1. High doses of hydroxytyrosol induce apoptosis in papillary and follicular thyroid cancer cells
G Toteda, S Lupinacci, D Vizza, R Bonofiglio, E Perri, M Bonofiglio, D Lofaro, A La Russa, F Leone, P Gigliotti, R A Cifarelli, A Perri J Endocrinol Invest. 2017 Feb;40(2):153-162. doi: 10.1007/s40618-016-0537-2. Epub 2016 Sep 3.
Purpose: Recent evidences indicates that hydroxytyrosol, one of the main olive oil phenols, possess antitumor effects because of its pro-oxidant properties and the capacity to inhibit proliferation and to promote apoptosis in several tumor cell lines, although most of the results were obtained for breast and digestive systems cancers. Methods: In this study, we evaluated the activities of hydroxytyrosol against papillary (TPC-1, FB-2) and follicular (WRO) thyroid cancer cell lines. Results: Cellular viability revealed that high doses of hydroxytyrosol reduced cancer cells viability concomitantly with a reduction of cyclin D1 expression and an up-regulation of cell cycle key modulator p21 levels. In the same experimental conditions, Annexin V-PI staining and DNA laddering revealed that hydroxytyrosol exerts proapoptotic effects on papillary and follicular cancer cells. Furthermore, by Western blot analysis, we observed that hydroxytyrosol treatment reduced thyroid cancer cells viability by promoting apoptotic cell death via intrinsic pathway. Conclusions: Collectively, our results demonstrated for the first time that in thyroid cancer cells hydroxytyrosol promoted apoptosis at higher doses with respect to other cancer cells lines. Therefore, further studies will reveal the mechanisms by which thyroid cancer cells are more resistant to the proapoptotic effect exerted by hydroxytyrosol as well as the potential application as novel target therapeutic in thyroid cancer.
2. Biologic activities of recombinant human-beta-defensin-4 toward cultured human cancer cells
O L Gerashchenko, E V Zhuravel, O V Skachkova, N N Khranovska, V V Filonenko, P V Pogrebnoy, M A Soldatkina Exp Oncol. 2013 Jun;35(2):76-82.
Aim: The aim of the study was in vitro analysis of biological activity of recombinant human beta-defensin-4 (rec-hBD-4). Methods: hBD-4 cDNA was cloned into pGEX-2T vector, and recombinant plasmid was transformed into E. coli BL21(DE3) cells. To purify soluble fusion GST-hBD-4 protein, affinity chromatography was applied. Rec-hBD-4 was cleaved from the fusion protein with thrombin, and purified by reverse phase chromatography on Sep-Pack C18. Effects of rec-hBD-4 on proliferation, viability, cell cycle distribution, substrate-independent growth, and mobility of cultured human cancer cells of A431, A549, and TPC-1 lines were analyzed by direct cell counting technique, MTT assay, flow cytofluorometry, colony forming assay in semi-soft medium, and wound healing assay. Results: Rec-hBD-4 was expressed in bacterial cells as GST-hBD-4 fusion protein, and purified by routine 3-step procedure (affine chromatography on glutathione-agarose, cleavage of fusion protein by thrombin, and reverse phase chromatography). Analysis of in vitro activity of rec-hBD-4 toward three human cancer cell lines has demonstrated that the defensin is capable to affect cell behaviour in concentration-dependent manner. In 1-100 nM concentrations rec-hBD-4 significantly stimulates cancer cell proliferation and viability, and promotes cell cycle progression through G2/M checkpoint, greatly enhances colony-forming activity and mobility of the cells. Treatment of the cells with 500 nM of rec-hBD-4 resulted in opposite effects: significant suppression of cell proliferation and viability, blockage of cell cycle in G1/S checkpoint, significant inhibition of cell migration and colony forming activity. Conclusion: Recombinant human beta-defensin-4 is biologically active peptide capable to cause oppositely directed effects toward biologic features of cancer cells in vitro dependent on its concentration.
3. DUSP6/MKP3 is overexpressed in papillary and poorly differentiated thyroid carcinoma and contributes to neoplastic properties of thyroid cancer cells
Debora Degl'Innocenti, et al. Endocr Relat Cancer. 2013 Jan 21;20(1):23-37. doi: 10.1530/ERC-12-0078. Print 2013 Feb.
Thyroid carcinomas derived from follicular cells comprise papillary thyroid carcinoma (PTC), follicular thyroid carcinoma, poorly differentiated thyroid carcinoma (PDTC) and undifferentiated anaplastic thyroid carcinoma (ATC). PTC, the most frequent thyroid carcinoma histotype, is associated with gene rearrangements that generate RET/PTC and TRK oncogenes and with BRAF-V600E and RAS gene mutations. These last two genetic lesions are also present in a fraction of PDTCs. The ERK1/2 pathway, downstream of the known oncogenes activated in PTC, has a central role in thyroid carcinogenesis. In this study, we demonstrate that the BRAF-V600E, RET/PTC, and TRK oncogenes upregulate the ERK1/2 pathway's attenuator cytoplasmic dual-phase phosphatase DUSP6/MKP3 in thyroid cells. We also show DUSP6 overexpression at the mRNA and protein levels in all the analysed PTC cell lines. Furthermore, DUSP6 mRNA was significantly higher in PTC and PDTC in comparison with normal thyroid tissues both in expression profile datasets and in patients' surgical samples analysed by real-time RT-PCR. Immunohistochemical and western blot analyses showed that DUSP6 was also overexpressed at the protein level in most PTC and PDTC surgical samples tested, but not in ATC, and revealed a positive correlation trend with ERK1/2 pathway activation. Finally, DUSP6 silencing reduced the neoplastic properties of four PTC cell lines, thus suggesting that DUSP6 may have a pro-tumorigenic role in thyroid carcinogenesis.
