The effects of lithium, metformin and everolimus substances on cell growth in 2D and 3D Ishikawa endometrial carcinoma cell culture

Öz

Purpose: Our aim is to study the effects of the single and combined treatments of Everolimus, Metformin, and Lithium Chloride in two-dimensional (2D, monolayer) and three-dimensional (3D, spheroid) cell cultures of Ishikawa cells, which comprise the endometrial cancer cell line. Materials and methods: As part of the study, the effects of single and combined forms of Everolimus, Metformin, and Lithium Chloride were determined on cell viability, invasion, colony formation and apoptosis, and PI3K/AKT/mTOR pathway. Cell viability was assessed using XTT assay. CASP3, CASP8, CASP9, FASL, FADD, TNF, TRADD, BAX, P53, PI3KCA, PI3KCB, PTEN, MTOR, AKT1 genes were evaluated with RT-PCR, apoptosis was evaluated by flow cytometry and 3D spheroid results were evaluated with invert microscope analysis. Results: Everolimus, metformin, and lithium's IC50 levels were found at 48 hours to be 37.46 nM, 48.59 mM, and 100 μM, respectively. It was determined that the invasive capacities of Ishikawa cells in treatment groups, as well as cell colony formation were significantly reduced. In addition, Ishikawa spheroid cells were significantly suppressed compared with the control groups. RT-PCR results revealed that substances and their combinations affect genes associated with PI3K/AKT/mTOR pathway and apoptosis. Flow cytometry results showed notably increased apoptosis by single and combined treatments. Conclusion: As a result, the single and combination forms of everolimus, metformin, and lithium have reduced cell proliferation, induced apoptosis, and decreased mTOR activation through various mechanisms in Ishikawa cells. However, our study has shown that Eve alone and triple combination therapy (Eve+Met+Lit) are more effective than other therapies in the treatment of endometrial cancer.

Anahtar Kelimeler

• Endometrial cancer
• PI3K/AKT/mTOR pathway
• everolimus
• metformin
• lithium

Lityum, metformin ve everolimus maddelerinin 2D ve 3D Ishikawa endometrial karsinom hücre kültüründe hücre büyümesi üzerine etkileri

Öz

Amaç: Amacımız endometrium kanser hücre hattı olan Ishikawa hücrelerinin iki boyutlu (2D, monolayer) ve üç boyutlu (3D, spheroid) hücre kültürlerinde Everolimus, Metformin ve Lityum Klorür'ün tekli ve kombine tedavilerinin etkilerini incelemektir. Gereç ve yöntem: Çalışma kapsamında, Everolimus, Metformin ve Lityum Klorürün tekli ve kombine formlarının hücre canlılığı, invazyon, koloni oluşumu ve apoptoz ve PI3K/AKT/mTOR yolağı üzerindeki etkileri belirlendi. Hücre canlılığı XTT testi kullanılarak değerlendirilmiştir. CASP3, CASP8, CASP9, FASL, FADD, TNF, TRADD, BAX, P53, PI3KCA, PI3KCB, PTEN, MTOR, AKT1 genleri RT-PCR ile, apoptoz Flow sitometri ile ve 3D sferoid sonuçları invert mikroskop analizi ile değerlendirildi. Bulgular: Everolimus, metformin ve lityumun IC50 seviyeleri 48 saatte sırasıyla 37.46 nM, 48.59 mM ve 100 μM olarak bulundu. Tedavi gruplarındaki Ishikawa hücrelerinin invazyon kapasitelerinin yanı sıra hücre koloni oluşumunun da önemli ölçüde azaldığı tespit edilmiştir. Ayrıca, Ishikawa sferoid hücreleri kontrol gruplarına kıyasla önemli ölçüde baskılanmıştır. RT-PCR sonuçları, maddelerin ve kombinasyonlarının PI3K/AKT/mTOR yolağı ve apoptoz ile ilişkili genleri etkilediğini ortaya koymuştur. Flow sitometri sonuçları tekli ve kombine tedavilerin apoptozu belirgin şekilde arttırdığını göstermiştir. Sonuç: Sonuç olarak, everolimus, metformin ve lityumun tekli ve kombinasyon formları, Ishikawa hücrelerinde çeşitli mekanizmalar yoluyla hücre çoğalmasını azaltmış, apoptozu indüklemiş ve mTOR aktivasyonunu azaltmıştır. Bununla birlikte, çalışmamız tek başına Eve ve üçlü kombinasyon tedavisinin (Eve+Met+Lit) endometriyal kanser tedavisinde diğer tedavilerden daha etkili olduğunu göstermiştir.

Anahtar Kelimeler

• Endometriyal kanser
• PI3K/AKT/mTOR yolağı
• everolimus
• metformin
• lityum

Kaynakça

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