Backgrounds and Objectives: All-trans retinoic acid (ATRA) which is a derivative of vitamin A, exert fundamental effects on regulation of cell growth, differenation and apoptosis. Recently, resistance to cisplatin and 5-fluorouracil developed in gastric adenocarcinoma and squamous cell carcinoma. In this study, we investigated the combination treatment of ATRA with cisplatin and 5-fluorouracil on gastric (AGS) and esophageal (KYSE-30) cancer cell lines. Materials and Methods: KYSE-30 and AGS cell lines were cultivated in RPMT-1640, including sub-toxic concentration of ATRA (13.6 and 3.6µM respectively) for 6 days. Then, treat with cisplatin and 5-fluorouracil for 72h. The effects of combination treatment tested by MTT assay, ethidium bromide/acridin orange (EB/AO) staining and flow cytometry. Results: Determination of IC50 (Inhibitory concentration of 50%) with MTT assay showed, that combination treatment had cytotoxic effect in both cell lines. IC50 value of the combined drugs was lower than the IC50 of drugs alone. (P<0/05) The percentage of apoptotic cells was increased in comparison to drugs alone in EB/AO staining. (P<0/05) Furthermore there was an indication that the combination of ATRA with cisplatin caused increased cell cycle arrest at G2/M in AGS and Kyse-30 cells. Also, ATRA and 5fu, induced increased the cell cycle arrest at G1/S phase in Kyse-30 and AGS cells (P<0/001). Conclusion: Our finding demonstrated that sub-toxic concentration of combination treatment of ATRA with cisplatin and 5-fluorouracil had additive cytotoxic effects on KYSE-30 and AGS cancer cell lines. References 1- Takaishi S, Okumura T, Tu S, et al. Identification of gastric cancer stem cells using the cell surface marker CD44. Stem Cells. 2009 27: 1006-20. 2- Enzinger P, Mayer R. Esophageal cancer. N Engl J Med. 2003 349: 2241-52. 3- Mohamadkhani A, Darvish Moghaddam S, Salmanroghani H, et al. Are the serum biomarkers pepsinogen I and II good predictors for the detection of subjects with atrophic gastritis in areas that have different gastric cancer incidence? Arch Iran Med. 2013 16: 208-12. 4- Ho YP, AuYeung SC, To KK. Platinum-based anticancer agents: innovative design strategies and biological perspectives. Med Res Rev. 2003 23: 633-55. 5- Gonzalez VM, Fuertes MA, Alonso C, Perez JM. Is cisplatin-induced cell death always produced by apoptosis. Mol Pharmacol. 2001 59: 657-63. 6- Shimizu M, Suzui M, Deguchi A, Lim JT, Weinstein IB. Effects of acyclic retinoid on growth, cell cycle control, epidermal growth factor receptor signaling, and gene expression in human squamous cell carcinoma cells. Clin Cancer Res. 2004 10: 1130-40. 7- Sacks PG, Harris D, Chou TC. Modulation of growth and proliferation in squamous cell carcinoma by retinoic acid: a rationale for combination therapy with chemotherapeutic agents. Int J Cancer. 1995 61: 409-15. 8- Petersson F, Colston K, Dalgleish A. Retinoic acid enhancest the cytotoxic effects of gemcitabine and cisplatin in pancreatic adenocarcinoma cells. Pancreas. 2001 23: 273-9. 9- Liu X, Chan SY, Ho PC. Comparison of the in vitro and in vivo effects of retinoids either alone or in combination with cisplatin and 5-fluorouracil on tumor development and metastasis of melanoma. Cancer Chemother Pharmacol. 2008 63: 167-74. 10- Nguyen GH, Murph MM, Chang JY. Cancer stem cell radioresistance and enrichment: where frontline radiation therapy may fail in lung and esophageal cancers. Cancers (Basel). 2011 3: 1232-52. 11- Soprano DR, Qin P, Soprano KJ. Retinoic acid receptors and cancers. Annu Rev Nutr. 2004 24: 201-21. 12- Dimberg A, Oberg F. Retinoic acid-induced cell cycle arrest of human myeloid cell lines. Leuk Lymphoma. 2003 44: 1641-50. 13- Liu S, Wu Q, Chen ZM, Su WJ. The effect pathway of retinoic acid through regulation of retinoic acid receptor alpha in gastric cancer cells. World J Gastroenterol. 2001 7: 662-6. 14- Lotan R, Xu XC, Lippman SM, et al. Suppression of retinoic acid receptor-beta in premalignant oral lesions and its up-regulation by isotretinoin. N Engl J Med. 1995 332: 1505-10. 15- Hengartner MO. The biochemistry of apoptosis. Nature. 2000 407: 770-6. 16- Dalirsani Z, Farajnia S, Javadzadeh Y, Mehdipour M, Koozegari S. The Effects of 5-fluorouracil alone and in combination with 13-cis retinoic asid and vitamin D3 on human oral squamous cell carcinoma lines. J Contemp Dent Practice. 2012 13: 345-50. 17- Hung S, Lee F, Su C, Tseng H. Effect of all-trans retinoic acid on the growth of two nasopharyngeal cancer cell lines and its treatment potential in combination with cisplatin. Eur Arch Otorhinolaryngol. 20124: 695-704. 18- Aebi S, Kroning R, Cenni B, et al. All-trans retinoic acid enhances cisplatin-induced apoptosis in human ovarian adenocarcinoma and in squamous head and neck cancer cells. Clin Cancer Res. 1997 3: 2033-8. 19- Hu l, Minden M, McCulloch E. Direct evidence for the participation of bcl-2 in the regulation by retinoic acid of the Ara-C sensitivity of leukemic stem cells. Leukemia. 1995 9: 1667-73. 20- Blagosklonny MV, Pardee AB. The restriction point of the cell cycle. Cell Cycle. 2002 1: 103-10. 21- Sorenson CM, Barry MA, Eastman A. Analysis of events associated with cell cycle arrest at G2 phase and cell death induced by cisplatin. J Natl Cancer Inst. 1990 82: 749-55. 22- Yoshikawa R, Kusunoki M, Yanagi H, et al. Dual antitumor effects of 5-fluorouracil on the cell cycle in colorectal carcinoma cells: a novel target mechanism concept for pharmacokinetic modulating chemotherapy. Cancer Res. 2001 61: 1029-37.