Effects on 5-Fluorouracil Induced Apoptosis and Cell Cycle Changes via Blocking Human Equilibrative Nucleoside Transporters in Pancreatic Cancer Cell Membrane_CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY

Authors：

GAO Yiming , ZHOU Yumin , SONG Haihu , FAN Ziping , LIU Hao.

Department of General Surgery, Baoshan Department of Shanghai First People’s Hospital, Shanghai 200940, China;

Corresponding author：

Keywords：

Cell apoptosis; Cell cycle; Nucleoside transporter; 5-fluorouracil; Dipyridamole

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ObjectiveTo investigate the effects of dipyridamole (DP), one of the human equilibrative nucleoside transporters (hENTs) blockers, on 5-fluorouracil (5-FU) induced cell apoptosis and cell cycle changes of the pancreatic cancer Panc-1 cell line. MethodsPancreatic cancer cell line Panc-1 was divided into hENTs blocked group and hENTs unblocked group. The hENTs blocked group was further divided into two subgroups according to the DP concentration, 5 μmol/L DP group and 10 μmol/L DP group. Each group was incubated in culture medium with or without 1.5×106 ng/L 5-FU for 24 h. Cell apoptosis and cell cycle were detected by flow cytometry. Results①The apoptosis results of each group： Incubated in culture medium with 1.5×106 ng/L 5-FU for 24 h, the apoptosis rates of 5 μmol/L DP group and 10 μmol/L DP group were higer than those of hENTs unblocked group （P lt;0.05）, and which of 10 μmol/L DP group was higer than that of 5 μmol/L DP group (P lt;0.05). Incubated in culture medium without 5-FU for 24 h, there were no significant differences of the apoptosis rates among three groups (P gt;0.05). ②The cell cycle results of each group: Incubated in culture medium with 1.5×106 ng/L 5-FU for 24 h, the percentages of S phase cells in the 5 μmol/L DP group and 10 μmol/L DP group were less than those of hENTs unblocked group (P lt;0.05). The percentage of S phase cell of 5 μmol/L DP group reduced to 87.09% and that of 10 μmol/L DP group reduced to 74.06% as compared with hENTs unblocked group. 5-FU had little influence on G2 phase (P gt;0.05) except for the percentage of G2 phase cells in the 5 μmol/L DP group increased significantly (P lt;0.05) as compared with the hENTs unblocked group. Incubated in culture medium without 5-FU for 24 h, there were no significant differences of the cell cycles among three groups (P gt;0.05). ConclusionsIn pancreatic cancer Panc-1 cell, DP could enhance the cytotoxicity of 5-FU by blocking hENTs. The enhanced cytotoxicity is related to elevation of 5-FU concentration in cells, and unrelated to DP itself.

Citation： GAO Yiming,ZHOU Yumin,SONG Haihu,FAN Ziping,LIU Hao.. Effects on 5-Fluorouracil Induced Apoptosis and Cell Cycle Changes via Blocking Human Equilibrative Nucleoside Transporters in Pancreatic Cancer Cell Membrane. CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY, 2011, 18(4): 396-400. doi: Copy

1.	刘胜利, 王晶敏, 鞠煌先, 等. 潘生丁抑制平衡型核苷转运蛋白对5氟尿嘧啶在胰腺癌细胞内浓聚的影响 [J]. 中国药科大学学报, 2003, 34(6): 544548.
2.	高毅明, 刘胜利. 阻断胰腺癌细胞膜hENTs对氟尿嘧啶细胞毒性的影响 [J]. 现代医学, 2006, 34(3): 149153.
3.	Tanaka M, Javle M, Dong X, et al. Gemcitabine metabolic and transporter gene polymorphisms are associated with drug toxicity and efficacy in patients with locally advanced pancreatic cancer [J]. Cancer, 2010, 116(22): 53255335.
4.	Santini D, Schiavon G, Vincenzi B, et al. Human equilibrative nucleoside transporter 1 (hENT1) levels predict response to gemcitabine in patients with biliary tract cancer (BTC) [J]. Curr Cancer Drug Targets, 2010 Jun 25 [Epub ahead of print].
5.	Santini D, Vincenzi B, Fratto ME, et al. Prognostic role of human equilibrative transporter 1 (hENT1) in patients with resected gastric cancer [J]. J Cell Physiol, 2010, 223(2): 384388.
6.	Maréchal R, Mackey JR, Lai R, et al. Human equilibrative nucleoside transporter 1 and human concentrative nucleoside transporter 3 predict survival after adjuvant gemcitabine therapy in resected pancreatic adenocarcinoma [J]. Clin Cancer Res, 2009, 15(8): 29132919.
7.	Kong W, Engel K, Wang J. Mammalian nucleoside transporters [J]. Curr Drug Metab, 2004, 5(1): 6384.
8.	Farrell JJ, Elsaleh H, Garcia M, et al. Human equilibrative nucleoside transporter 1 levels predict response to gemcitabine in patients with pancreatic cancer [J]. Gastroenterology, 2009, 136(1): 187195.
9.	Michalski CW, Erkan M, Sauliunaite D, et al. Ex vivo chemosensitivity testing and gene expression profiling predict response towards adjuvant gemcitabine treatment in pancreatic cancer [J]. Br J Cancer, 2008, 99(5): 760767.
10.	Santini D, Vincenzi B, Fratto ME, et al. Prognostic role of human equilibrative transporter 1 (hENT1) in patients with resected gastric cancer [J]. J Cell Physiol, 2010, 223(2): 384388.
11.	冯燮林, 彭俊平, 李桂芳, 等. 胰腺癌区域灌注化疗的并发症及其防治 [J]. 中国普外基础与临床杂志, 2000, 7(6): 367369.
12.	Andersson R, Aho U, Nilsson BI, et al. Gemcitabine chemoresistance in pancreatic cancer: molecular mechanisms and potential solutions [J]. Scand J Gastroenterol, 2009, 44(7): 782786.
13.	MolinaArcas M, Marcé S, Villamor N, et al. Equilibrative nucleoside transporter2 (hENT2) protein expression correlates with ex vivo sensitivity to fludarabine in chronic lymphocytic leukemia (CLL) cells [J]. Leukemia, 2005, 19(1): 6468.
14.	Paproski RJ, Ng AM, Yao SY, et al. The role of human nucleoside transporters in uptake of 3’deoxy3’fluorothymidine [J]. Mol Pharmacol， 2008, 4(5): 13721380.
15.	Damaraju VL, Damaraju S, Young JD, et al. Nucleoside anticancer drugs: the role of nucleoside transporters in resistance to cancer chemotherapy [J]. Oncogene, 2003, 22(47): 75247536.
16.	King KM, Damaraju VL, Vickers MF, et al. A comparison of the transportability, and its role in cytotoxicity, of clofarabine, cladribine, and fludarabine by recombinant human nucleoside transporters produced in three model expression systems [J]. Mol Pharmacol, 2006, 69(1): 346353.
17.	Zhang J, Sun X, Smith KM, et al. Studies of nucleoside transporters using novel autofluorescent nucleoside probes [J]. Biochemistry, 2006, 45(4): 10871098.
18.	Ritzel MW, Ng AM, Yao SY, et al. Molecular identification and characterization of novel human and mouse concentrative Na+nucleoside cotransporter proteins (hCNT3 and mCNT3) broadly selective for purine and pyrimidine nucleosides (system cib) [J]. J Biol Chem, 2001, 276(4): 29142927.
19.	Mackey JR, Mani RS, Selner M, et al. Functional nucleoside transporters are required for gemcitabine influx and manifestation of toxicity in cancer cell lines [J]. Cancer Res, 1998, 58(19): 43494357.
20.	Yao SY, Sundaram M, Chomey EG, et al. Identification of Cys140 in helix 4 as an exofacial cysteine residue within the substratetranslocation channel of rat equilibrative nitrobenzylthioinosine (NBMPR)insensitive nucleoside transporter rENT2 [J]. Biochem J, 2001, 353(Pt 2): 387393.
21.	Goda AE, Yoshida T, Horinaka M, et al. Mechanisms of enhancement of TRAIL tumoricidal activity against human cancer cells of different origin by dipyridamole [J]. Oncogene, 2008, 27(24): 34353445.
22.	Schrier SM, van Tilburg EW, van der Meulen H, et al. Extracellular adenosineinduced apoptosis in mouse neuroblastoma cells: studies on involvement of adenosine receptors and adenosine uptake [J]. Biochem Pharmacol, 2001, 61(4): 417425.
23.	Kanno S, Hiura T, Ohtake T, et al. Characterization of resistance to cytosine arabinoside (AraC) in NALM6 human B leukemia cells [J]. Clin Chim Acta, 2007, 377(12): 144149.
24.	Visser F, Vickers MF, Ng AM, et al. Mutation of residue 33 of human equilibrative nucleoside transporters 1 and 2 alters sensitivity to inhibition of transport by dilazep and dipyridamole [J]. J Biol Chem, 2002, 277(1): 395401.
25.	Galmarini CM, Mackey JR, Dumontet C. Nucleoside analogues and nucleobases in cancer treatment [J]. Lancet Oncol, 2002, 3(7): 415424.
26.	Gerstin KM, Dresser MJ, Giacomini KM. Specificity of human and rat orthologs of the concentrative nucleoside transporter, SPNT [J]. Am J Physiol Renal Physiol, 2002, 283(2): F344F349.

1. 刘胜利, 王晶敏, 鞠煌先, 等. 潘生丁抑制平衡型核苷转运蛋白对5氟尿嘧啶在胰腺癌细胞内浓聚的影响 [J]. 中国药科大学学报, 2003, 34(6): 544548.
2. 高毅明, 刘胜利. 阻断胰腺癌细胞膜hENTs对氟尿嘧啶细胞毒性的影响 [J]. 现代医学, 2006, 34(3): 149153.
3. Tanaka M, Javle M, Dong X, et al. Gemcitabine metabolic and transporter gene polymorphisms are associated with drug toxicity and efficacy in patients with locally advanced pancreatic cancer [J]. Cancer, 2010, 116(22): 53255335.
4. Santini D, Schiavon G, Vincenzi B, et al. Human equilibrative nucleoside transporter 1 (hENT1) levels predict response to gemcitabine in patients with biliary tract cancer (BTC) [J]. Curr Cancer Drug Targets, 2010 Jun 25 [Epub ahead of print].
5. Santini D, Vincenzi B, Fratto ME, et al. Prognostic role of human equilibrative transporter 1 (hENT1) in patients with resected gastric cancer [J]. J Cell Physiol, 2010, 223(2): 384388.
6. Maréchal R, Mackey JR, Lai R, et al. Human equilibrative nucleoside transporter 1 and human concentrative nucleoside transporter 3 predict survival after adjuvant gemcitabine therapy in resected pancreatic adenocarcinoma [J]. Clin Cancer Res, 2009, 15(8): 29132919.
7. Kong W, Engel K, Wang J. Mammalian nucleoside transporters [J]. Curr Drug Metab, 2004, 5(1): 6384.
8. Farrell JJ, Elsaleh H, Garcia M, et al. Human equilibrative nucleoside transporter 1 levels predict response to gemcitabine in patients with pancreatic cancer [J]. Gastroenterology, 2009, 136(1): 187195.
9. Michalski CW, Erkan M, Sauliunaite D, et al. Ex vivo chemosensitivity testing and gene expression profiling predict response towards adjuvant gemcitabine treatment in pancreatic cancer [J]. Br J Cancer, 2008, 99(5): 760767.
10. Santini D, Vincenzi B, Fratto ME, et al. Prognostic role of human equilibrative transporter 1 (hENT1) in patients with resected gastric cancer [J]. J Cell Physiol, 2010, 223(2): 384388.
11. 冯燮林, 彭俊平, 李桂芳, 等. 胰腺癌区域灌注化疗的并发症及其防治 [J]. 中国普外基础与临床杂志, 2000, 7(6): 367369.
12. Andersson R, Aho U, Nilsson BI, et al. Gemcitabine chemoresistance in pancreatic cancer: molecular mechanisms and potential solutions [J]. Scand J Gastroenterol, 2009, 44(7): 782786.
13. MolinaArcas M, Marcé S, Villamor N, et al. Equilibrative nucleoside transporter2 (hENT2) protein expression correlates with ex vivo sensitivity to fludarabine in chronic lymphocytic leukemia (CLL) cells [J]. Leukemia, 2005, 19(1): 6468.
14. Paproski RJ, Ng AM, Yao SY, et al. The role of human nucleoside transporters in uptake of 3’deoxy3’fluorothymidine [J]. Mol Pharmacol， 2008, 4(5): 13721380.
15. Damaraju VL, Damaraju S, Young JD, et al. Nucleoside anticancer drugs: the role of nucleoside transporters in resistance to cancer chemotherapy [J]. Oncogene, 2003, 22(47): 75247536.
16. King KM, Damaraju VL, Vickers MF, et al. A comparison of the transportability, and its role in cytotoxicity, of clofarabine, cladribine, and fludarabine by recombinant human nucleoside transporters produced in three model expression systems [J]. Mol Pharmacol, 2006, 69(1): 346353.
17. Zhang J, Sun X, Smith KM, et al. Studies of nucleoside transporters using novel autofluorescent nucleoside probes [J]. Biochemistry, 2006, 45(4): 10871098.
18. Ritzel MW, Ng AM, Yao SY, et al. Molecular identification and characterization of novel human and mouse concentrative Na+nucleoside cotransporter proteins (hCNT3 and mCNT3) broadly selective for purine and pyrimidine nucleosides (system cib) [J]. J Biol Chem, 2001, 276(4): 29142927.
19. Mackey JR, Mani RS, Selner M, et al. Functional nucleoside transporters are required for gemcitabine influx and manifestation of toxicity in cancer cell lines [J]. Cancer Res, 1998, 58(19): 43494357.
20. Yao SY, Sundaram M, Chomey EG, et al. Identification of Cys140 in helix 4 as an exofacial cysteine residue within the substratetranslocation channel of rat equilibrative nitrobenzylthioinosine (NBMPR)insensitive nucleoside transporter rENT2 [J]. Biochem J, 2001, 353(Pt 2): 387393.
21. Goda AE, Yoshida T, Horinaka M, et al. Mechanisms of enhancement of TRAIL tumoricidal activity against human cancer cells of different origin by dipyridamole [J]. Oncogene, 2008, 27(24): 34353445.
22. Schrier SM, van Tilburg EW, van der Meulen H, et al. Extracellular adenosineinduced apoptosis in mouse neuroblastoma cells: studies on involvement of adenosine receptors and adenosine uptake [J]. Biochem Pharmacol, 2001, 61(4): 417425.
23. Kanno S, Hiura T, Ohtake T, et al. Characterization of resistance to cytosine arabinoside (AraC) in NALM6 human B leukemia cells [J]. Clin Chim Acta, 2007, 377(12): 144149.
24. Visser F, Vickers MF, Ng AM, et al. Mutation of residue 33 of human equilibrative nucleoside transporters 1 and 2 alters sensitivity to inhibition of transport by dilazep and dipyridamole [J]. J Biol Chem, 2002, 277(1): 395401.
25. Galmarini CM, Mackey JR, Dumontet C. Nucleoside analogues and nucleobases in cancer treatment [J]. Lancet Oncol, 2002, 3(7): 415424.
26. Gerstin KM, Dresser MJ, Giacomini KM. Specificity of human and rat orthologs of the concentrative nucleoside transporter, SPNT [J]. Am J Physiol Renal Physiol, 2002, 283(2): F344F349.

CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY

Effects on 5-Fluorouracil Induced Apoptosis and Cell Cycle Changes via Blocking Human Equilibrative Nucleoside Transporters in Pancreatic Cancer Cell Membrane

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