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

Year 2023, Volume: 30 Issue: 4, 672 - 679, 30.12.2023

Ali Haydar Kaygusuz Fatma Sogutlu Çığır Biray Avcı

https://doi.org/10.17343/sdutfd.1361012

Abstract

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.

Keywords

Epigallocatechin gallate, Gemcitabine, microRNA, Pancreatic cancer

Ethical Statement

Yok

Supporting Institution

Bilimsel Araştırma Proje (BAP) Ege Üniversitesi

Project Number

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

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

Abstract

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.

Keywords

Epigallokateşin gallat, Gemsitabin, mikroRNA, Pankreas kanseri

Project Number

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

References

• 1. Sung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, et al. Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA Cancer J Clin. 2021;71(3):209-49.
• 2. Lu CH, Chen WT, Hsieh CH, Kuo YY, Chao CY. Thermal cycling- hyperthermia in combination with polyphenols, epigallocatechin gallate and chlorogenic acid, exerts synergistic anticancer effect against human pancreatic cancer PANC-1 cells. PLoS One 2019;14(5):e0217676.
• 3. Jin J, Teng C, Li T. Combination therapy versus gemcitabine monotherapy in the treatment of elderly pancreatic cancer: a meta-analysis of randomized controlled trials. Drug Des Devel Ther. 2018;12:475-80.
• 4. Castañeda AM, Meléndez CM, Uribe D, Pedroza-Díaz J. Synergistic effects of natural compounds and conventional chemotherapeutic agents: recent insights for the development of cancer treatment strategies. Heliyon. 2022;8(6):e09519.
• 5. Amrutkar M, Gladhaug IP. Pancreatic Cancer Chemoresistance to Gemcitabine. Cancers (Basel). 2017;9(11).
• 6. Gan RY, Li HB, Sui ZQ, Corke H. Absorption, metabolism, anti-cancer effect and molecular targets of epigallocatechin gallate (EGCG): An updated review. Crit Rev Food Sci Nutr. 2018;58(6):924-41.
• 7. Azimi H, Khakshur AA, Abdollahi M, Rahimi R. Potential New Pharmacological Agents Derived From Medicinal Plants for the Treatment of Pancreatic Cancer. Pancreas. 2015;44(1):11-5.
• 8. Wei R, Penso NEC, Hackman RM, Wang Y, Mackenzie GG. Epigallocatechin-3-Gallate (EGCG) Suppresses Pancreatic Cancer Cell Growth, Invasion, and Migration partly through the Inhibition of Akt Pathway and Epithelial-Mesenchymal Transition: Enhanced Efficacy when Combined with Gemcitabine. Nutrients. 2019;11(8).
• 9. Wei R, Hackman RM, Wang Y, Mackenzie GG. Targeting Glycolysis with Epigallocatechin-3-Gallate Enhances the Efficacy of Chemotherapeutics in Pancreatic Cancer Cells and Xenografts. Cancers (Basel). 2019;11(10).
• 10. Wei R, Wirkus J, Yang Z, Machuca J, Esparza Y, Mackenzie GG. EGCG sensitizes chemotherapeutic-induced cytotoxicity by targeting the ERK pathway in multiple cancer cell lines. Arch Biochem Biophys. 2020;692:108546.
• 11. Namima D, Fujihara S, Iwama H, Fujita K, Matsui T, Nakahara M, et al. The Effect of Gemcitabine on Cell Cycle Arrest and microRNA Signatures in Pancreatic Cancer Cells. In Vivo. 2020;34(6):3195-203.
• 12. Kanteti R, Batra SK, Lennon FE, Salgia R. FAK and paxillin, two potential targets in pancreatic cancer. Oncotarget. 2016;7(21):31586-601.
• 13. Martinelli P, Carrillo-de Santa Pau E, Cox T, Sainz B, Jr., Dusetti N, Greenhalf W, et al. GATA6 regulates EMT and tumour dissemination, and is a marker of response to adjuvant chemotherapy in pancreatic cancer. Gut. 2017;66(9):1665-76.
• 14. Park YG, Lee KH, Lee JK, Lee KT, Choi DW, Choi SH, et al. [MicroRNA expression pattern in intraductal papillary mucinous neoplasm]. Korean J Gastroenterol. 2011;58(4):190-200.
• 15. Fan J, Li H, Nie X, Yin Z, Zhao Y, Chen C, et al. MiR-30c- 5p ameliorates hepatic steatosis in leptin receptor-deficient (db/db) mice via down-regulating FASN. Oncotarget. 2017;8(8):13450-63.
• 16. Lu Y, Wu X, Wang J. Correlation of miR-425-5p and IL-23 with pancreatic cancer. Oncol Lett. 2019;17(5):4595-9.
• 17. Permuth-Wey J, Chen YA, Fisher K, McCarthy S, Qu X, Lloyd MC, et al. A genome-wide investigation of microRNA expression identifies biologically-meaningful microRNAs that distinguish between high-risk and low-risk intraductal papillary mucinous neoplasms of the pancreas. PLoS One. 2015;10(1):e0116869.
• 18. Fukuhisa H, Seki N, Idichi T, Kurahara H, Yamada Y, Toda H, et al. Gene regulation by antitumor miR-130b-5p in pancreatic ductal adenocarcinoma: the clinical significance of oncogenic EPS8. J Hum Genet. 2019;64(6):521-34.
• 19. Khan MA, Zubair H, Srivastava SK, Singh S, Singh AP. Insights into the Role of microRNAs in Pancreatic Cancer Pathogenesis: Potential for Diagnosis, Prognosis, and Therapy. Adv Exp Med Biol. 2015;889:71-87.
• 20. Wang W, Liu B, Sun S, Lan L, Chen Y, Han S, et al. Downregulation of miR-486-5p Enhances the Anti-Tumor Effect of 5-Fluorouracil on Pancreatic Cancer Cells. Onco Targets Ther. 2020;13:1649-59.
• 21. Wu M, Li W, Huang F, Sun J, Li KP, Shi J, et al. Comprehensive Analysis of the Expression Profiles of Long Non-Coding RNAs with Associated ceRNA Network Involved in the Colon Cancer Staging and Progression. Sci Rep. 2019;9(1):16910.
• 22. Fan MJ, He PJ, Lin XY, Yang CR, Li CZ, Xing LG. MicroRNA- 324-5p affects the radiotherapy response of cervical cancer via targeting ELAV-like RNA binding protein 1. Kaohsiung J Med Sci. 2020;36(12):965-72.