Applied Microbiology and Biotechnology

, Volume 97, Issue 10, pp 4393–4401 | Cite as

Tumor-targeting Salmonella typhimurium, a natural tool for activation of prodrug 6MePdR and their combination therapy in murine melanoma model

Biotechnologically Relevant Enzymes and Proteins


The PNP/6-methylpurine 2′-deoxyriboside (6MePdR) system is an efficient gene-directed enzyme prodrug therapy system with significant antitumor activities. In this system, Escherichia coli purine nucleoside phosphorylase (ePNP) activates nontoxic 6MePdR into potent antitumor drug 6-methylpurine (6MeP). The Salmonella typhimurium PNP (sPNP) gene has a 96-% sequence homology in comparison with ePNP and also has the ability to convert 6MePdR to 6MeP. In this study, we used tumor-targeting S. typhimurium VNP20009 expressing endogenous PNP gene constitutively to activate 6MePdR and a combination treatment of bacteria and prodrug in B16F10 melanoma model. The conversion of 6MePdR to 6MeP by S. typhimurium was analyzed by HPLC and the enzyme activity of sPNP was confirmed by in vitro (tetrazolium-based colorimetric assay) MTT cytotoxicity assay. After systemic administration of VNP20009 to mice, the bacteria largely accumulated and specifically delivered endogenous sPNP in the tumor. In comparison with VNP20009 or 6MePdR treatment alone, combined administration of VNP20009 followed by 6MePdR treatment significantly delayed the growth of B16F10 tumor and increased the CD8+ T-cell infiltration. In summary, our results demonstrated that the combination therapy of S. typhimurium and prodrug 6MePdR is a promising strategy for cancer therapy.


Salmonella typhimurium Tumor therapy Purine nucleoside phosphorylase Bacteria/prodrug therapy system 



The authors are grateful to grants from: the Doctoral Station Science Foundation from the Chinese Ministry of Education (200802840023), the National Key Basic Research Program from Ministry of Science and Technology (2011CB933502), the Jiangsu Provincial Nature Science Foundation (BK2010046, BZ2010074, BK2011228, BZ2011048), the Chinese National Nature Sciences Foundation (30821006, 50973046, 31071196, 30730030), Bureau of Science and Technology of Changzhou (CN20100016, CZ20100008, CJ20115006, CE20115034, CZ20110028), and the Department of Science and Technology of Wujin District, Changzhou (WS201004).


The authors have no conflicts of interest.


  1. Avogadri F, Martinoli C, Petrovska L, Chiodoni C, Transidico P, Bronte V, Longhi R, Colombo MP, Dougan G, Rescigno M (2005) Cancer immunotherapy based on killing of Salmonella-infected tumor cells. Cancer Res 65:3920–3927CrossRefGoogle Scholar
  2. Bharara S, Sorscher EJ, Gillespie GY, Lindsey JR, Hong JS, Curlee KV, Allan PW, Gadi VK, Alexander SA, Secrist JA 3rd, Parker WB, Waud WR (2005) Antibiotic-mediated chemoprotection enhances adaptation of E. coli PNP for herpes simplex virus-based glioma therapy. Hum Gene Ther 16:339–347CrossRefGoogle Scholar
  3. Cai X, Zhou J, Lin J, Sun X, Xue X, Li C (2005) Experimental studies on PNP suicide gene therapy of hepatoma. J Huazhong Univ Sci Technolog Med Sci 25:178–181CrossRefGoogle Scholar
  4. Chen G, Wei DP, Jia LJ, Tang B, Shu L, Zhang K, Xu Y, Gao J, Huang XF, Jiang WH, Hu QG, Huang Y, Wu Q, Sun ZH, Zhang JF, Hua ZC (2009) Oral delivery of tumor-targeting Salmonella exhibits promising therapeutic efficacy and low toxicity. Cancer Sci 100:2437–2443CrossRefGoogle Scholar
  5. Cheng CM, Lu YL, Chuang KH, Hung WC, Shiea J, Su YC, Kao CH, Chen BM, Roffler S, Cheng TL (2008) Tumor-targeting prodrug-activating bacteria for cancer therapy. Cancer Gene Ther 15:393–401CrossRefGoogle Scholar
  6. Deharvengt S, Wack S, Aprahamian M, Hajri A (2005) Transcriptional tumor-selective promoter targeting of E. coli purine nucleoside phosphorylase for pancreatic cancer suicide gene therapy. J Gene Med 7:672–680CrossRefGoogle Scholar
  7. Friedlos F, Lehouritis P, Ogilvie L, Hedley D, Davies L, Bermudes D, King I, Martin J, Marais R, Springer CJ (2008) Attenuated Salmonella targets prodrug activating enzyme carboxypeptidase G2 to mouse melanoma and human breast and colon carcinomas for effective suicide gene therapy. Clin Cancer Res 14:4259–4266CrossRefGoogle Scholar
  8. Fu W, Lan H, Li S, Han X, Gao T, Ren D (2008a) Synergistic antitumor efficacy of suicide/ePNP gene and 6-methylpurine 2′-deoxyriboside via Salmonella against murine tumors. Cancer Gene Ther 15:474–484CrossRefGoogle Scholar
  9. Fu W, Lan H, Liang S, Gao T, Ren D (2008b) Suicide gene/prodrug therapy using Salmonella-mediated delivery of Escherichia coli purine nucleoside phosphorylase gene and 6-methoxypurine 2′-deoxyriboside in murine mammary carcinoma 4T1 model. Cancer Sci 99:1172–1179CrossRefGoogle Scholar
  10. Gadi VK, Alexander SD, Kudlow JE, Allan P, Parker WB, Sorscher EJ (2000) In vivo sensitization of ovarian tumors to chemotherapy by expression of E. coli purine nucleoside phosphorylase in a small fraction of cells. Gene Ther 7:1738–1743CrossRefGoogle Scholar
  11. Ganai S, Arenas RB, Forbes NS (2009) Tumour-targeted delivery of TRAIL using Salmonella typhimurium enhances breast cancer survival in mice. Br J Cancer 101:1683–1691CrossRefGoogle Scholar
  12. Hersey P, Zhang XD (2009) Treatment combinations targeting apoptosis to improve immunotherapy of melanoma. Cancer Immunol Immunother 58:1749–1759CrossRefGoogle Scholar
  13. Huang QL, Chen C, Chen YZ, Gong CG, Cao L, Wang J, Hua ZC (2006) Application to immunoassays of the fusion protein between protein ZZ and enhanced green fluorescent protein. J Immunol Methods 309:130–138CrossRefGoogle Scholar
  14. Hughes BW, Wells AH, Bebok Z, Gadi VK, Garver RI Jr, Parker WB, Sorscher EJ (1995) Bystander killing of melanoma cells using the human tyrosinase promoter to express the Escherichia coli purine nucleoside phosphorylase gene. Cancer Res 55:3339–3345Google Scholar
  15. Jia LJ, Wei DP, Sun QM, Jin GH, Li SF, Huang Y, Hua ZC (2007) Tumor-targeting Salmonella typhimurium improves cyclophosphamide chemotherapy at maximum tolerated dose and low-dose metronomic regimens in a murine melanoma model. Int J Cancer 121:666–674CrossRefGoogle Scholar
  16. Jia LJ, Xu HM, Ma DY, Hu QG, Huang XF, Jiang WH, Li SF, Jia KZ, Huang QL, Hua ZC (2005) Enhanced therapeutic effect by combination of tumor-targeting Salmonella and endostatin in murine melanoma model. Cancer Biol Ther 4:840–845CrossRefGoogle Scholar
  17. Jiang Z, Zhao P, Zhou Z, Liu J, Qin L, Wang H (2007) Using attenuated Salmonella typhi as tumor targeting vector for MDR1 siRNA delivery. Cancer Biol Ther 6:555–560CrossRefGoogle Scholar
  18. Kikuchi E, Menendez S, Ozu C, Ohori M, Cordon-Cardo C, Logg CR, Kasahara N, Bochner BH (2007) Delivery of replication-competent retrovirus expressing Escherichia coli purine nucleoside phosphorylase increases the metabolism of the prodrug, fludarabine phosphate and suppresses the growth of bladder tumor xenografts. Cancer Gene Ther 14:279–286CrossRefGoogle Scholar
  19. King I, Bermudes D, Lin S, Belcourt M, Pike J, Troy K, Le T, Ittensohn M, Mao J, Lang W, Runyan JD, Luo X, Li Z, Zheng LM (2002) Tumor-targeted Salmonella expressing cytosine deaminase as an anticancer agent. Hum Gene Ther 13:1225–1233CrossRefGoogle Scholar
  20. Le UM, Yanasarn N, Lohr CV, Fischer KA, Cui Z (2008) Tumor chemo-immunotherapy using gemcitabine and a synthetic dsRNA. Cancer Biol Ther 7:440–447CrossRefGoogle Scholar
  21. Lee CH, Hsieh JL, Wu CL, Hsu PY, Shiau AL (2011) T cell augments the antitumor activity of tumor-targeting Salmonella. Appl Microbiol Biotechnol 90:1381–1388CrossRefGoogle Scholar
  22. Lee CH, Wu CL, Tai YS, Shiau AL (2005) Systemic administration of attenuated Salmonella choleraesuis in combination with cisplatin for cancer therapy. Mol Ther 11:707–716CrossRefGoogle Scholar
  23. Liao Z, Huang C, Zhou F, Xiong J, Bao J, Zhang H, Sun W, Xie C, Zhou Y (2009) Radiation enhances suicide gene therapy in radioresistant laryngeal squamous cell carcinoma via activation of a tumor-specific promoter. Cancer Lett 283:20–28CrossRefGoogle Scholar
  24. Low KB, Ittensohn M, Luo X, Zheng LM, King I, Pawelek JM, Bermudes D (2004) Construction of VNP20009: a novel, genetically stable antibiotic-sensitive strain of tumor-targeting Salmonella for parenteral administration in humans. Methods Mol Med 90:47–60Google Scholar
  25. Nowak AK, Lake RA, Marzo AL, Scott B, Heath WR, Collins EJ, Frelinger JA, Robinson BW (2003) Induction of tumor cell apoptosis in vivo increases tumor antigen cross-presentation, cross-priming rather than cross-tolerizing host tumor-specific CD8 T cells. J Immunol 170:4905–4913Google Scholar
  26. Pawelek JM, Low KB, Bermudes D (1997) Tumor-targeted Salmonella as a novel anticancer vector. Cancer Res 57:4537–4544Google Scholar
  27. Platt J, Sodi S, Kelley M, Rockwell S, Bermudes D, Low KB, Pawelek J (2000) Antitumour effects of genetically engineered Salmonella in combination with radiation. Eur J Cancer 36:2397–2402CrossRefGoogle Scholar
  28. Robertson BC, Hoffee PA (1973) Purification and properties of purine nucleoside phosphorylase from Salmonella typhimurium. J Biol Chem 248:2040–2043Google Scholar
  29. Saccheri F, Pozzi C, Avogadri F, Barozzi S, Faretta M, Fusi P, Rescigno M (2010) Bacteria-induced gap junctions in tumors favor antigen cross-presentation and antitumor immunity. Sci Transl Med 2:44ra57CrossRefGoogle Scholar
  30. Sorscher EJ, Peng S, Bebok Z, Allan PW, Bennett LL Jr, Parker WB (1994) Tumor cell bystander killing in colonic carcinoma utilizing the Escherichia coli DeoD gene to generate toxic purines. Gene Ther 1:233–238Google Scholar
  31. Todryk S, Melcher A, Bottley G, Gough M, Vile R (2001) Cell death associated with genetic prodrug activation therapy of colorectal cancer. Cancer Lett 174:25–33CrossRefGoogle Scholar
  32. Toso JF, Gill VJ, Hwu P, Marincola FM, Restifo NP, Schwartzentruber DJ, Sherry RM, Topalian SL, Yang JC, Stock F, Freezer LJ, Morton KE, Seipp C, Haworth L, Mavroukakis S, White D, MacDonald S, Mao J, Sznol M, Rosenberg SA (2002) Phase I study of the intravenous administration of attenuated Salmonella typhimurium to patients with metastatic melanoma. J Clin Oncol 20:142–152CrossRefGoogle Scholar
  33. Yagil E, Beacham IR (1975) Uptake of adenosine 5′-monophosphate by Escherichia coli. J Bacteriol 121:401–405Google Scholar
  34. Zhang L, Gao L, Zhao L, Guo B, Ji K, Tian Y, Wang J, Yu H, Hu J, Kalvakolanu DV, Kopecko DJ, Zhao X, Xu DQ (2007) Intratumoral delivery and suppression of prostate tumor growth by attenuated Salmonella enterica serovar typhimurium carrying plasmid-based small interfering RNAs. Cancer Res 67:5859–5864CrossRefGoogle Scholar
  35. Zhao M, Yang M, Li XM, Jiang P, Baranov E, Li S, Xu M, Penman S, Hoffman RM (2005) Tumor-targeting bacterial therapy with amino acid auxotrophs of GFP-expressing Salmonella typhimurium. Proc Natl Acad Sci U S A 102:755–760CrossRefGoogle Scholar
  36. Zhou JH, Tang B, Liu XL, He DW, Yang DT (2007) hTERT-targeted E. coli purine nucleoside phosphorylase gene/6-methylpurine deoxyribose therapy for pancreatic cancer. Chin Med J (Engl) 120:1348–1352Google Scholar

Copyright information

© Springer-Verlag 2012

Authors and Affiliations

  • Guo Chen
    • 1
    • 4
  • Bo Tang
    • 1
    • 2
    • 4
    • 5
  • Bing-Ya Yang
    • 1
    • 4
  • Jian-Xiang Chen
    • 1
    • 4
  • Jia-Hua Zhou
    • 3
  • Jia-Huang Li
    • 1
    • 4
  • Zi-Chun Hua
    • 1
    • 2
    • 4
    • 5
  1. 1.The State Key Laboratory of Pharmaceutical BiotechnologyNanjing UniversityNanjingPeople’s Republic of China
  2. 2.Changzhou High-Tech Research Institute of Nanjing UniversityChangzhouPeople’s Republic of China
  3. 3.Department of Biliary–Pancreatic SurgeryZhongda Hospital of Southeast UniversityNanjingPeople’s Republic of China
  4. 4.Department of Biochemistry, College of Life SciencesNanjing UniversityNanjingPeople’s Republic of China
  5. 5.Jiangsu TargetPharma Laboratories Inc.ChangzhouPeople’s Republic of China

Personalised recommendations