台灣為胃癌好發率高的國家，奧沙利鉑為其中之一具有活性的抗癌藥物用來治療晚期胃癌。但是治療過程中病人癌細胞衍生抗藥性，使癌細胞對於奧沙利鉑的治療失去效果，進而導致化學治療失敗與降低病人的存活。因此，發展可行的治療方案以逆轉鉑類化療藥物的抗藥性是迫切需要的。鐵離子在細胞的增生上扮演著重要的角色，因此發展鐵螯合劑來治療癌症是一個新的抗癌策略。最近研究發現新合成的鐵螯合劑Dp44mT是個有潛力的抗癌藥物，除了能對抗各種組織衍生的腫瘤外，也能對抗具貝福癌妥(etoposide)和敏克瘤(vincristine)抗藥性的腫瘤。儘管Dp44mT具治療化療抗藥衍生腫瘤之優勢，但其是否能夠有效地逆轉鉑類化療藥物的抗藥性目前仍是未知。我們實驗室先前利用TSGH人類胃癌細胞株建立了具有奧沙利鉑抗藥性的細胞株TSGH-S3 (S3)，並發現S3對鉑類化療藥物的抗藥性機制，包括增加銅排出蛋白ATP7A與降低銅吸收蛋白hCTR1的表現，以及增加細胞對DNA的修復能力。在本研究中我們利用這株奧沙利鉑抗藥性細胞株S3作為模式細胞，研究Dp44mT在逆轉鉑類化療藥物抗藥性所扮演的角色及機制之探討。首先我們檢測細胞對於鉑類化療藥物及Dp44mT的敏感性。結果發現，和TSGH相比，S3對奧沙利鉑和順鉑有很高的抗藥性，IC50的差異達73.5和8.9倍，此外S3細胞對Dp44mT也有交叉抗藥性，IC50差異達35倍。進一步isobologram分析結果顯示，Dp44mT與奧沙利鉑或順鉑合併使用，對於奧沙利鉑抗藥性細胞株S3具有加乘毒殺的作用，但是在非抗藥性細胞株TSGH只有加成的影響；此結果顯示Dp44mT能逆轉鉑類抗癌藥物在奧沙利鉑抗藥性細胞株之抗藥性。由於增加銅排出蛋白ATP7A及降低銅轉運蛋白hCTR1在奧沙利鉑抗藥性癌細胞中扮演著重要的角色，因此我們藉由西方點墨法分析Dp44mT處裡後銅轉運蛋白在S3的表現。結果顯示在抗藥性細胞株裡，鐵螯合劑Dp44mT會使得銅吸收蛋白hCTR1在S3細胞的表現有劑量和時間依存性的增加情形，然而銅排出蛋白ATP7A沒有很顯著的改變。接著我們探討鐵螯合劑Dp44mT如何調控銅吸收蛋白hCTR1的表現。由即時定量聚合酶連鎖反應分析證實鐵螯合劑Dp44mT可增加銅吸收蛋白hCTR1的mRNA，顯示hCTR1在S3的轉錄抑制可以藉由Dp44mT的處理而復原。此外，我們也發現Dp44mT能夠延長銅吸收蛋白hCTR1在奧沙利鉑抗藥性細胞株S3的半衰期。最後，我們設計三種不同合併給藥的順序，以釐清奧沙利鉑與Dp44mT的加乘毒殺作用是否為給藥順序依賴性的交互作用。結果顯示，先加入鐵螯合劑Dp44mT後再加入奧沙利鉑或同時給藥能夠有效地增加奧沙利鉑在奧沙利鉑抗藥性細胞株S3的細胞毒性；反之先加奧沙利鉑再加入鐵螯合劑Dp44mT只有加成的影響。綜合上述的結果，我們首先證實Dp44mT和奧沙利鉑在奧沙利鉑抗藥株中具有加乘毒性之交互作用，其機制為Dp44mT能增加銅吸收蛋白hCTR1的基因表現與延長hCTR1蛋白的穩定性，藉由Dp44mT對hCTR1的調控使此合併給藥的加乘作用具有給藥順序依賴性。綜而言之，我們認為合併使用螯合劑Dp44mT與奧沙利鉑，可作為治療復發或對奧沙利鉑治療產生抗藥性的胃癌病患的新穎治療策略，值得後續進一步的研究。

Gastric cancer is the predominant cancers found in Taiwan. Oxaliplatin is one of active agent for the treatment of advanced gastric cancer, however, patients will ultimately develop drug resistance which compromise patient’s survival. Thus, discovery of the potential therapeutic regimens to reverse platinum resistance is warranted. Iron plays a fundamental role in cellular proliferation. Recently, iron chelators, including Dp44mT, are being examined as a potential class of pharmaceutical agents to battle different types of cancers. In addition, the ability to overcome tumor resistance to established chemotherapeutics, including etoposide and vincristine, have also been indicated as an important advantage of Dp44mT. However, the effect of Dp44mT in reversing platinum resistance is not known. We previously have established an oxaliplatin-resistant human gastric cancer cell line, TSGH-S3 (S3), from its parental TSGH. The mechanisms responsible for platinum resistance of S3 cells, at least in part, are through upregulation of copper efflux transporter ATP7A, downregulation of copper influx transporter hCTR1, and enhancement of DNA repair capacity. In this study, we used this resistant subline to investigate the role and the underlying mechanisms of Dp44mT in improving the therapeutic efficacy of platinums in oxaliplatin-resistant cells.Firstly, we evaluated the drug sensitivity of platinum drugs and Dp44mT by growth inhibitory assay. The result demonstrated that the S3 cells were more resistance to oxaliplatin and cisplatin with approximately 73.5- and 8.9-fold of IC50 values, respectively, when compared to parental TSGH cells. In addition, the S3 cells were also cross-resistant to iron chelator, Dp44mT, with an IC50 values was approximately 35-fold. Further study demonstrated that combination treatment with Dp44mT and oxaliplatin or cisplatin exerts the synergistic interaction in S3 cells, yet only additive effect was observed in parental TSGH cells, suggesting that Dp44mT possesses chemosensitization potential to oxaliplatin in oxaliplatin-resistant cells. Because increased copper efflux transporter ATP7A and decreased copper uptake transporter hCTR1 are responsible for oxaliplatin resistance in S3 cells, we then examined the protein level of copper transporters in Dp44mT-treated S3 cells by Western blot analysis. The result showed that Dp44mT upregulated the protein level of copper influx transporter hCTR1 in S3 cells with concentration- and time-dependent manner, whereas the expression level of copper efflux transporter ATP7A was only changed slightly after Dp44mT treatment. In parallel with the change in the protein level, Dp44mT also increased the mRNA level of hCTR1 in S3 cells by quantitative RT-PCR analysis. This result suggested that the transcriptional repression of hCTR1 in S3 cells might be rescued by Dp44mT. In addition to enhanced hCTR1 transcript by Dp44mT, we also found that this compound prolonged the half-life of hCTR1 protein in S3 cells. Moreover, S3 cells were treated with three different schedules to determine whether the synergistic interaction of oxaliplatin with Dp44mT was schedule-dependent. The result demonstrated that a synergistic interaction was observed for Dp44mT prior to oxaliplatin or co-treated of Dp44mT and oxaliplatin exerts best synergism effect more then pre-treated with oxaliplatin which displayed only additive effect.Taken together, we demonstrated for the first time that Dp44mT exerts synergistic interaction with oxaliplatin in a schedule-dependent manner in oxaliplatin-resistant cells, which is through restoring the level of copper uptake transporter, hCTR1, indicating that combined use of Dp44mT with oxaliplatin may have potential to treat gastric cancer patients whom refractory to oxaliplatin treatment.

1. Krejs, G.J., Gastric cancer: epidemiology and risk factors. Dig Dis, 2010. 28(4-5): p. 600-3.
2. Wagner, A.D., et al., Chemotherapy for advanced gastric cancer. Cochrane Database Syst Rev, 2010(3): p. CD004064.
3. Wohrer, S.S., M. Raderer, and M. Hejna, Palliative chemotherapy for advanced gastric cancer. Ann Oncol, 2004. 15(11): p. 1585-95.
4. Souglakos, J., et al., Combination of irinotecan (CPT-11) plus oxaliplatin (L-OHP) as first-line treatment in locally advanced or metastatic gastric cancer: a multicentre phase II trial. Ann Oncol, 2004. 15(8): p. 1204-9.
5. Benson, A.B., Advanced gastric cancer: an update and future directions. Gastrointest Cancer Res, 2008. 2(4 Suppl): p. S47-53.
6. Woll, E., et al., Biweekly oxaliplatin and irinotecan chemotherapy in advanced gastric cancer. A first-line multicenter phase II trial of the Arbeitsgemeinschaft Medikamentose Tumortherapie (AGMT). Anticancer Res, 2008. 28(5B): p. 2901-5.
7. Choy, H., C. Park, and M. Yao, Current status and future prospects for satraplatin, an oral platinum analogue. Clin Cancer Res, 2008. 14(6): p. 1633-8.
8. Ho Yp Fau - Au-Yeung, S.C.F., K.K.W. Au-Yeung Sc Fau - To, and K.K. To, Platinum-based anticancer agents: innovative design strategies and biological perspectives. (0198-6325 (Print)).
9. Cepeda, V., et al., Biochemical mechanisms of cisplatin cytotoxicity. Anticancer Agents Med Chem, 2007. 7(1): p. 3-18.
10. Eriguchi, M., et al., A molecular biological study of anti-tumor mechanisms of an anti-cancer agent Oxaliplatin against established human gastric cancer cell lines. Biomed Pharmacother, 2003. 57(9): p. 412-5.
11. Graham, M.A., et al., Clinical pharmacokinetics of oxaliplatin: a critical review. Clin Cancer Res, 2000. 6(4): p. 1205-18.
12. Helleman, J., et al., Impaired cisplatin influx in an A2780 mutant cell line: evidence for a putative, cis-configuration-specific, platinum influx transporter. Cancer Biol Ther, 2006. 5(8): p. 943-9.
13. Takahashi, T., et al., Dynamic alteration of gene expression induced by anticancer-agent exposure in gastric cancer cell lines. Oncol Rep, 2009. 21(2): p. 451-9.
14. Wang and Z. Guo, The role of sulfur in platinum anticancer chemotherapy. Anticancer Agents Med Chem, 2007. 7(1): p. 19-34.
15. Hall, M.D., et al., The role of cellular accumulation in determining sensitivity to platinum-based chemotherapy. Annu Rev Pharmacol Toxicol, 2008. 48: p. 495-535.
16. Ishida, S., et al., Uptake of the anticancer drug cisplatin mediated by the copper transporter Ctr1 in yeast and mammals. Proc Natl Acad Sci U S A, 2002. 99(22): p. 14298-302.
17. Ishida, S., et al., Enhancing tumor-specific uptake of the anticancer drug cisplatin with a copper chelator. Cancer Cell, 2010. 17(6): p. 574-83.
18. Hector, S., et al., In vitro studies on the mechanisms of oxaliplatin resistance. Cancer Chemother Pharmacol, 2001. 48(5): p. 398-406.
19. Chen, C.C., et al., Combined modalities of resistance in an oxaliplatin-resistant human gastric cancer cell line with enhanced sensitivity to 5-fluorouracil. Br J Cancer, 2007. 97(3): p. 334-44.
20. Fu, S., et al., Overcoming Platinum Resistance through the Use of a Copper-Lowering Agent. Mol Cancer Ther, 2012.
21. Larson, C.A., et al., The role of the mammalian copper transporter 1 in the cellular accumulation of platinum-based drugs. Mol Pharmacol, 2009. 75(2): p. 324-30.
22. Farhat, F.S., J. Kattan, and M.G. Ghosn, Role of capecitabine and irinotecan combination therapy in advanced or metastatic gastric cancer. Expert Rev Anticancer Ther, 2010. 10(4): p. 541-8.
23. Hentze, M.W., M.U. Muckenthaler, and N.C. Andrews, Balancing acts: molecular control of mammalian iron metabolism. Cell, 2004. 117(3): p. 285-97.
24. Batts, K.P., Iron overload syndromes and the liver. Mod Pathol, 2007. 20 Suppl 1: p. S31-9.
25. Franz, K.J., Application of inorganic chemistry for non-cancer therapeutics. Dalton Trans, 2012. 41(21): p. 6333-4.
26. Zhou, T., et al., Design of iron chelators with therapeutic application. Dalton Trans, 2012. 41(21): p. 6371-89.
27. Richardson, D.R., Iron chelators as therapeutic agents for the treatment of cancer. Crit Rev Oncol Hematol, 2002. 42(3): p. 267-81.
28. Franchini, M., et al., Safety and efficacy of subcutaneous bolus injection of deferoxamine in adult patients with iron overload. Blood, 2000. 95(9): p. 2776-9.
29. Kalinowski, D.S. and D.R. Richardson, The evolution of iron chelators for the treatment of iron overload disease and cancer. Pharmacol Rev, 2005. 57(4): p. 547-83.
30. Yuan, J., D.B. Lovejoy, and D.R. Richardson, Novel di-2-pyridyl-derived iron chelators with marked and selective antitumor activity: in vitro and in vivo assessment. Blood, 2004. 104(5): p. 1450-8.
31. Le, N.T. and D.R. Richardson, Iron chelators with high antiproliferative activity up-regulate the expression of a growth inhibitory and metastasis suppressor gene: a link between iron metabolism and proliferation. Blood, 2004. 104(9): p. 2967-75.
32. Richardson, D.R., et al., Dipyridyl thiosemicarbazone chelators with potent and selective antitumor activity form iron complexes with redox activity. J Med Chem, 2006. 49(22): p. 6510-21.
33. Whitnall, M., et al., A class of iron chelators with a wide spectrum of potent antitumor activity that overcomes resistance to chemotherapeutics. Proc Natl Acad Sci U S A, 2006. 103(40): p. 14901-6.
34. Rao, V.A., et al., The iron chelator Dp44mT causes DNA damage and selective inhibition of topoisomerase IIalpha in breast cancer cells. Cancer Res, 2009. 69(3): p. 948-57.
35. Lord, C.J. and A. Ashworth, The DNA damage response and cancer therapy. Nature, 2012. 481(7381): p. 287-94.
36. Fuertes, M.A., C. Alonso, and J.M. Perez, Biochemical modulation of Cisplatin mechanisms of action: enhancement of antitumor activity and circumvention of drug resistance. Chem Rev, 2003. 103(3): p. 645-62.
37. Fan, T.J., et al., Caspase family proteases and apoptosis. Acta Biochim Biophys Sin (Shanghai), 2005. 37(11): p. 719-27.
38. Burger, H., et al., Drug transporters of platinum-based anticancer agents and their clinical significance. Drug Resist Updat, 2011. 14(1): p. 22-34.
39. Cvetkovic, R.S. and L.J. Scott, Dexrazoxane : a review of its use for cardioprotection during anthracycline chemotherapy. Drugs, 2005. 65(7): p. 1005-24.
40. Kelland, L., The resurgence of platinum-based cancer chemotherapy. Nat Rev Cancer, 2007. 7(8): p. 573-84.
41. Hanane, A., et al., Anticancer Drug Metabolism: Chemotherapy Resistance and New Therapeutic Approaches. 2012.
42. Sau, A., et al., Glutathione transferases and development of new principles to overcome drug resistance. Arch Biochem Biophys, 2010. 500(2): p. 116-22.
43. Howell, S.B., et al., Copper Transporters and the Cellular Pharmacology of the Platinum-Containing Cancer Drugs. Mol Pharmacol, 2010. 77(6): p. 887-94.
44. Liang, Z.D., et al., Specificity protein 1 (sp1) oscillation is involved in copper homeostasis maintenance by regulating human high-affinity copper transporter 1 expression. Mol Pharmacol, 2012. 81(3): p. 455-64.
45. Fu, S., et al., Overcoming Platinum Resistance through the Use of a Copper-Lowering Agent. Mol Cancer Ther, 2012. 11(6): p. 1221-5.
46. Lovejoy, D.B., et al., Antitumor activity of metal-chelating compound Dp44mT is mediated by formation of a redox-active copper complex that accumulates in lysosomes. Cancer Res, 2011. 71(17): p. 5871-80.
47. Wang, D. and S.J. Lippard, Cellular processing of platinum anticancer drugs. Nat Rev Drug Discov, 2005. 4(4): p. 307-20.
48. Basu, A. and S. Krishnamurthy, Cellular responses to Cisplatin-induced DNA damage. J Nucleic Acids, 2010. 2010.
49. Giaccone, G., Clinical perspectives on platinum resistance. Drugs, 2000. 59 Suppl 4: p. 9-17; discussion 37-8.