Objectives: This study evaluated the combined effects of protocatechuic acid (PCA) and 5-fluorouracil (5-FU) on gastric adenocarcinoma (AGS)cells.Materials and Methods: The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, colony formation assay, flow cytometry technique,real-time quantitative polymerase chain reaction, and Western blotting were used to investigate cytotoxic effects, colony formation, apoptosis, p53gene expression, and Bcl-2 protein level in AGS cells treated with 5-FU and PCA.Results: Our results demonstrated that PCA (500 µM) alone or in combination with 5-FU (10 µM) inhibited AGS cell proliferation, inhibited a colonyformation, and increased apoptosis compared with untreated control cells. Moreover, the combined 5-FU/PCA exposure led to upregulation of p53and downregulation of Bcl-2 protein when compared to the untreated control cells.Conclusion: The results demonstrate that the combined 5-FU/PCA may promote antiproliferative and pro-apoptotic effects with the inhibition ofcolony formation in AGS cells. The mechanisms by which the combined 5-FU/PCA exposure exerts its effects are associated with upregulation ofp53 gene expression and downregulation of Bcl-2 level. Therefore, the combination of 5-FU with PCA not only could be a promising approach topotentially reduce the dose requirements of 5-FU but also could promote apoptosis via p53 and Bcl-2 signaling pathways.Key words: Apoptosis, 5-fluorouracil, protocatechuic acid, gastric cancer, combination, colony formation
Amaç: Bu çalışma protokateşuik asit (PCA) ve 5-florourasilin (5-FU) gastrik adenokarsinoma (AGS) hücreleri üzerine kombine etkisini değerlendirmiştir. Gereç ve Yöntemler: 3-(4,5-dimetiltiazol-2-il)-2,5-difeniltetrazolium bromid yöntemi, koloni oluşturma yöntemi, akış sitometrisi tekniği, gerçek zamanlı kantitatif polimeraz zincir reaksiyonu ve Western blot yöntemleri 5-FU ve PCA’ya maruz kalan AGS hücrelerinde sitotoksisite, koloni oluşumu, apoptoz, p53 gen ekspresyonu ve Bcl-2 düzeylerini belirlemek için kullanılmıştır. Bulgular: Sonuçlarımız PCA’nın tek başına (500 µM) veya 5-FU (10 µM) ile kombinasyonunun uygulama yapılmamış kontrol hücrelerine göre AGS hücre proliferasyonunu ve koloni oluşturmunu inhibe ettiğini ve apoptozu artırdığını göstermiştir. Ek olarak, uygulama yapılmamış kontrol hücrelerine göre kombine 5-FU/PCA maruziyeti p53 düzeylerini artırmış ve Bcl-2 düzeylerini artırmıştır. Sonuç: Sonuçlar göstermiştir ki AGS hücrelerinde kombine 5-FU/PCA maruziyeti koloni oluşumunu inhibe ederek antiproliferatif ve pro-apoptotik etkiler başlatabilir. Kombine 5-FU/PCA maruziyetinin etki gösterme mekanizması p53 geninin artması ve Bcl-2 geninin azalması ile ilişkili olabilir.
1. Teng H, Huang Q, Chen L. Inhibition of cell proliferation and triggering of apoptosis by agrimonolide through MAP kinase (ERK and p38) pathways in human gastric cancer AGS cells. Food Funct. 2016;7:4605- 4613.
2. Mohammadi M, Zarghami N, Hedayati M, Ghaemmaghami S. Synergistic effects of resistin and visfatin as adipocyte derived hormones on telomerase gene expression in AGS gastric cancer cell line. Acta Med Iran. 2017;55:621-627.
3. Zheng LC, Yang MD, Kuo CL, Lin CH, Fan MJ, Chou YC, Lu HF, Huang WW, Peng SF, Chung JG. Norcantharidin-induced apoptosis of AGS human gastric cancer cells through reactive oxygen species production, and caspase- and mitochondria-dependent signaling pathways. Anticancer Res. 2016;36:6031-6042.
4. Noordhuis P, Holwerda U, Van der Wilt C, Van Groeningen C, Smid K, Meijer S, Pinedo HM. 5-Fluorouracil incorporation into RNA and DNA in relation to thymidylate synthase inhibition of human colorectal cancers. Ann Oncol. 2004;15:1025-1032.
5. Wagner AD, Grothe W, Haerting J, Kleber G, Grothey A, Fleig WE. Chemotherapy in advanced gastric cancer: a systematic review and metaanalysis based on aggregate data. J Clin Oncol. 2006;24:2903-2909.
6. Hauner K, Maisch P, Retz M. Nebenwirkungen der Chemotherapie. Der Urologe. 2017;56:472-479.
7. de Oliveira Júnior RG, Ferraz CAA, da Silva Almeida JRG, Grougnet R, Thiéry V, Picot L. Sensitization of tumor cells to chemotherapy by natural products: a systematic review of preclinical data and molecular mechanisms. Fitoterapia. 2018;21.
8. Sato Y, Tsurumi T. Genome guardian p53 and viral infections. Rev Med Virol. 2013;23:213-220.
9. Jiang L, Sheikh MS, Huang Y. Decision making by p53: life versus death. Mol Cell Pharmacol. 2010;2:69-77.
10. Fresco P, Borges F, Diniz C, Marques M. New insights on the anticancer properties of dietary polyphenols. Med Res Rev. 2006;26:747-766.
11. Sak K. Cytotoxicity of dietary flavonoids on different human cancer types. Pharmacogn Rev. 2014;8:122-146.
12. Tanaka T, Kojima T, Kawamori T, Mori H. Chemoprevention of digestive organs carcinogenesis by natural product protocatechuic acid. Cancer. 1995;75:1433-1439.
13. Khan AK, Rashid R, Fatima N, Mahmood S, Mir S, Khan S, Jabeen N, Murtaza G. Pharmacological activities of protocatechuic acid. Acta Pol Pharm. 2015;72:643-650.
14. Masella R, Santangelo C, D’archivio M, LiVolti G, Giovannini C, Galvano F. Protocatechuic acid and human disease prevention: biological activities and molecular mechanisms. Curr Med Chem. 2012;19:2901- 2917.
15. Yoshimizu N, Otani Y, Saikawa Y, Kubota T, Yoshida M, Furukawa T, Kumai K, Kameyama K, Fujii M, Yano M, Sato T, Ito A, Kitajima M. Anti-tumour effects of nobiletin, a citrus flavonoid, on gastric cancer include: antiproliferative effects, induction of apoptosis and cell cycle deregulation. Aliment Pharmacol Ther. 2004;1:95-101.
16. Kapp N, Stander XX, Stander BA. Synergistic in vitro effects of combining an antiglycolytic, 3-bromopyruvate, and a bromodomain-4 inhibitor on U937 myeloid leukemia cells. Anticancer Drugs. 2018;29:429-439.
17. Koraneekit A, Limpaiboon T, Sangka A, Boonsiri P, Daduang S, Daduang J. Synergistic effects of cisplatin-caffeic acid induces apoptosis in human cervical cancer cells via the mitochondrial pathways. Oncol Lett. 2018;15:7397-7402.
18. Franken NA, Rodermond HM, Stap J, Haveman J, Van Bree C. Clonogenic assay of cells in vitro. Nat Protoc. 2006;1:2315-2319.
19. Pauzi AZM, Yeap SK, Abu N, Lim KL, Omar AR, Aziz SA, Chow ALT, Subramani T, Tan SG, Alitheen NB. Combination of cisplatin and bromelain exerts synergistic cytotoxic effects against breast cancer cell line MDA-MB-231 in vitro. Chin Med. 2016;11:46.
20. Sun Y, Zhang D, Mao M, Lu Y, Jiao N. Roles of p38 and JNK protein kinase pathways activated by compound cantharidin capsules containing serum on proliferation inhibition and apoptosis of human gastric cancer cell line. Exp Ther Med. 2017;14:1809-1817.
21. Heidarian E, Keloushadi M, Ghatreh-Samani K, Jafari-Dehkordi E. Gallic acid inhibits invasion and reduces IL-6 gene expression, pSTAT3, pERK1/2, and pAKT cellular signaling proteins in human prostate cancer DU-145 cells. Int J Cancer Manag. 2017;10:e9163.
22. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976;72:248-254.
23. Mahlberg R, Lorenzen S, Thuss-Patience P, Heinemann V, Pfeiffer P, Mohler M. New perspectives in the treatment of advanced gastric cancer: S-1 as a novel oral 5-FU therapy in combination with cisplatin. Chemotherapy. 2017;62:62-70.
24. Longley DB, Harkin DP, Johnston PG. 5-fluorouracil: mechanisms of action and clinical strategies. Nat Rev Cancer. 2003;3:330-338.
25. Sun W, Sanderson PE, Zheng W. Drug combination therapy increases successful drug repositioning. Drug Discov Today. 2016;21:1189-1195.
26. Yin MC, Lin CC, Wu HC, Tsao SM, Hsu CK. Apoptotic effects of protocatechuic acid in human breast, lung, liver, cervix, and prostate cancer cells: potential mechanisms of action. J Agric Food Chem. 2009;57:6468-6473.
27. Tsao SM, Hsia TC, Yin MC. Protocatechuic acid inhibits lung cancer cells by modulating FAK, MAPK, and NF-kappaB pathways. Nutr Cancer. 2014;66:1331-1341.
28. Shukla S, Mehta A. Anticancer potential of medicinal plants and their phytochemicals: a review. Braz J Bot. 2015;38:199-210.
29. Satelli A, Li S. Vimentin in cancer and its potential as a molecular target for cancer therapy. Cell Mol Life Sci. 2011;68:3033-3046.
30. Hour TC, Chen J, Huang CY, Guan JY, Lu SH, Pu YS. Curcumin enhances cytotoxicity of chemotherapeutic agents in prostate cancer cells by inducing p21WAF1/CIP1 and C/EBPβ expressions and suppressing NFkappaB activation. Prostate. 2002;51:211-218.
31. Fulda S, Debatin KM. Sensitization for anticancer drug-induced apoptosis by the chemopreventive agent resveratrol. Oncogene. 2004;23:6702-6711.
32. Braicu C, Gherman CD, Irimie A, Berindan-Neagoe I. Epigallocatechin3-Gallate (EGCG) inhibits cell proliferation and migratory behaviour of triple negative breast cancer cells. J Nanosci Nanotechnol. 2013;13:632- 637.
33. He B, Wei W, Liu J, Xu Y, Zhao G. Synergistic anticancer effect of curcumin and chemotherapy regimen FP in human gastric cancer MGC-803 cells. Oncol Lett. 2017;14:3387-3394.
34. Zhang L, Chen H, Wang M, Song X, Ding F, Zhu J, Liu X. Effects of glabridin combined with 5-fluorouracil on the proliferation and apoptosis of gastric cancer cells. Oncol Lett. 2018;15:7037-7045.
35. Guan S, Jiang B, Bao Y, An L. Protocatechuic acid suppresses MPP+- induced mitochondrial dysfunction and apoptotic cell death in PC12 cells. Food Chem Toxicol. 2006;44:1659-1666.
36. Liu YM, Jiang B, Bao YM, An LJ. Protocatechuic acid inhibits apoptosis by mitochondrial dysfunction in rotenone-induced PC12 cells. Toxicol In Vitro. 2008;22:430-437.
37. Lin HH, Chen JH, Huang CC, Wang CJ. Apoptotic effect of 3, 4-dihydroxybenzoic acid on human gastric carcinoma cells involving JNK/p38 MAPK signaling activation. Int J Cancer. 2007;120:2306-2316.
38. Tseng TH, Kao TW, Chu CY, Chou FP, Lin WL, Wang CJ. Induction of apoptosis by hibiscus protocatechuic acid in human leukemia cells via reduction of retinoblastoma (RB) phosphorylation and Bcl-2 expression. Biochem Pharmacol. 2000;60:307-315.
39. Petak I, Tillman DM, Houghton JA. p53 dependence of Fas induction and acute apoptosis in response to 5-fluorouracil-leucovorin in human colon carcinoma cell lines. Clin Cancer Res. 2000;6:4432-5541.
40. Bunz F, Hwang PM, Torrance C, Waldman T, Zhang Y, Dillehay L, Williams J, Lengauer C, Kinzler KW, Vogelstein B. Disruption of p53 in human cancer cells alters the responses to therapeutic agents. J Clin Invest. 1999;104:263-269.
41. Zhang L, Yu J, Park BH, Kinzler KW, Vogelstein B. Role of BAX in the apoptotic response to anticancer agents. Science. 2000;290:989-992.
42. Xu GY, Tang XJ. Troxerutin (TXN) potentiated 5-Fluorouracil (5- Fu) treatment of human gastric cancer through suppressing STAT3/ NF-kappaB and Bcl-2 signaling pathways. Biomed Pharmacother. 2017;92:95-107.
43. Wagner H, Ulrich-Merzenich G. Synergy research: approaching a new generation of phytopharmaceuticals. Phytomedicine. 2009;16:97-110. 44. Tiwari M. Apoptosis, angiogenesis and cancer therapies. J Can Res Ther. 2012;1:3.
45. Hussain SA, Sulaiman AA, Balch C, Chauhan H, Alhadidi QM, Tiwari AK. Natural polyphenols in cancer chemoresistance. Nutr Cancer. 2016;68:879-891.
46. Lin HH, Huang HP, Huang CC, Chen JH, Wang CJ. Hibiscus polyphenolrich extract induces apoptosis in human gastric carcinoma cells via p53 phosphorylation and p38 MAPK/FasL cascade pathway. Mol Carcinog. 2005;43:86-99.
47. Boueroy P, Hahnvajanawong C, Boonmars T, Saensa-ard S, Wattanawongdon W, Kongsanthia C, Salao K, Wongwajana S, Anantachoke N, Reutrakul V. Synergistic effect of forbesione from Garcinia hanburyi in combination with 5-Fluorouracil on cholangiocarcinoma. Asian Pac J Cancer Prev. 2017;18:3343-3351.
48. Liu Y, Bi T, Dai W, Wang G, Qian L, Shen G, Gao Q. Lupeol enhances inhibitory effect of 5-fluorouracil on human gastric carcinoma cells. Naunyn Schmiedebergs Arch Pharmacol. 2016;389:477-484.
49. Xu H, Yang T, Liu X, Tian Y, Chen X, Yuan R, Su S, Lin X, Du G. Luteolin synergizes the antitumor effects of 5-fluorouracil against human hepatocellular carcinoma cells through apoptosis induction and metabolism. Life Sci. 2016;144:138-147.
50. Wang L, Feng J, Chen X, Guo W, Du Y, Wang Y, Zang W, Zhang S, Zhao G. Myricetin enhance chemosensitivity of 5-fluorouracil on esophageal carcinoma in vitro and in vivo. Cancer Cell Int. 2014;14:71.