PAK4, MCF-7 hücrelerinde E-kaderini baskılayarak PKC-bağımlı invaziv potansiyeli destekler

Year 2020, Volume: 77 Issue: 1, 107 - 116, 01.03.2020

Suray Pehlivanoğlu Çiğdem Aydın Acar

Abstract

Amaç: Normal meme epitel hücrelerinde p21 ile aktive edilen kinaz 4 PAK4 overekspresyonu sağlandığında tek başına tümörigenezis sürecini başlatabilmektedir. Son zamanlarda yapılan araştırmalar, PAK4’ün meme kanserinde önemli bir onkojenik faktör olabileceğini ileri sürmektedir. Bu çalışmada, PAK4 overeksprese eden ve etmeyen MCF-7 meme kanseri hücrelerinde protein kinaz C aktivasyonu ve inhibisyonuna bağlı hücrelerin migrasyon kabiliyeti ve hücre-hücre kontaktını sağlayan E-Kaderin ekspresyon düzeylerinin araştırılması amaçlandı. Yöntem: Çalışmada, meme kanseri modeli olarak MCF7 hücre hattı kullanıldı. MCF7 hücrelerinde PAK4 plasmid kullanılarak yabanıl-tip insan PAK4 geninin ektopik ekspresyonu sağlandı. Kontrol vektör olarak ise p3XFLAGCMV-10 plazmiti kullanıldı. PKC inhibitörü olarak RO318220 ve PKC aktivatörü olarak ise phorbol 12-myristate-13- acetate PMA / TPA kullanıldı. PAK4 plazmid ve kontrol vektör ile transfekte edilen her iki gruptaki hücreler %0,2 FBS, %10 FBS, RO318220 5μM ve TPA 200 nM olacak şekilde 48 saat süre ile kültüre edildi. Meme kanseri hücrelerinde hücre migrasyonu, Oris Hücre migrasyon deneyi ile değerlendirildi. E-kaderin ekspresyonunun değerlendirilmesi için Western blot yöntemi kullanıldı.Bulgular: Yüksek PAK4 ekspresyonu sağlanan MCF7 hücrelerinde mezenkimal-benzer fenotipin meydana geldiği ve podozomal yapıların sayılarının ve uzunluklarının arttığı belirlendi. Ayrıca TPA ile PKC aktivasyonu sağlanan hücrelerde PAK4 overekspresyonuna bağlı hücre migrasyonunda artış görüldü. Fakat PKC ile indüklenen invaziv etkiler PKC inhibitörü olan RO318220 ile muamele edilen hücrelerde bloke edildi. Bunun yanısıra, PAK4 overeksprese eden hücrelerde kontrole göre E-kaderin ekspresyonunda baskılanma meydana geldiği belirlendi. Sonuç: E-kaderin, hücre-hücre kontaktını sağlayan ve hücre göçünü engelleyen temel yapılardan biridir. Birlikte değerlendirildiğinde, bu bulgular PKC ile aktive edilen PAK4 sinyalinin meme kanseri progresyonuna katkıda bulunduğunu göstermektedir. Bu nedenle, sonuçlarımız PKC-PAK4 sinyal yolağının inhibe edilmesinin meme kanseri tedavisi için potansiyel bir terapötik yaklaşım olabileceğini göstermektedir.

Keywords

Meme kanseri, PAK4, Protein kinaz C

PAK4 promotes invasive potential of MCF-7 cells in PKC-dependent manner through downregulation of E-Cadherin

Abstract

Objective: The p21-activated kinase 4 PAK4 overexpression is sufficient to initiate the tumorigenesis process in normal breast epithelial cells. Recent studies suggested that PAK4 could be an important oncogenic factor in breast cancer. The aim of this study was to investigate the migration ability of cells due to protein kinase C activation and inhibition and the expression levels of E-cadherin which provides cell-cell contact in MCF-7 breast cancer cells that PAK4 overexpressing and nonoverexpressing cells.Methods: MCF7 cell line was used as a breast cancer model. Ectopic expression of the wild-type human PAK4 gene was achieved using PAK4 plasmid in MCF7 cells. Plasmid p3XFLAG-CMV-10 was used as control vector. RO318220 was used as PKC inhibitor and phorbol 12-myristate-13-acetate PMA / TPA was used as PKC activator. Cells in both groups transfected with PAK4 plasmid and control vector were cultured for 0.2 h FBS, 10% FBS, RO318220 5μM and TPA 200 nM for 48 hours. Cell migration in breast cancer cells was evaluated by Oris cell migration assay. Western blot method was used to evaluate the expression of E-cadherin.Results: It was determined that mesenchymallike phenotype was formed and the number and length of podosomal structures were increased in these PAK4 overexpressed cells. In addition, PKC activation via TPA treatment increased cell migration due to PAK4 overexpression. However, PKC-induced invasive effects were blocked by the PKC kinase inhibitor RO318220. In addition, PAK4 overexpression leads to downregulation of E-cadherin compared to control. Conclusion: E-cadherin is one of the basic structures that provide cell-cell contacts and prevent cell migration. Taken together, these findings suggest that PKC-activated PAK4 signalling contributes to breast cancer progression. Therefore, our results show that inhibition of the PKC-PAK4 signaling pathway may be a potential therapeutic approach for the treatment of breast cancer

Keywords

Breast cancer, PAK4, Protein kinase C

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