The Effect of 5-Florouracil and Resveratrol Combination on Apoptosis in A549 Human Lung Cancer Cell Line

Abstract

Objective: 5-Fluorouracil (5-FU) is a chemotherapy drug used in various cancers including breast, stomach, esophageal and pancreatic cancer. Resveratrol (RES) is a well known non-flavon polyphenolic compound that has chemotherapeutic effect in various types of cancer. Despite the fact that effects of 5-FU and RES alone have been extensively studied in the past few years, the effect of 5-FU combined with RES in the A549 cell line remains unknown. In this study, we investigated the effect of combination of RES and 5-FU on apoptotic pathway in A549 lung cancer cell line.

Material and Methods: The effect of of 5-FU and RES combination on A549 lung cancer cells was examined by cell viability using Water Soluble Tetrazolium-1 (WST-1) assays, by ROS level using 2’,7’–dichlorofluorescin diacetate (DCFDA) assay and by the ability of cells to form colonies by clonogenic assay, pro-apoptotic Bax and TIGAR protein expression using Western Blotting analysis.

Results: Combination of 5-FU and RES significantly decreased cell viability and clonogenic potential of A549 cell line. Furthermore, Western Blotting analysis showed that 5-FU and RES combination caused pro-apoptotic Bax protein to increase, while TIGAR protein expression level decreased in concentration-dependent manner.

Conclusion: Our results suggested that resveratrol can increase the anti-tumor effect of 5-FU in A549 cell line.

Keywords

• 5-Florouracil,Resveratrol,A549,TIGAR

5-FLOROURACİL VE RESVERATROL KOMBİNASYONUNUN İNSAN AKCİĞER KANSERİ A549 HÜCRE HATTINDA APOPTOZİS ÜZERİNE ETKİSİ

Abstract

Amaç: 5-Florourasil (5-FU), göğüs, mide, özofagus ve pankreas kanseri dahil olmak üzere çeşitli kanser türlerinde kullanılan bir kemoterapi ilacıdır. Resveratrol (RES), flavon olmayan polifenolik bir bileşiktir ve çeşitli kanser türlerinde kemoterapötik etkisi bulunmaktadır. 5-FU ve RES'in etkilerinin tek başına kapsamlı bir şekilde araştırılmasına rağmen, RES ile kombine edilen 5-FU'nun A549 hücre hattı üzerine etkisi hala bilinmemektedir. Bu çalışmada, A549 akciğer kanseri hücre hattında 5-FU ve RES kombinasyonunun apopitotik yolak üzerindeki etkisi araştırılmıştır.

Gereç ve Yöntemler: 5-FU ve RES kombinasyonunun A549 akciğer kanseri hücreleri üzerindeki etkisini belirlemek için, Water Soluble Tetrazolium-1 (WST-1) deneyi ile hücre canlılığı, 2’,7’–dichlorofluorescin diacetate (DCFDA) kiti ile ROS seviyesi, klonojenik deney ile hücrelerin koloni oluşturma kabiliyeti, Western blot ile pro-apoptotik Bax ve TP53 kaynaklı glikoliz ve apopitozis düzenleyicisi (TIGAR) proteinlerin ekspresyon düzeyleri analiz edilmiştir.

Bulgular: 5-FU ve RES kombinasyonu, A549 hücre hattının hücre canlılığını ve koloni oluşturma potansiyelini önemli ölçüde azaltmıştır. Dahası, Western Blot analizi, 5-FU ve RES kombinasyonunun pro-apopitotik Bax proteininin artmasına neden olduğunu, TIGAR protein ekspresyon seviyesinin ise konsantrasyona bağlı bir şekilde azaldığını göstermiştir.

Sonuç: Sonuçlarımız, Resveratrolün A549 hücre dizisinde 5-FU'nun anti-tümör etkisini artırabileceğini göstermiştir.

Keywords

• 5-Florouracil,Resveratrol,A549,TIGAR

References

1. 1. Fitzgerald JB, Schoeberl B, Nielsen UB, Sorger PK. Systems biology and combination therapy in the quest for clinical efficacy. Nat Cheml Biol. 2006;2(9):458-66.
2. 2. Wang W, Yang SM, Su Y, Xiao ZY, Wang CY, Li XF et al. Enhanced antitumor effect of combined triptolide and ionizing radiation. Clin Cancer Res. 2007;13(16):4891-9.
3. 3. Okon T, Schwartzberg L. Are we winning the war on cancer. J Oncol Pract. 2011;7(3):62-4.
4. 4. Ueda M, Kumagai K, Ueki K, Inoki C, Orino I, Ueki M. Growth inhibition and apoptotic cell death in uterine cervical carcinoma cells induced by 5-fluorouracil. Int J Cancer. 1997;71(4):668-74.
5. 5. Tsai CM, Hsiao SH, Frey CM, Chang KT, Perng RP, Gazdar AF et al. Combination cytotoxic effects of cis-diamminedichloroplatinum (II) and 5-fluorouracil with and without leucovorin against human non-small cell lung cancer cell lines. Cancer Res. 1993;53(5):1079-84.
6. 6. Stewart JR, Artime MC, O’Brian CA. Resveratrol: a candidate nutritional substance for prostate cancer prevention. J Nutr. 2003;133(7):2440-3.
7. 7. Aggarwal BB, Bhardwaj A, Aggarwal RS, Seeram NP, Shishodia S et al. Role of resveratrol in prevention and therapy of cancer: preclinical and clinical studies. Anticancer Res. 2004;24(5A):2783-840.
8. 8. Patel KR, Brown VA, Jones DJ, Britton RG, Hemingway D, Miller AS et al. Clinical pharmacology of resveratrol and its metabolites in colorectal cancer patients. Cancer Res. 2010;70(19):7392-9.

9. 9. Green DR. Apoptotic pathways: ten minutes to dead. Cell. 2005;121(5):671-4.
10. 10. Kim DH, Park KW, Chae IG, Kundu J, Kim EH, Kundu JK et al. Carnosic acid inhibits STAT3 signaling and induces apoptosis through generation of ROS in human colon cancer HCT116 cells. Mol Carcinog. 2016;55(6):1096-110.
11. 11. Gottlieb E, Vousden KH. p53 regulation of metabolic pathways. Cold Spring Harb Perspect Biol. 2010;2(4):1-14.
12. 12. Kondoh H, Lleonart ME, Gil J, Wang J, Degan P, Peters G et al. Glycolytic enzymes can modulate cellular life span. Cancer Res. 2005;65(1):177-85.
13. 13. Bensaad K, Tsuruta A, Selak MA, Vidal MN, Nakano K, Bartrons R et al. TIGAR, a p53-inducible regulator of glycolysis and apoptosis. Cell. 2006;126(1):107-20.
14. 14. Won KY, Lim SJ, Kim GY, Kim YW, Han SA, Song JY et al. Regulatory role of p53 in cancer metabolism via SCO2 and TIGAR in human breast cancer. Human Pathol. 2012;43(2):221-8.
15. 15. Oliva CR, Moellering DR, Gillespie GY, Griguer CE. Acquisition of chemoresistance in gliomas is associated with increased mitochondrial coupling and decreased ROS production. PLoS ONE. 2011;6(9):e24665.
16. 16. Tunç İ, Zergeroğlu S, Daylan B, Aydoğdu T, Mollamahmutoğlu L. Serviks Kanserlerinde BCL-2 ve BAX Protein Expresyonları ve Prognostik Faktörlerle Korelasyonu. Türk Jinekolojik Onkoloji Dergisi. 2004;7(4):140-8.
17. 17. Tseng YS, Tzeng CC, Chiu AWH, Lin CH, Won SJ, Wu IC et al. Ha-ras overexpression mediated cell apoptosis in the presense of 5-fluorouracil. Exp Cell Res. 2003;288(2):403-14.
18. 18. Huang CJ, Yang SH, Huang SM, Lin CM, Chien CC, Chen YC et al. A predicted protein, KIAA0247, is a cell cycle modulator in colorectal cancer cell sunder 5-FU treatment. J Transl Med. 2011;9(1):82-9.
19. 19. Garvin S, Ollinger K, Dabrosin C. Resveratrol induces apoptosis and inhibits angiogenesiss in human breast cancer xenografts in vivo. Cancer Lett. 2006;231(1):113-22.
20. 20. Emmett MS, Dewing D, Pritchard-Jones RO. Angiogenesis and melanoma-from basic science to clinical trials. Am J Cancer Res. 2011;1(7):852-68.
21. 21. Hwang JT, Ha J, Park, OJ. Combination of 5‑fluorouracil and genistein induces apoptosis synergistically in chemo‑resistant cancer cells through the modulation of AMPK and COX‑2 signaling pathways. Biochem Biophys Res Commun. 2005;332(2):434-40.
22. 22. Colin D, Gimazane A, Lizard G, Izard JC, Solary E, Latruffe N et al. Effects of resveratrol analogs on cell cycle progression, cell cycle associated protein sand 5fluorouracil sensitivity in human derived colon cancer cells. Int J Cancer. 2009;124(12):2780-8.
23. 23. Lee SH, Koo BS, Park S.Y, Kim YM. Anti-angiogenic effects of resveratrol in combination with 5-fluorouracil on B16 murine melanoma cells. Mol Med Rep. 201512(2):2777-83.
24. 24. Bensaad K, Cheung E C, Vousden K H. Modulation of intracellular ROS levels by TIGAR controls autophagy. EMBO J. 2009;28(19):3015-26.
25. 25. Landriscina M, Maddalena F, Laudiero G, Esposito F. Adaptation to oxidative stress, chemoresistance, and cell survival. Antioxid Redox Signal. 2009;11(11):2701-16.
26. 26. Kumar B, Iqbal MA, Singh RK, Bamezai RN. Resveratrol inhibits TIGAR to promote ROS induced apoptosis and autophagy. Biochimie. 2015;118:26-35.
27. 27. Yu H, Xie J, Li B, Sun Y, Gao Q, Ding Z et al. TIGAR regulates DNA damage and repair through pentose phosphate pathway and Cdk5-ATM pathway. Sci Rep. 2015;5:9853.
28. 28. Zhao M, Fan J, Liu Y, Yu Y, Xu J, Wen Q. Oncogenic role of the TP53-induced glycolysis and apoptosis regulator in nasopharyngeal carcinoma through NF-κB pathway modulation. Int J Oncol. 2016;48(2):756-64.
29. 29. Gross A, McDonnell JM, Korsmeyer SJ. BCL-2 family members and the mitochondria in apoptosis. Genes Dev. 1999;13:1899-911.
30. 30. Broughton BR, Reutens DC, Sobey CG. Apoptotic mechanisms after cerebral ischemia. Stroke. 2009;40(5):331-9.
31. 31. Hildeman DA, Mitchell T, Aronow B, Wojciechowski S, Kappler J, Marrack P. Control of Bcl-2 expression by reactive oxygen species. Proc Natl Acad Sci USA. 2003;100(25):15035-40.
32. 32. Li D, Ueta E, Kimura T, Yamamoto T, Osaki T. Reactive oxygen species (ROS) control the expression of Bcl-2 family proteins by regulating their phosphorylation and ubiquitination. Cancer Sci. 2004;95(8):644-50.
33. 33. Luanpitpong S, Chanvorachote P, Stehlik C, Tse W, Callery PS, Wang L. Regulation of apoptosis by Bcl-2 cysteine oxidationin human lung epithelial cells. Mol Biol Cell. 2013;24(6):858-69.