sdü tıp fak derg

Medical Journal of Süleyman Demirel University

2602-2109

Süleyman Demirel Üniversitesi

10.17343/sdutfd.1361012

Cell Development, Proliferation and Death Epigenetics

Hücre Gelişimi, Proliferasyon ve Ölümü Epigenetik

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

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

https://orcid.org/0000-0002-5139-0411

Kaygusuz

Ali Haydar

EGE ÜNİVERSİTESİ, EGE TIP FAKÜLTESİ

https://orcid.org/0000-0002-1210-7660

Sogutlu

Fatma

EGE ÜNİVERSİTESİ, EGE TIP FAKÜLTESİ

https://orcid.org/0000-0001-8251-4520

Biray Avcı

Çığır

EGE ÜNİVERSİTESİ, TIP FAKÜLTESİ

12 30 2023

30 4 672 679 09 15 2023 12 18 2023

1994

Süleyman Demirel Üniversitesi Tıp Fakültesi Dergisi

Pancreatic cancer is a leading cause of cancer-relateddeaths in developed countries, with a 5-year averagesurvival rate of less than 5%, making it a malignantdisease. Gemcitabine (GEM), an FDA-approvedpyrimidine antimetabolite, is widely used in pancreaticcancer treatment. However, due to its targeting ofall dividing cells, severe side effects are frequentlyobserved in patients undergoing GEM treatment forpancreatic cancer. Consequently, meta-analyses haveshown that the combination of GEM with other activecompounds significantly improves the 1-year survivalrate of pancreatic cancer patients. Epigallocatechin-3-gallate (EGCG), an active compound found ingreen tea (Camellia sinensis), has proven anticanceractivity in pancreatic cancer. Subsequent studies havedemonstrated that EGCG enhances the sensitivity ofpancreatic cancer cells to GEM. However, amongthe studies conducted to date, the impact of thecombination of EGCG and GEM on the expressionof critical microRNAs, which act as key epigeneticregulators in pancreatic cancer pathology, has notbeen investigated. This study aims to determine thecytotoxic and apoptotic effects of the combination ofGEM and EGCG on PANC1 cells and to examine itseffectiveness on the expression levels of microRNAsinvolved in cancer progression.Material and MethodCytotoxicity of GEM and EGCG in PANC1 cells wasassessed using the WST-1 assay, and combinationeffects were analyzed using isobologram analysis.Apoptosis analysis was performed using the AnnexinV method. miRNA isolation was conducted with themiRNeasy Kit, followed by cDNA synthesis usingthe miScript II Reverse Transcription Kit. Changesin the expression of miRNAs involved in cancercell proliferation, apoptosis, and metastasis wereexamined using real-time qRT-PCR analysis.ResultsThe IC50 values for GEM at 24, 48, and 72 hours weredetermined as 72.85 μM, 26.55 μM, and 9.38 μM,respectively. EGCG's IC50 values at 24, 48, and 72hours were determined as 64.36 μM, 48.34 μM, and19.73 μM, respectively. When combined at a 2:3 ratio(GEM: EGCG) at 24 and 72 hours, a synergistic effectwas observed, while at 48 hours, a strong synergisticdrug interaction was observed. At a concentration ofonly 26.55 μM, the group treated with GEM showeda 4.2-fold increase in apoptosis compared to thecontrol group receiving fresh medium. In contrast,the combination treatment (EGCG: 4.71 μM, GEM:3.14 μM) resulted in a remarkable 12.04-fold increasein apoptosis. After combination treatment, theexpression of tumor suppressor miRNAs, miR-137,and miR-130a-3p, increased, while the expression ofoncogenic 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.ConclusionEGCG can sensitize pancreatic cancer to GEMthrough epigenetic mechanisms, shedding light onnovel therapeutic approaches.

AmaçPankreas kanser, gelişmiş ülkelerde kansere bağlıölümlerin önde gelen nedenlerinden biri olup, 5 yıllıkortalama sağkalım oranının %5'ten az olduğu malignbir hastalıktır. Gemsitabin (GEM), FDA onaylı bir pirimidinantimetaboliti olup, pankreas kanser tedavisindeyaygın olarak kullanılmaktadır. Ancak, tüm bölünenhücreleri hedef alması sebebiyle, GEM tedavisi görenpankreas kanseri hastalarında ciddi yan etkiler sıklıklagözlemlenmektedir. Sonuç olarak, meta-analizler,GEM'in diğer aktif bileşiklerle kombinasyonunun,pankreas kanser hastalarının 1 yıllık sağkalım oranınıönemli ölçüde artırdığını göstermiştir. Epigallokateşin-3-gallat (EGCG), yeşil çayda (Camellia sinensis)bulunan aktif bir bileşik olup, pankreas kanserindeantikanser aktivitesi kanıtlanmıştır. Devamındaki çalışmalardada EGCG’nin pankreas kanseri hücrelerininGEM’e karşı hassasiyetini arttırdığı gösterilmiştir.Ancak, bugüne kadar yapılan çalışmalar arasında,EGCG ve GEM kombinasyonunun pankreatik kanserpatolojisinde kritik bir epigenetik düzenleyici olanmikroRNA ifadesinin üzerine etkisi incelenmemiştir.Bu çalışmada, GEM ve EGCG kombinasyonununPANC1 hücrelerindeki sitotoksik ve apoptotik etkilerininbelirlemesi ve kanser progresyonunda rol oynayanmikroRNA'ların ifade düzeyleri üzerindeki etkinliğininincelenmesi amaçlanmıştır.Gereç ve YöntemGEM ve EGCG'nin PANC1 hücrelerindeki sitotoksisitesiWST-1 testi kullanılarak değerlendirildi vekombinasyon etkileri izobologram analizi kullanılarakanaliz edildi. Apoptoz analizi, Annexin V yöntemikullanılarak yapıldı. MikroRNA izolasyonu miRNeasyKiti ile gerçekleştirildi ve miScript II Ters TranskriptazKiti kullanılarak cDNA sentezi yapıldı. Kanser hücresiproliferasyonu, apoptozis ve metastazda rol oynayanmikroRNA'ların ifadesindeki değişiklikler, gerçek zamanlıqRT-PCR analizi kullanılarak incelendi.BulgularGEM'in 24, 48 ve 72 saatteki IC50 değerleri sırasıyla72.85 μM, 26.55 μM ve 9.38 μM olarak belirlendi.EGCG'nin 24, 48 ve 72 saatteki IC50 değerleri sırasıyla64.36 μM, 48.34 μM ve 19.73 μM olarak belirlendi.24 ve 72 saatte GEM:EGCG oranı 2:3 olarakbirleştirildiğinde sinerjistik bir etki gözlemlenirken, 48saatte güçlü sinerjistik bir ilaç etkileşimi gözlendi. Sadece26.55 μM konsantrasyonda GEM ile tedavi edilengrupta taze ortam verilen kontrol grubuna kıyaslaapoptozda 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 baskılayıcımikroRNA'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.SonuçEGCG, pankreatik kanseri GEM'e epigenetik mekanizmalararacılığıyla hassaslaştırabilir ve yeni terapötikyaklaşımların ışığını göstermektedir.

Epigallocatechin gallate Gemcitabine microRNA Pancreatic cancer

Epigallokateşin gallat Gemsitabin mikroRNA Pankreas kanseri

Bilimsel Araştırma Proje (BAP) Ege Üniversitesi

Ege University Research Foundation (Project no: TYL-2019-20764)

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