Tumor-targeting Salmonella Typhimurium Improves Anti-tumor Efficacy Of EPNP/MePdR System In Murine Tumors

Advances in bacteriology and molecular biology have widened the scope of bacterial approaches to cancer therapy and salmonella has recently been accepted as delivery agents for anticancer drugs.S.typhimurium is a facultative anaerobe,growing well in both aerobic and anaerobic conditions.To develop a clinically safe product that could serve as gene delivery vector,Salmonellae was attenuated by mutations in metabolic pathways to limit the pathogenesis of such bacteria.Several papers report that the tumor may provide additional nutrients and make the selective tumor accumulation of attenuated salmonella in a variety of animal models.Attenuated salmonella has been shown to transfer exogenous gene expression plasmids to mammalian host cells in vitro and in vivo.The ease of generating such vehicles makes it a highly attractive area for further research.ePNP/MePdR system has extraordinary strong potentials as an gene-directed enzyme-prodrug therapy(GDEPT) systems.It is reported that Escherichia coli purine nucleoside phosphorylase(ePNP) gene could cleave nucleoside prodrugs,such as 6-methylpurine 2'-deoxyriboside(MePdR) to a toxic substance named 6-methylpurine (MeP).Mep performs high toxicity to both proliferating and non-proliferating cancer cells by inhibiting synthesis of DNA,RNA and protein.Since prokaryotic PNP enzymes differ fundamentally in sequence,structure,and function from their eukaryotic counterparts;MePdR and related compounds are very poor substrates for mammalian. Furthermore,MeP released from the cells can enter nearby cells via the purine transport system,resulting in high bystander killing of tumor cells.It is also demonstrated that ePNP/MePdR system may have a great bystander effect against non-proliferating tumors, such as glioma,hepatoma,pancreatic cancer,ovarian cancer,prostate cancer,melanoma and other human cancers.In summary,ePNP/MePdR system shows an excellent future in the treatment of cancer.This paper is the first report to exploit a new attenuated S.typhimurium named SC36 as an antitumor agent to transfer an eukaryotic expression vector carrying Escherichia coli purine nucleoside phosphorylase(ePNP) gene within tumors in mice.In our research,a new mutant SC36(his G aro A cys purâ… ) with low residual virulence was gained by DES mutagenesis of SL3261.SC36 was used as a vehicle for oral gene therapy against murine tumors,such as mouse lewis lung carcinoma LL/2 and murine melanoma B16F10 cells.To fully explore the potentially synergistic antitumor effect of ePNP/MePdR system via orally attenuated S.typhimurium as a specific gene agent,we constructed a recombinant plasmid expressing ePNP gene named pEGFP-CI-ePNP. Subsequently,eukaryotic expression vector containing exogenous genes,such as E.Coli Purine Nucleoside Phosphorylase(ePNP),murine interleukin- 12(mIL- 12) and Cytosine deaminase(CD),was used to transform attenuated Salmonella(SC36),and such transformants were administered orally to C57BL/6 mice.Then we evaluated the antitumor effect of ePNP/MePdR system alone or associated with other therapeutic strategies,such as CD/5-FC and cytokine mIL-12.The antitumor activities in vitro and in vivo were also addressed in LL/2 and B16F10 cells by MTT assay and flow cytometry.In vivo test,oral administration of the SC36 led to preferential accumulation in murine tumors.Then tumor growth and animal survival were assessed for oral administration of ePNP/MePdR system via SC36 in tumor-bearing mice.Our data support the idea that ePNP/MePdR system significantly suppressed the aggressive growth of murine tumors by attenuated S.typhimurium.We speculate that salmonella has the positive synergistic antitumor properties and appears to be a promising strategy to improve ePNP/MePdR bystander killing of solid tumors.Moreover,combination with other therapeutic strategies,such as CD/5-FC system and cytokine mIL-12,ePNP/MePdR system may be more useful for tumor regression.