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

Abstract

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.

Keywords

• 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

Abstract

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.

Keywords

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

References

1. Arnold M, Sierra MS, Laversanne M, Soerjomataram I, Jemal A, Bray F. Global patterns and trends in colorectal cancer incidence and mortality. Gut. 2017;66(4):683-691. DOI:10.1136/gutjnl-2015-310912
2. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2020. CA Cancer J Clin. 2020;70(1):7-30. DOI:10.3322/caac.21590
3. Morgado-Díaz JA. Colorectal cancer: From prevention to personalized medicine. World J Gastroenterol. 2022;28(3):233-254. DOI:10.3748/wjg.v28.i3.233
4. Brenner H, Kloor M, Pox CP. Colorectal cancer. Lancet. 2014;383(9927):1490-1502. DOI:10.1016/S0140-6736(13)61649-9
5. Mota J, Lima AMM, Gomes JIS, et al. Klotho in cancer: Potential diagnostic and prognostic applications. Diagnostics (Basel). 2023;13(21):3357. DOI:10.3390/diagnostics13213357
6. Ortega MA, Boaru DL, De Leon Oliva D, et al. The impact of Klotho in cancer: From development and progression to therapeutic potential. Genes (Basel). 2025;16(2):128. DOI:10.3390/genes16020128
7. Li X, Huang L, Peng J, Liang L, Shi D, Zheng H, Cai S. Klotho suppresses growth and invasion of colon cancer cells through inhibition of IGF1R-mediated PI3K/AKT pathway. Int J Oncol. 2014;45(2):611-618. DOI:10.3892/ijo.2014.2430
8. Xie B, Zhou J, Shu G, Liu DC, Chen J. Restoration of Klotho gene expression induces apoptosis and autophagy in gastric cancer cells: Tumor suppressive role of Klotho in gastric cancer. Cancer Cell Int. 2013;13:18. DOI:10.1186/1475-2867-13-18

9. Xie B, Zhou J, Shu G, Liu DC, Chen J, Yuan L. Overexpression of Klotho suppresses liver cancer progression and induces cell apoptosis by negatively regulating Wnt/β-catenin signaling pathway. World J Surg Oncol. 2015;13:71. DOI:10.1186/s12957-015-0717-0
10. Rubinstein TA, et al. Klotho is a tumor suppressor in human colorectal cancer. Clin Cancer Res. 2018;24(20):5091-5101. DOI:10.1158/1078-0432.CCR-17-3509
11. Soykan MN, Gunes S. Overexpression of Klotho gene using CRISPR/Cas9 induces apoptosis and inhibits cell motility in human colorectal cancer cells. Biotechnol J. 2024;19(2):e2300496. DOI:10.1002/biot.202300496
12. Tang X, Wang Y, Fan Z, Ji G, Wang M, Lin J, Huang S, Meltzer SJ. Klotho: A tumor suppressor and modulator of the Wnt/β-catenin pathway in human hepatocellular carcinoma. Lab Invest. 2016;96(2):197-205. DOI:10.1038/labinvest.2015.86
13. Sachdeva A, Gouge J, Kontovounisios C, Nikolaou S, Ashworth A, Lim K, Chong I. Klotho and the treatment of human malignancies. Cancers (Basel). 2020;12(6):1665. DOI:10.3390/cancers12061665
14. Zhang Y, Liu X, Xu Z, et al. Proteomic profiling reveals Klotho suppresses invasion and metastasis by inhibiting the Wnt/β-catenin axis in lung adenocarcinoma. Sci Rep. 2024;14:52616. DOI:10.1038/s41598-024-52616-0
15. Cabral-Pacheco GA, Garza-Veloz I, Castruita-De la Rosa C, et al. The roles of matrix metalloproteinases and their inhibitors in human diseases. Int J Mol Sci. 2020;21(24):9739. DOI:10.3390/ijms21249739
16. Vandooren J, Van den Steen PE, Opdenakker G. The roles of matrix metalloproteinases and their inhibitors in human diseases. Cell Signal. 2020;32:123-134. DOI:10.1016/j.cellsig.2019.109570
17. Saw S, Weiss A, Khokha R, Waterhouse PD. Metalloproteases: On the watch in the hematopoietic niche. Trends Immunol. 2019;40(12):1138-1153. DOI:10.1016/j.it.2019.09.006
18. Olejnik A, Krzywonos-Zawadzka A, Sambor I, Mysiak A, Bil-Lula I. Klotho protein alleviates heart ischemia/reperfusion injury and oxidative stress through regulation of the NOS/MMP pathway. Sci Rep. 2025;15(1):32299. DOI:10.1038/s41598-025-18021-x
19. Maekawa Y, Ishikawa K, Yasuda O, et al. Klotho suppresses TNF-alpha-induced expression of adhesion molecules in the endothelium and attenuates NF-kappaB activation. Endocrine. 2009;35(3):341-346. DOI:10.1007/s12020-009-9181-3
20. Zhao X, Han D, Zhao C, et al. New insights into the role of Klotho in inflammation and fibrosis: Molecular and cellular mechanisms. Front Immunol. 2024;15:1454142. DOI:10.3389/fimmu.2024.1454142
21. Chen P, Tang Y, Luo L, et al. Lower serum Klotho level and higher systemic immune-inflammation index: An inverse correlation. BMC Geriatr. 2023;23:650. DOI:10.1186/s12877-023-04349-4
22. Gunes S, Uysal O, Soykan MN, Eker Sariboyaci A. Investigation of apoptotic effect of Klotho protein on human colorectal cancer cells via TRAIL death receptors. ADYU J Sci. 2022;12(2):324-337. DOI:10.37094/adyujsci.1212028
23. Pan J, Zhong J, Gan LH, Chen SJ, et al. Klotho, an anti-senescence related gene, is frequently inactivated through promoter hypermethylation in colorectal cancer. Tumour Biol. 2011;32(4):729-735. DOI:10.1007/s13277-011-0174-5
24. Wang Y, Zhang L, Wu GR, et al. Klotho suppresses the growth of human colorectal cancer cells by modulating the IGF1R and Wnt/β-catenin pathways. Biomed Pharmacother. 2020;128:110275. DOI:10.1016/j.biopha.2020.110275
25. Chen B, Ma X, Liu S, Zhao W, Wu J. Inhibition of lung cancer cell growth, motility and induction of apoptosis by Klotho, a novel secreted Wnt antagonist. Cancer Biol Ther. 2012;13(12):1221-1228. DOI:10.4161/cbt.21636
26. Hu Y, Xu L, Zhang J, et al. Klotho suppresses tumor progression via inhibiting PI3K/Akt/GSK3β/Snail signaling in renal cell carcinoma. Cancer Sci. 2013;104(6):663-671. DOI:10.1111/cas.12132
27. Luo Z, et al. Klotho protein: A multifaceted regulator in aging and cancer dynamics. Cell Mol Life Sci. 2025;82:Article number pending. DOI:10.1007/s00018-024-4521-0
28. Shiozawa J, Ito M, Nakayama T, et al. Expression of matrix metalloproteinase-1 in human colorectal carcinoma. Mod Pathol. 2000;13(9):925-933. DOI:10.1038/modpathol.3880179
29. Sunami E, Tsuno N, Osada T, et al. MMP-1 is a prognostic marker for hematogenous metastasis of colorectal cancer. Oncologist. 2000;5(2):108-114. DOI:10.1634/theoncologist.5-2-108
30. Bendardaf R, Buhmeida A, Ristamäki R, Syrjänen K, Pyrhönen S. MMP-1 (collagenase-1) expression in primary colorectal cancer and its metastases. Scand J Gastroenterol. 2007;42(12):1473-1478. DOI:10.1080/00365520701443879
31. Said A, Raufman JP, Xie G. The role of matrix metalloproteinases in colorectal cancer. Cancers (Basel). 2014;6(1):366-375. DOI:10.3390/cancers6010366
32. Orlichenko LS, Radisky DC. Matrix metalloproteinases stimulate epithelial-mesenchymal transition during tumor development. Clin Exp Metastasis. 2008;25(6):593-600. DOI:10.1007/s10585-008-9169-y
33. Nieto MA, Huang RY, Jackson RA, Thiery JP. EMT: 2016. Cell. 2016;166(1):21-45. DOI:10.1016/j.cell.2016.06.028
34. Shibue T, Weinberg RA. EMT, CSCs, and drug resistance: The mechanistic link and clinical implications. Nat Rev Clin Oncol. 2017;14(10):611-629. DOI:10.1038/nrclinonc.2017.44
35. Huang MY, Chang HJ, Chung FY, et al. MMP-13 is a potential prognostic marker for colorectal cancer. Oncol Rep. 2010;24(5):1241-1247. DOI:10.3892/or_00000961
36. Lim YJ, Kim SM, Lee JY. Klotho attenuates intestinal inflammation via modulation of epithelial TNF-α signaling. Int J Mol Sci. 2022;23(7):3725. DOI:10.3390/ijms23073725