Effects of r-Klotho on MMP/TIMP Balance and TNF-α in Colon Cancer and Normal Colon Epithelial Cells

Öz

Objective: To evaluate the effects of recombinant klotho protein (r-klotho) on human colon adenocarcinoma cells (Caco-2) and normal colon epithelial cells (CCD 841 CoN), with a focus on extracellular matrix (ECM) remodeling and inflammation-related gene expression. Materials and Methods: Caco-2 and CCD 841 CoN cells were cultured in four experimental groups: CCD 841 CoN; Caco-2; r-klotho-treated CCD 841 CoN; and r-klotho-treated Caco-2. r-klotho was administered at 0.3 μg/mL, selected from a previous dose–response study as the concentration yielding the lowest relative viability in Caco-2 cells within the tested range, for 24 and 48 hours. Expression levels of MMP-1, MMP-3, MMP-13, TIMP-1, TIMP-3, and TNF-α were analysed using quantitative real-time PCR (qRT-PCR) and evaluated by the ΔΔCt method with GAPDH as the reference gene. Results: In Caco-2 cells, r-klotho significantly reduced MMP-1 and TIMP-1 expression (p<0.05 and p<0.01), while significantly increasing MMP-3, TIMP-3, and TNF-α expression (p<0.001 and p<0.01). In CCD 841 CoN cells, r-klotho caused significant increases in MMP-1, MMP-3, TIMP-1, TIMP-3, and TNF-α expression (p<0.05–0.001). Increases in TIMP-3 and TNF-α became more pronounced with more prolonged exposure. Conclusion: Our findings suggest that klotho exerts cell-type-specific, bidirectional, and time-dependent effects on ECM remodeling and inflammatory response–related gene expression. These results indicate that r-klotho may represent a potential candidate for modulating tumour microenvironmental and inflammatory processes relevant to colorectal cancer progression. Keywords: Colon cancer, gene expression, klotho, MMP, TIMP, TNF-α.

Anahtar Kelimeler

• Klotho
• colon cancer
• MMP
• TIMP
• TNF-α
• gene expression

r-Klotho’nun Kolon Kanseri ve Normal Kolon Epitel Hücrelerinde MMP/TIMP Dengesi ve TNF-α Üzerine Etkileri

Öz

Amaç: Bu çalışma, rekombinant klotho (r klotho) proteininin insan kolon adenokarsinom hücreleri (Caco 2) ve normal kolon epitel hücreleri (CCD 841 CoN) üzerindeki etkilerini, özellikle hücre dışı matriks (ECM) yeniden yapılanması ve inflamasyonla ilişkili genlerin ekspresyonu açısından değerlendirmeyi amaçlamaktadır. Gereç ve Yöntem: Caco-2 ve CCD 841 CoN hücreleri dört ayrı deney grubu oluşturularak kültüre edilmiştir: CCD 841 CoN, Caco-2, r-klotho uygulanan CCD 841 CoN ve r-klotho uygulanan Caco-2. Önceki doz-yanıt çalışmamızda belirlenen 0,3 μg/mL r-klotho, 24 ve 48 saat süreyle uygulanmıştır. MMP-1, MMP-3, MMP-13, TIMP-1, TIMP-3 ve TNF-α genlerinin ekspresyon düzeyleri kantitatif gerçek zamanlı PCR (qRT-PCR) yöntemiyle analiz edilmiş ve GAPDH referans geni kullanılarak ΔΔCt yöntemiyle değerlendirilmiştir. Bulgular: Caco-2 hücrelerinde r-klotho uygulaması, MMP-1 ve TIMP-1 genlerinin ekspresyonunu anlamlı şekilde azaltırken (p<0.05 ve p<0.01), MMP-3, TIMP-3 ve TNF-α genlerinin ekspresyonunu belirgin şekilde artırmıştır (p<0.001; p<0.01). CCD 841 CoN hücrelerinde ise r-klotho uygulaması, MMP-1, MMP-3, TIMP-1, TIMP-3 ve TNF-α gen ekspresyonlarında anlamlı artışlara yol açmıştır (p<0.05–0.001). Uygulama süresi uzadıkça özellikle TIMP-3 ve TNF-α’daki artışların daha belirgin hale geldiği saptanmıştır. Sonuç: Bulgularımız, klotho proteininin ECM yeniden yapılanması ve inflamatuvar yanıtların düzenlenmesinde hücre tipine özgü, çift yönlü ve zaman bağımlı bir rol üstlendiğini göstermektedir. Bu sonuçlar, klothonun yalnızca yaşlanma karşıtı bir protein değil, aynı zamanda kolorektal kanser progresyonunda tümör mikroçevresi ve inflamatuvar süreçlerin modülasyonu için umut vadeden bir terapötik hedef olabileceğini ortaya koymaktadır. Anahtar Kelimeler: Kolon kanseri, gen ekspresyonu, klotho, MMP, TIMP, TNF-α.

Anahtar Kelimeler

• Klotho
• kolon kanseri
• MMP
• TIMP
• TNF-α
• gen ekspresyonu

Kaynakça

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