EPIGALLOCATECHIN GALLATE SENSITIZES PANCREATIC CANCER CELLS TO GEMCITABINE BY MODULATING MICRORNA EXPRESSION PROFILE

Öz

Pancreatic cancer is a leading cause of cancer-related deaths in developed countries, with a 5-year average survival rate of less than 5%, making it a malignant disease. Gemcitabine (GEM), an FDA-approved pyrimidine antimetabolite, is widely used in pancreatic cancer treatment. However, due to its targeting of all dividing cells, severe side effects are frequently observed in patients undergoing GEM treatment for pancreatic cancer. Consequently, meta-analyses have shown that the combination of GEM with other active compounds significantly improves the 1-year survival rate of pancreatic cancer patients. Epigallocatechin- 3-gallate (EGCG), an active compound found in green tea (Camellia sinensis), has proven anticancer activity in pancreatic cancer. Subsequent studies have demonstrated that EGCG enhances the sensitivity of pancreatic cancer cells to GEM. However, among the studies conducted to date, the impact of the combination of EGCG and GEM on the expression of critical microRNAs, which act as key epigenetic regulators in pancreatic cancer pathology, has not been investigated. This study aims to determine the cytotoxic and apoptotic effects of the combination of GEM and EGCG on PANC1 cells and to examine its effectiveness on the expression levels of microRNAs involved in cancer progression. Material and Method Cytotoxicity of GEM and EGCG in PANC1 cells was assessed using the WST-1 assay, and combination effects were analyzed using isobologram analysis. Apoptosis analysis was performed using the Annexin V method. miRNA isolation was conducted with the miRNeasy Kit, followed by cDNA synthesis using the miScript II Reverse Transcription Kit. Changes in the expression of miRNAs involved in cancer cell proliferation, apoptosis, and metastasis were examined using real-time qRT-PCR analysis. Results The IC50 values for GEM at 24, 48, and 72 hours were determined as 72.85 μM, 26.55 μM, and 9.38 μM, respectively. EGCG's IC50 values at 24, 48, and 72 hours were determined as 64.36 μM, 48.34 μM, and 19.73 μM, respectively. When combined at a 2:3 ratio (GEM: EGCG) at 24 and 72 hours, a synergistic effect was observed, while at 48 hours, a strong synergistic drug interaction was observed. At a concentration of only 26.55 μM, the group treated with GEM showed a 4.2-fold increase in apoptosis compared to the control group receiving fresh medium. In contrast, the combination treatment (EGCG: 4.71 μM, GEM: 3.14 μM) resulted in a remarkable 12.04-fold increase in apoptosis. After combination treatment, the expression of tumor suppressor miRNAs, miR-137, and miR-130a-3p, increased, while the expression of oncogenic miRNAs, including miR-27a-3p, miR-425- 5p, miR-183-5p, miR-187-3p, miR-21-5p, miR-324-5p, and miR-486-5p, decreased. Conclusion EGCG can sensitize pancreatic cancer to GEM through epigenetic mechanisms, shedding light on novel therapeutic approaches.

Anahtar Kelimeler

• Epigallocatechin gallate
• Gemcitabine
• microRNA
• Pancreatic cancer

EPİGALLOKATEŞİN GALLAT, PANKREAS KANSER HÜCRELERİNİ GEMSİTABİN’E KARŞI MİKRORNA İFADE PROFİLİNİ DEĞİŞTİREREK HASSASLAŞTIRMAKTADIR

Öz

Amaç Pankreas kanser, gelişmiş ülkelerde kansere bağlı ölümlerin önde gelen nedenlerinden biri olup, 5 yıllık ortalama sağkalım oranının %5'ten az olduğu malign bir hastalıktır. Gemsitabin (GEM), FDA onaylı bir pirimidin antimetaboliti olup, pankreas kanser tedavisinde yaygın olarak kullanılmaktadır. Ancak, tüm bölünen hücreleri hedef alması sebebiyle, GEM tedavisi gören pankreas kanseri hastalarında ciddi yan etkiler sıklıkla gözlemlenmektedir. Sonuç olarak, meta-analizler, GEM'in diğer aktif bileşiklerle kombinasyonunun, pankreas kanser hastalarının 1 yıllık sağkalım oranını önemli ölçüde artırdığını göstermiştir. Epigallokateşin- 3-gallat (EGCG), yeşil çayda (Camellia sinensis) bulunan aktif bir bileşik olup, pankreas kanserinde antikanser aktivitesi kanıtlanmıştır. Devamındaki çalışmalarda da EGCG’nin pankreas kanseri hücrelerinin GEM’e karşı hassasiyetini arttırdığı gösterilmiştir. Ancak, bugüne kadar yapılan çalışmalar arasında, EGCG ve GEM kombinasyonunun pankreatik kanser patolojisinde kritik bir epigenetik düzenleyici olan mikroRNA ifadesinin üzerine etkisi incelenmemiştir. Bu çalışmada, GEM ve EGCG kombinasyonunun PANC1 hücrelerindeki sitotoksik ve apoptotik etkilerinin belirlemesi ve kanser progresyonunda rol oynayan mikroRNA'ların ifade düzeyleri üzerindeki etkinliğinin incelenmesi amaçlanmıştır. Gereç ve Yöntem GEM ve EGCG'nin PANC1 hücrelerindeki sitotoksisitesi WST-1 testi kullanılarak değerlendirildi ve kombinasyon etkileri izobologram analizi kullanılarak analiz edildi. Apoptoz analizi, Annexin V yöntemi kullanılarak yapıldı. MikroRNA izolasyonu miRNeasy Kiti ile gerçekleştirildi ve miScript II Ters Transkriptaz Kiti kullanılarak cDNA sentezi yapıldı. Kanser hücresi proliferasyonu, apoptozis ve metastazda rol oynayan mikroRNA'ların ifadesindeki değişiklikler, gerçek zamanlı qRT-PCR analizi kullanılarak incelendi. Bulgular GEM'in 24, 48 ve 72 saatteki IC50 değerleri sırasıyla 72.85 μM, 26.55 μM ve 9.38 μM olarak belirlendi. EGCG'nin 24, 48 ve 72 saatteki IC50 değerleri sırasıyla 64.36 μM, 48.34 μM ve 19.73 μM olarak belirlendi. 24 ve 72 saatte GEM:EGCG oranı 2:3 olarak birleştirildiğinde sinerjistik bir etki gözlemlenirken, 48 saatte güçlü sinerjistik bir ilaç etkileşimi gözlendi. Sadece 26.55 μM konsantrasyonda GEM ile tedavi edilen grupta taze ortam verilen kontrol grubuna kıyasla apoptozda 4.2 kat artış meydana geldiği saptanırken, kombinasyon uygulaması (EGCG: 3.14 μM, GEM: 4.71 μM) apoptozda 12.04 kat artışa neden olmuştur. Kombinasyon tedavisi sonrası, tümör baskılayıcı mikroRNA'ların, miR-137 ve miR-130a-3p, ifadesi artarken, onkogenik mikroRNA'ların, miR-27a-3p, miR- 425-5p, miR-183-5p, miR-187-3p, miR-21-5p, miR- 324-5p ve miR-486-5p, ifadesi azaldı. Sonuç EGCG, pankreatik kanseri GEM'e epigenetik mekanizmalar aracılığıyla hassaslaştırabilir ve yeni terapötik yaklaşımların ışığını göstermektedir.

Anahtar Kelimeler

• Epigallokateşin gallat
• Gemsitabin
• mikroRNA
• Pankreas kanseri

Kaynakça

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