Melatoninin MCF-7 Hücre Kültüründeki Apoptoz Aktivasyonunun ve Sitotoksisitesinin Polimeraz Zincir Reaksiyonu (PCR), MTT Hücre Canlılık Testi ve İmmunsitokimya Yöntemleriyle Araştırılması

Öz

Amaç: Dünyada ve Türkiye'de en sık görülen kanser türlerinden biri olan meme kanseri, kanser nedenli ölüm oranları arasında ilk sıralarda yer almaktadır. Bu nedenle bu hastalığın tedavisine yönelik yeni araştırmalar yapılmaktadır. Biz de güçlü bir antioksidan olan melatoninin kanserle ilişkisini ve kanser hastalığının çözümüne katkısını araştırmayı amaçladık. Yöntemler: Çalışmamızda melatoninin sitotoksik dozlarını ve IC50 değerini belirleyebilmek için MCF-7 hücre hattına 10 nM–100.000 nM aralığında melatonin konsantrasyonları uygulandı. Melatonin uygulamasının ardından MTT analizi ile hücre canlılığı tespiti yapıldı ve melatonin için etkin dozlar belirlendi. MCF-7 hücreleri, 5 konsantrasyon (10, 100, 1000, 10.000 ve 100.000 nM) melatonin dozu ile 24 saat inkübasyon işlemine tabi tutuldu. Tüm gruplarda melatonin sitotoksisitesi, TAS, TOS, apoptotik aktivite (Bax ve p53 immünpozitifliği) ve p53 geninin ekspresyon düzeyleri incelendi. Bulgular: 24 saatlik inkübasyon sonunda MCF-7 hücrelerine uygulanan melatoninin hücre proliferasyonunu inhibe ederek sitotoksik etki yaptığı, p53 gen ekspresyonunu ve Bax protein sentezini artırdığı tespit edildi. İmmünsitokimyasal boyamada Bax ve p53 immünpozitifliğinin arttığı belirlendi. Ayrıca melatonin tedavisi TAS'ı artırıp TOS'u azalttı. Sonuç: Bu etkiler, melatoninin p53 gen ekspresyonu ve Bax proteinindeki artışa bağlı olarak apoptoz aktivasyonunu indüklediğini ve dolayısıyla kanser tedavisinde kullanılabilecek yardımcı bir ajan olabileceğini göstermektedir.

Anahtar Kelimeler

• Apoptoz
• Bax
• Meme Kanseri
• Melatonin
• p53

The Investigastion of Apoptosis Activation and Cyotoxicity of Melatonin in MCF-7 Cell Culture Through Polimerase Chain Reaction (PCR), MTT Cell Viability Assay and Immunocytochemical Methods

Abstract

Objective: Breast cancer, one of the most common cancer types in the world and in Turkey, ranks first among cancer-related deaths. For this reason, new researches are carried out for the treatment of this disease. We aimed to investigate the relationship of melatonin, a powerful antioxidant, with cancer and its contribution to the solution of cancer. Methods: In our study, melatonin concentrations in the range of 10 nM–100,000 nM were applied to the MCF-7 cell line to determine the cytotoxic doses and IC50 value of melatonin. After melatonin application, cell viability was determined by MTT analysis and effective doses for melatonin were determined. MCF-7 cells were incubated for 24 hours with 5 concentrations (10, 100, 1000, 10,000 and 100,000 nM) doses of melatonin. Melatonin cytotoxicity, TAS, TOS, apoptotic activity (Bax and p53 immunpositivity) and p53 gene expression levels were examined in all groups. Results: At the end of the 24-hour incubation, it was determined that melatonin administered to MCF-7 cells inhibited cell proliferation and had a cytotoxic effect, increasing p53 gene expression and Bax protein synthesis. It was determined that Bax and p53 immunopositivity increased in immunocytochemical staining. In addition, melatonin treatment increased TAS and decreased TOS. Conclusion: These effects show that melatonin induces apoptosis activation due to the increase in p53 gene expression and Bax protein, and thus may be an adjunctive agent that can be used in cancer treatment.

Keywords

• Apoptosis
• Bax
• Breast cancer
• Melatonin
• p53

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