ADAMTS13 expression in human chondrosarcoma cells induced by insulin

Abstract

Objectives: A Disintegrin-like Metalloproteinase with Thrombospondin Motifs (ADAMTS) proteins is a proteinase enzyme group that primarily located in the extracellular matrix (ECM). Insulin has been known to stimulate proteoglycan biosynthesis in chondrosarcoma chondrocytes and thereby the levels of ADAMTS proteins. The aim of this study is to evaluate the time-dependent effects of insulin on the ADAMTS13 expression in OUMS-27 human chondrosarcoma cell line to test the hypothesis that insulin diminishes ADAMTS13 expression because of its anabolic effects. Methods: To test this hypothesis OUMS-27 cells were cultured in Dulbecco\'s modified Eagle\' medium (DMEM) containing 10μg/mL insulin. The medium containing insulin was changed every other day up to 11th day. Cells were harvested at 1, 3, 7, and 11th days and protein and RNA isolations were performed at the proper times. The levels of RNA expression of ADAMTS13 was quantified by qRT-PCR using appropriate primers while protein levels was detected by Western blot technique using anti-ADAMTS13 antibody. Results: Although there was a decrease in both RNA and protein levels in insulin-applied groups compared to the control cells, it was not statistically significant. Conclusion: Under the light of our findings, it is suggested that insulin does not participate in regulation of ADAMTS13 in OUMS-27 chondrosarcoma cells. J Clin Exp Invest 2014; 5 (2): 226-232

Keywords

• Insulin; ADAMTS13, chondrosarcoma, OUMS-27, RNA

Insulin tarafindan indüklenmiş insan kondrosarkom hücrelerinde ADAMTS13 ekspresyonu

Öz

Amaç: A Disintegrin-like Metalloproteinase with Thrombospondin Motifs (ADAMTS) proteinleri esas olarak ekstraselüler matrikste (ECM) bulunan bir ptoteinaz enzim grubudur. İnsülinin kondrosarkom kondrositlerinde proteoglikan sentezini stimüle ettiği uzun yıllardır bilinmekteydi. Dolayısıyla bu çalışmanın amacı insülinin, OUMS-27 insan kondrosarkom hücre kültüründeki zaman bağımlı etkilerini değerlendirmek ve insülinin anabolik etkilerinden dolayı ADAMTS13 ekspresyonunu azaltma ihtimali hipotezini test etmektir. Yöntemler: Bu hipotezi test etmek için OUMS-27 hücrelerinin, 10μg/mL insulin içeren Dulbecco\'s modified Eagle besiyerinde (DMEM) kültürü yapıldı. 11. güne kadar iki günde bir besiyeri ile birlikte insülin takviyesi yapıldı. Hücreler 1, 3, 7 ve 11. günlerde harvest edilerek uygun zamanlarda protein ve RNA izolasyonu yapıldı. ADAMTS13 RNA ekspresyonu uygun primerler kullanılarak qRT-PCR yöntemi ile ölçüldü ve anti-ADAMTS13 antikorları kullanılarak Western blot yöntemi ile protein miktarı ölçüldü. Bulgular: Kontrol grubu ile karşılaştırıldığında insülin uygulanan hücre gruplarında, hem RNA hem de protein miktarı azaldı ancak bu istatistiksel olarak anlamlı değildi. Sonuç: Bu bulgular ışığında insülinin OUMS-27 kondrosarkom hücrelerinde ADAMTS13 regülasyonuna pozitif ya da negative herhangi bir etki yapmadığı sonucu ortaya çıkmıştır.

Anahtar Kelimeler

• İnsulin, ADAMTS13, Kondrosarkom, OUMS-27, RNA

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