Evaluation of apoptotic effects of recombinant klotho protein on human colorectal cancer cells

Abstract

Colorectal cancer is the most common type of gastrointestinal cancer in the world. Klotho is an anti-aging glycosylated transmembrane protein that plays an important role in regulating phosphate and calcium homeostasis in the body and acts as a hormone. Klotho not only functions as a "suppressor of aging", but also regulates cell survival and proliferation and acts as a potential tumor suppressor that plays a role in cancer metastasis. Mutations in the gene expressing the klotho protein have been identified in the pathogenesis of various cancers, including lung, liver, breast, pancreatic, thyroid, ovarian, kidney, and colon cancer. In this study, recombinant klotho (r-klotho) protein, which plays an important role in cell proliferation, tumor migration and invasion, was detected in apoptosis-resistant human colorectal cancer cells (Caco-2) with different concentrations (0.075µg/mL, 0.15µg/mL, 0.3µg/mL, 0.6µg/mL, and 0.9µg/mL) and hours (24 and 48 hours). It was aimed to investigate the possible cytotoxic and apoptotic effects on the healthy colon (CCD 841 CoN) and cancer cells of exogenous application by adding to the culture medium. For this purpose, in the present study, for the first time, colorectal cancer cells were treated with r-klotho. The in vitro viability effects of r-klotho on cancer and healthy cells were evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and flow cytometry analyses of active caspase-3/7. Our findings revealed that r-klotho works as a tumor suppressor by suppressing cell proliferation and inducing cell apoptosis in human colorectal cancer cells. Consequently, klotho protein can be used as a potential antitumor agent for the development of new strategies in therapeutic interventions for colorectal cancer.

Keywords

• Colorectal cancer
• recombinant klotho
• cytotoxicity
• caspase-3/7
• apoptosis

Rekombinant klotho proteininin insan kolorektal kanser hücreleri üzerindeki apoptotik etkilerinin değerlendirilmesi

Abstract

Kolorektal kanser dünyada en sık görülen gastrointestinal kanser türüdür. Klotho, bedende fosfat ve kalsiyum homeostazının düzenlenmesinde önemli rol oynayan, hormon görevi de gören yaşlanma karşıtı glikozile edilmiş transmembran bir proteindir. Klotho sadece bir "yaşlanma baskılayıcı" olarak işlev görmez, aynı zamanda hücrenin hayatta kalmasını ve çoğalmasını da düzenler ve kanser metastazında rol oynayan potansiyel bir tümör baskılayıcı olarak görev yapar. Bu durum, kanser ve yaşlanmanın benzer moleküler özellikleri paylaştığı düşünüldüğünde, şaşırtıcı değildir. Akciğer, karaciğer, meme, pankreas, tiroid, over, böbrek ve kolon kanseri de dahil çeşitli kanserlerin patogenezinde, klotho proteinini eksprese eden gende, mutasyonlar tespit edilmiştir. Bu çalışmada hücre proliferasyonunda, tümör migrasyon ve invazyonunda önemli bir rol üstlenen rekombinant klotho (r-klotho) proteininin, apoptoza dirençli insan kolorektal kanser hücrelerine (Caco-2) farklı konsantrasyon (0,075µg/mL, 0,15µg/mL, 0,3µg/mL, 0,6µg/mL ve 0,9µg/mL) ve saatlerde (24 ve 48 saat) ekzojen olarak kültür besiyerine eklenerek uygulanmasının sağlıklı kolon (CCD 841 CoN) ve kanser hücreleri üzerindeki olası sitotoksik ve apoptotik etkilerini araştırmak amaçlandı. Bu amaç doğrultusunda ilk olarak kolorektal kanser hücrelerinin r-klotho ile muamelesi gerçekleştirildi. R-klothonun kanser ve sağlıklı hücreler üzerindeki in vitro canlılığa etkileri 3-(4,5-dimetiltiazol-2-il)-2,5-difeniltetrazolium bromid (MTT) ve aktif kaspaz-3/7’nin flow sitometri analizleri ile değerlendirildi. Bulgularımız r-klothonun insan kolorektal kanser hücrelerinde hücre proliferasyonunu baskılamak ve hücre apoptozunu indüklemek yoluyla bir tümör baskılayıcı olarak çalıştığını ortaya koymuştur. Sonuç olarak klotho proteini, kolorektal kanser için terapötik girişimlerde yeni stratejilerin geliştirilebilmesi için, potansiyel antitümör bir ajan olarak kullanılabilir.

Keywords

• Kolekteral kanser
• Rekombinant klotho
• sitotoksisite
• kaspaz 3/7
• apoptoz

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