 exhibits peak levels of expression in the liver, thus indicating that an increased production of CCL20 may contribute to the selective recruitment of CCR6-expressing cancer cells in CRC. Furthermore, we could demonstrate that CRC patients who developed liver metastases express significantly more CCL20 and CCL21 in the liver in comparison with an unaffected control group. Therefore, our findings strongly suggest an association between CCL20/CCR6 expression in human CRC and the promotion of colorectal liver metastasis.
Asunto(s)
Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/secundario , Receptores de Quimiocina/metabolismo , Western Blotting , Femenino , Humanos , Masculino , Estadificación de Neoplasias , ARN Mensajero/análisis , ARN Mensajero/metabolismo , Receptores CCR6 , Receptores de Quimiocina/análisis , Receptores de Quimiocina/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Distribución Tisular , Regulación hacia ArribaRESUMEN
Careful normalization is essential for the accurate quantitation of mRNA levels in biopsy-sized tissue samples. Commonly, normalization of the target gene with an endogenous standard, mainly housekeeping genes (HKGs), is applied. However, differences in the expression levels of endogenous reference genes have been reported between different tissues and pathological states. Therefore, we were challenged to identify a set of endogenous reference genes whose mRNA expression levels would not change significantly between normal and cancerous tissues. Quantitative real-time PCR (Q-RT-PCR) analysis was applied to evaluate the variability in gene expression among 21 classical housekeeping genes in colorectal, pancreatic, esophageal and gastric cancer as well as in liver metastases in comparison to the corresponding normal tissue. Our results indicated that some housekeeping genes were candidates with relatively stable gene expression in several of the investigated tissues but for most of the HKGs under investigation our data have revealed distinct differences in the extent of variability in gene expression between the different tissues and pathological states. However, for each of the five tissues investigated we found a group of genes that were expressed at a constant level thus representing a panel of candidates that we can recommend as housekeeping genes in the respective tissue types. In summary, our results can be used as guidance for other scientists studying various carcinomas for tissue-specific selection of the optimal housekeeping gene (HKG) to be used in normalizing target gene expression.