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    • br Materials and methods br Results br

    2019-10-23


    Materials and methods
    Results
    Discussion The nitric oxide generating enzymes of the NOS family comprise three isoforms: nNOS, iNOS and eNOS [31]. Expression of eNOS is mainly found in endothelial pyruvate dehydrogenase kinase inhibitor where it physically promotes vasodilation [32]. Conversely, evidence also indicates that eNOS is expressed in cancer where it plays a role in cell proliferation, anti-apoptosis, angiogenesis, invasion and metastasis [16], [22], [17], [33], [31]. Regulation of the expression of eNOS revealed that ephrin-A1 and VEGF interact with eNOS through the PI3K/AKT pathway [23], [25]. A recent study indicates that alteration of the receptor tyrosine kinases, as well as the downstream kinases, is involved in the development of CCA. Among the downstream kinases, eNOS is predominantly activated in CCA cell lines and CCA tissues. Besides activation of eNOS, EphA3 and VEGFR3 are also activated [4]. Therefore, the expression and clinical significance of eNOS, as well as its upstream regulator proteins including VEGFR3, EphA3 with the specific ligands VEGFC and ephrin-A1, were investigated in the present study. As detailed in Section 3.2, immunohistochemical staining increased for the analyzed proteins during development of CCA in the hamster model, indicating their increased abundance. Results from previous studies demonstrate that eNOS is strongly expressed in the lung tissues of mice that were transplanted with C3L5 cells. These have a high metastatic potential when compared to C10, which is rather weakly metastatic [34]. This observation suggests that eNOS and P-NOS, as well as their upstream regulator proteins, are associated with cholangiocarcinogenesis and may play an important role in the activation CCA. Therefore, we explored the expression of eNOS and P-eNOS and their upstream regulator proteins in human CCA tissues. IHC staining in human CCA tissue arrays (n=171) was performed. Interestingly, our results indicated that VEGFR3, VEGFC, EphA3 and ephrin-A1 can all act as specific upstream regulators of eNOS. We then demonstrated that eNOS and P-eNOS, as well as their upstream regulator proteins, are significantly associated with the metastatic status of CCA patients. Our results are corroborated by IHC staining in oral squamous cell carcinoma (OSCC), which reveals that eNOS and VEGF are significantly correlated with nodal metastasis [35]. The clinico-pathological significance of EphA3 with lymph node metastasis has been shown for colorectal carcinoma [36]. However, for colorectal cancer there is a positive, but not significant, correlation between ephrin-A1 and lymph node metastasis [37]. Moreover, VEGFC is also significantly associated with non-papillary CCA. IHC of VEGFC increases significantly in papillary thyroid cancer [38]. We next showed a high, concurrent abundance of eNOS with its upstream regulator proteins. We demonstrated, for the first time, that a high concurrent abundance of eNOS with VEGFR3, VEGFC, EphA3, and ephrin-A1 was significantly associated with metastasis in CCA patients. Apart from this, a high concurrent abundance of eNOS with VEGFR3 and ephrin-A1 was also significantly associated with non-papillary CCA. A previous study suggested that eNOS is a key regulator in angiogenic processes [39], [33]. We also found that a high abundance of eNOS and P-eNOS was significantly associated with high MVD.
    Acknowledgment This work was supported by an Invitation Research Grant (Grant No. I57231 and IN58135), a Scholarship of the Liver Fluke and Cholangiocarcinoma Research Center (LFCRC001/2556) to MS, a Research Assistantship Grant of the Faculty of Medicine, Khon Kaen University (Grant No. AS57202) and grants from the Higher Education Research Promotion and National Research University Project of Thailand (NRU 592005), Office of the Higher Education Commission, through the Center of Excellence in Specific Health Problems in Greater Mekong Sub-region cluster (SHeP-GMS), Khon Kaen University allocated to WL. We thank Professor Trevor N. Petney for editing the MS via the Publication Clinic KKU, Thailand.