Effects of cholinergic compounds and TNF-alpha on human erythroleukemia K562 cell proliferation and caspase expression

Yıl 2019, Cilt: 32 Sayı: 1, 20 - 26, 29.01.2019

Zehra Kanlı Banu Aydın Hulya Cabadak

https://doi.org/10.5472/marumj.518797

Cited By: 2

Öz

Objective: The purpose of this study was to investigate if

stimulating auto-paracrine muscarinic receptor signalling pathway

could change human erythroleukemia K562 cell proliferation and

caspase 3, 8 and 9 expression levels. To better understand the role of

muscarinic receptors in cell signalling mechanism, we investigated

the effects of several compounds on human erythroleukemia

K562 cell proliferation and caspase 3, 8 and 9 expression. These

compounds were M3 muscarinic receptor agonist, pilocarpine, proinflammatory

cytokine, tumor necrosis factor (TNF)-alpha, and

the wortmannin which is a phosphoinositide 3-kinase inhibitor.

Materials and Methods: Cell proliferation and cell viability

were evaluated by the trypan blue exclusion test and 5-Bromo-2-

deoxy-uridine (BrdU) Labelling and Detection Kits. Caspase 3, 8

and 9 expression levels were determined by immunoblot analysis.

Results: Both pilocarpine and TNF-alpha caused a small increase

in human erythroleukemia K562 cell proliferation. However, when

all the compounds were treated together, proliferation of human

erythroleukemia K562 cells increased significantly when compared to

untreated control cells. TNF-alpha and wortmannin treatment increased

caspase 3 and caspase 8 expression patterns significantly in human

erythroleukemia K562 cells. TNF-alpha and wortmannin treatment

increased caspase 9 expression level (P>0.05) but it was not significant.

Conclusion: These findings partly demonstrated that M₃

muscarinic receptor mediated an increase in K562 cell proliferation.

Pilocarpine prevented TNF-alpha and wortmannin induced

caspase 3 and 8 expression and indirectly showed apoptosis in

human erythroleukemia K562 cells.

Anahtar Kelimeler

M3 muscarinic receptors, Cytokine, Pilocarpine, Caspases, Erythroleukemia K562 cells

Kolinerjik bileşiklerin ve TNF-alfanın insan eritrolösemi K562 hücre çoğalmasına ve kaspaz ekspresyonu üzerine etkileri

Öz

Amaç: Bu çalışmanın amacı, otoparakrin M3 muskarinik

reseptör sinyal yolağının uyarılmasının, insan eritrolösemi K562

hücrelerinin çoğalmasında ve kaspaz 3, 8 ve 9 ekspresyon seviyeleri

üzerinde etkisi olup olmadığını araştırmaktır. Hücre sinyal

ileti mekanizmasında muskarinik reseptörlerin rolünü daha iyi

anlamak üzere, çeşitli bileşiklerin insan eritrolösemisi K562 hücre

proliferasyonu ve kaspaz 3, 8 ve 9 ekspresyon seviyeleri üzerindeki

etkilerini araştırdık. Bu bileşikler, M₃ muskarinik reseptör agonisti,

pilokarpin, pro-enflamatuvar sitokin, tümör nekroz faktör (TNF)-

alfa ve fosfoinositid 3-kinaz inhibitörü wortmanindir.

Gereçler ve Yöntemler: Hücre çoğalması ve hücre canlılığı,

tripan mavisi testi ve 5-Bromo-2-deoxy-uridine (BrdU) İşaretleme

ve Belirleme kitleri ile değerlendirildi. Kaspaz 3, 8 ve 9 ekspresyon

seviyeleri immunoblot analizi ile belirlendi.

Bulgular: Hem pilokarpin, hem de TNF-alfa, insan eritrolösemi

K562 hücre çoğalmasında çok az artışa neden oldu. Ancak, tüm

bileşikler birlikte muamele edildiğinde, insan eritrolösemi K562

hücrelerinin çoğalması, muamele edilmeyen kontrol hücrelerine

göre anlamlı olarak arttı. TNF-alfa ve wortmanin ile muamele

edilen K562 hücrelerinde kaspaz 3 ve kaspaz 8 ekspresyonu

seviyelerinde anlamlı değişim belirlendi. TNF-alfa ve wortmannin

muamelesi kaspaz 9 ekspresyon seviyesini arttırdı (P> 0.05) ancak

anlamlı değildi.

Sonuç: Bu bulgular, kısmen M3 muskarinik reseptör aracılı

K562 hücre çoğalmasında artış olduğunu göstermektedir.

Pilokarpin, insan eritrolösemi K562 hücrelerinde, TNF-alfa ve

wortmanin ile uyarılan kaspaz 3 ve 8 ekspresyonunu önledi ve

dolaylı olarak apoptozu gösterdi.

Anahtar Kelimeler

M3 muskarinik reseptorler, Sitokin, Pilokarpin, Kaspaz, Eritrolösemi K562 hücreleri

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