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The Roles And Mechanisms Of Corticotropin Releasing Factor Receptor 2 In The Growth Of Human Tumors

Posted on:2011-11-19Degree:DoctorType:Dissertation
Country:ChinaCandidate:J J WangFull Text:PDF
GTID:1114330302955592Subject:Pharmacology
Abstract/Summary:PDF Full Text Request
Corticotropin-releasing factor (CRF) family is composed of CRF, urocortin 1 (UCN 1), UCN 2, and UCN 3, which are identified as about 40-amino acid neuropeptides and bind to their two known receptors, CRF receptor 1 (CRFR1) and CRFR2. These receptors are G-protein coupled, and their activation can mediate responses via protein kinase signaling pathways. Both CRF receptors are distributed in central nervous system (CNS) and periphery, but the two subtypes display quite different pharmacological profiles. It has become apparent that CRF pathway and its components are involved in a wide array of physiological and potentially pathophysiological processes, therefore, the CRF pathway presents is a very promising clinical therapeutic target. Reubi et al. reported that CRFR1 and CRFR2 expressed in many human cancers. CRF/UCN was also found to distribute in many types of cancers, such as pituitary adenoma, prostatic carcinoma, and endometrial carcinoma. Furthermore, series of literatures reported CRF/UCN has anticancer effects via CRFR1. Though there are few reports about the relationship between CRFR2 and tumors, the roles of CRFR2 in tumoral formation and development is being more and more followed with interest.In this thesis, we investigated the roles and mechanisms of CRFR2 in the formation and development of human tumors. Different type human tumor tissues were used to identify whether they expressed the CRF receptors. Human small lung cancer (SCLC) tissues were found the expression of CRF receptors, CRFR2 only. Therefore, SCLC cells were used to study the direct effect of CRFR2 on tumor cells. Because CRFR2 was recognized as a tonic suppressor of angiogenesis, we also study the role of CRFR2 in tumor angiogenesis with human hepatocelluar carcinoma (HCC), since it is such a hypervascular tumor and there is no effective chemotherapy for this cancer.In the first chapter of thesis, we investigated the direct effect of CRFR2 on human SCLC cells. CRFR2 was found to be expressed in the human SCLC tissue and SCLC cell lines (NCI-H446 and NCI-H1688). Therefore, the direct effect of CRFR2 on the human SCLC was investigated by using CRFR2 angonists UCN 1 and UCN 2. Based on our observations, UCNs could directly inhibit the proliferation of SCLC cell lines NCI-H446 and NCI-H1688 with a dose-dependent manner, meanwhile, UCNs also could promote the apoptosis of SCLC cells. These anti-proliferative and apoptosis promotive effects could be abolished by pretreatment with CRFR2 antagonist anti-sauvagine-30 (anti-Svg-30). These results indicated that UCNs could inhibit the growth of SCLC cells, and this effect was mediated by activation of CRFR2. Interestingly, treatment with UCNs could inhibit p38 and Akt phosphorylation, while didn't affect the phosphorylation of ERK1/2 and JNK1/2 in human SCLC cells, implying that the effect of CRFR2 on SCLC cells might be mediated by inhibiting the phosphorylation of p38 and Akt, but not ERK1/2 or JNK1/2. Furthermore, the secretion levels of vascular endothelial growth factor (VEGF) in SCLC cells were measured, activation of CRFR2 by UCNs significantly decreased the VEGF secretion level, but pretreatment with p38 activator anisomysin or Akt activator PIP2 could abolish the UCNs'inhibition on the secretion of VEGF. Taken together, UCNs activated CRFR2 to inhibit the phosphorylation of p38 and Akt, and these effects mediated the down-regulation of VEGF secretion, finally to inhibit the growth of human SCLC cells. Therefore, we concluded that CRFR2 may be a potential anticancer target in SCLC. Moreover, its ligands might become new endocrine biologic response modifiers to neuroendocrine SCLC.Evidence indicated that activation of CRFR2 resulted in a tonic suppression of angiogenesis and remodeling the juvenile and adult vasculature, and UCN inhibited the proliferation of vascular smooth muscle cells (VSMCs) via CRFR2. Our previous data also showed that UCN could reduce the viability of rat VSMCs. Moreover, as an endothelial cell (EC)-specific mitogen and a major mediator of pathological angiogenesis, VEGF level was also decreased in rat VSMCs by UCN treatment via CRFR2. These reports highly imply that CRFR2 may play an inhibitory role in tumor angiogenesis and thus tumor growth by targeting ECs in tumors. In the second chapter of this thesis, we investigated the potential roles of CRFR2 in the tumor angiogenesis. Because HCC is charactered by its hypervasculature and no effective chemotherapy for this cancer, we used HCC as model to study the roles of CRFR2 in the tumor angiogenesis. Though there was no expression of CRF receptors in human HCC cells in tissues, the vessels and ECs in the HCC were found to express CRF receptors, CRFR2 mainly. In the model of HCC tumor-bearing nude mice, administration with UCN could block the growth of HCC in vivo. Significant reduction in tumor microvessel density (MVD) was also found in UCN-treated tumor-bearing nude mice. Since HCC cells didn't express CRF receptors, it was believed that tumor suppression was attributed to the inhibition of angiogenesis via activation of CRFR2 by UCN. In the in vitro model, UCN significantly inhibited human HCC angiogenesis in the three-dimensional angiogenesis assay with dose-dependent manner; this effect was reversed by CRFR2 antagonist, anti-Svg-30, but not by CRFR1 antagonist NBI-27914, demonstrating that it's CRFR2 that involved in the effect of inhibition of tumor angiogenesis. Furthermore, UCN also inhibited the proliferation and promoted the apoptosis of human umbilical vein endothelial cells (HUVEC), anti-Svg-30 also abolished these effects, while CRFR1 antagonist NBI-27914 can not. These data showed that UCN activated CRFR2 on the ECs to inhibit their growth. Moreover, UCN could down-regulate VEGF expression in serum and tumor tissue of HCC-bearing nude mice, which could be abolished by anti-Svg-30. In conclusion, UCN, acting on CRFR2, down-regulated the VEGF expression of ECs in tumor tissue, then, inhibited tumor angiogenesis, finally, inhibited the growth of HCC in vivo. We believed that activation of CRFR2 has antiangiogenic properties, which is a potential anticancer target. Since VEGF is responsible for the increased angiogenic activity of HCC, UCN could regulate this growth factor to inhibit tumor growth by CRFR2.Based on our observations, activation of CRFR2 produced dual effects on tumor growth: it can directly inhibit the growth of cancer cells which express CRFR2; moreover, it blocks the tumor growth by inhibiting tumor angiogenesis in vivo. The mechanism of these effects is attributed to the down-regulation of VEGF. Therefore, it is reasonable to believe that CEFR2 may be a new promising tumor therapeutic target. UCNs, as CRFR2 natural agonists, may be potential tumor biological therapeutic agents.
Keywords/Search Tags:CRF receptor 2(CRFR2), urocortin (UCN), small cell lung cancer (SCLC), hepatocellar carcinoma (HCC), tumor growth, tumor angiogenesis, vascular endothelial growth factor (VEGF), endothelial cells (ECs)
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