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  • br Results and discussion br

    2020-05-28


    Results and discussion
    Conclusion Recent studies showed that EGFR signaling is involved in inflammatory response in several inflammatory conditions. The normal inflammatory macrophage hyPerFUsion™ high-fidelity PCR Kit show EGFR-dependent production of inflammatory mediators. Nevertheless, the development of EGFR inhibitors as anti-inflammatory agents has not been reported yet. In this study, we have synthesized a series of amides (4) and structurally related urea derivative (5). While the amide derivatives (4) exhibited EGFR inhibitory activity, the urea derivative (5) was devoid from any significant EGFR inhibition up to 100 µM concentration. These derivatives were employed in cellular assays for anti-inflammatory activity in LPS-induced macrophages. Two macrophage cell lines were employed; peritoneal macrophages and RAW 264.7 macrophages. While the EGFR signaling pathway is intact in normal peritoneal macrophages, the transformed RAW 264.7 macrophages have modified EGFR signaling. Thus, EGFR inhibitors might reduce production of inflammatory mediators in peritoneal macrophages but not in RAW 264.7 macrophages. This might help to confirm that the elicited anti-inflammatory activity is mediated through EGFR inhibition. On contrast, the profile of non-EGFR inhibitor decoy compound might be different from EGFR inhibitors. The results of the anti-inflammatory assay showed that all of amide series exhibited high specific anti-inflammatory response against LPS-stimulated production of NO in peritoneal macrophages through inhibition of EGFR. Among them, compound 4d was the most potent eliciting IC50 value of 20.8 µM and showed high protection percent (18.9% remaining increased NO production at 40 µM). In addition, compound 4b presented high protection against increased LPS-stimulated NO production (18.5% remaining increased NO production at 40 µM) and IC50 value of 25.2 µM. We selected the most potent compound 4d to conduct further molecular mechanistic studies. Compound 4d inhibited the production of multiple inflammatory cytokines in macrophage cells including IL-1β, IL-6, and TNF-α. Furthermore, the EGFR inhibitor compound 4d attenuated NF-κB activation by dephosphorylating p65. It might be concluded that compound 4d might serve as a lead compound for development of a class of novel EGFR inhibitors as anti-inflammatory therapeutics. Furthermore, this study highlighted that the use of RAW 264.7 macrophages as a sole screen to judge the anti-inflammatory activity may result in loss of molecules with potential anti-inflammatory activity if their mechanism of action is mediated through the altered pathways in the transformed RAW 264.7 macrophage cells.
    Experimental
    Introduction Basal cell carcinoma (BCC) is the most frequent type among non-melanoma skin tumours and head and neck region is the most common site (80% of cases) (Rubin et al., 2005; Rogers et al., 2010). Their clinical presentations are mostly associated with the histologic subtype of the tumour. Nodular and superficial BCC\'s are the most prevalent subtypes usually presents as chronic ulcerations with distinct borders while the morphea form type, also known as infiltrative basal-cell carcinoma, typically appears as indurated, whitish, scar-like plaque with indistinct margins (Rubin et al., 2005). The metastasis of these tumours is accepted as unusual, but they have risk of recurrence especially in lesions with multiple risk factors. Lesions of head have lower five-year cure rate than lesions of other body parts. Tumour diameter higher than 2 cm, location of the tumour, an aggressive histopathologic pattern and perineural or perivascular involvement are the main risk factors for the recurrence. Also, subclinical extension or indistinct margins of the tumour causes residual positive margins after surgical excision and these kinds of lesions have a higher recurrence rate than tumours with distinct borders (Walling et al., 2004). Surgical excision is the principle treatment method for obtaining the best oncologic results, while various methods including cryotherapy, radiotherapy, and photodynamic therapy can be performed in clinical practice. There are some difficulties in surgical practice. Because of the cosmetic characteristics of the face region, obtaining the best cosmetic and oncologic result at the same time is the main challenge for head and neck surgeons. Hence, some histopathologic and surgical approaches have been defined for reducing defect size and successful excision with clear margins. Among them Mohs microscopic surgery accepted as most successful one for especially recurrent basal-cell carcinoma. In this technique 100% of the peripheral and deep surgical margins of excised lesion are examined with horizontal frozen-section specimens4.Three-dimensional (3D) histology another surgical method that were defined with the aim of reducing recurrence rates, all of lateral and deep margins of excised lesions are examined with a routine paraffin procedure. The five-year cure rate for this method is around %1 (Shriner et al., 1998; Häfner et al., 2011).