# Microbes as Medicines: Harnessing the Power of Bacteria in Advancing Cancer Treatment
## 2 Mechanism of Tumor Suppression
### Bacterial Directed Enzyme Prodrug Therapy (BDEPT)  
The BDEPT approach involves engineering bacteria to carry the gene encoding for a prodrug converting enzyme. The gene directed enzyme prodrug therapy involves two steps. In the first step, the engineered bacterial vector is delivered into the tumor. The bacteria then preferentially proliferate in the hypoxic and necrotic tumor where the enzyme is expressed. In the second step, the prodrug is administered systemically and upon accumulation at the tumor site, the expressed enzyme catalyzes the conversion of the prodrug into its active cytotoxic form. The in situ generation of the active drug at the tumor site promotes tumor selective cytotoxicity and reduces the chances of systemic toxicity [47; 76]. In the BDPET approach, the enzyme expressing bacteria stimulate the killing of neighboring tumor cells that do not express the enzyme (known as bystander effect). The active metabolite of the prodrug can be transported to the neighboring tumor cells by passive diffusion or can be transported through gap junctions [77]. Thus, the colony of the bacterial cells in tumor stroma can cause tumor regression without invading into the cancer cells. The design of the BDEPT system is critical and factors such as enzyme orientations, enzyme kinetics, toxicity of the prodrug/active pharmaceutical ingredient to the bacterium, and pharmacokinetic properties of the drug should be considered. Selection of an appropriate bacterial carrier is vital considering the level of invasiveness--intracellular or extracellular, wild type or recombinant carrier, and nature of the bacterium--faculative or obligate anaerobic bacteria [78]. Several prodrug/enzyme systems have been investigated, with commonly employed activating enzymes being cytosine deaminase (CD), nitroreductase, thymidine kinase, and cytochrome P450 enzymes. The CD enzyme catalyzes deamination of the prodrug, 5-fluorocytosine (5-FC) into 5-fluorouracilacil (5-FU) which interferes with DNA and RNA synthesis. CD was expressed in bacterial species, _Bifidobacterium longum_ and Clostridal species--C. _beijerintckii_, _C. acetobutylicum_, and _C. sporogenes_[79]. In an ongoing Phase I-II study (NCT01562626), 5-FC in conjunction with recombinant _Bifidobacterium longum_ (APS001F) modified to produce CD is being evaluated for its efficacy and safety to treat solid tumors [80]. In another study by [PERSON] et al., _E. coli_ was engineered to express CD at elevated temperatures [9]. The engineered bacteria were encapsulated in magnetic nanoparticles and expression was controlled by alternating magnetic field-induced hyperthermia. Combination of this system with 5-FC demonstrated cytotoxicity on glioma cells, prostate cancer, and breast cancer cell lines [9]. [PERSON] et al. investigated the therapeutic efficacy of BDEPT by genetically engineering _E. coli_ to carry DH5\(\alpha\)-lux/8G plasmid containing \(\beta\)-glucuronidase enzyme encoding gene. This gene helped in the activation of natural, non-toxic product glycyrrhizic acid to active glycyrrhetinic acid in TME of colon carcinoma mouse model. Results were indicative of effective bacterial tumor colonization and proliferation in TME as observed from the fluorescence emitted by the bacteria. The combined treatment greatly increased apoptosis and reduced the tumor growth [81]. _Escherichia coli_ Nissle 1917 was reprogrammed by [PERSON] et al. to promote its binding to heparan sulfate proteoglycan on colorectal cancer cells and secrete the enzyme myrosinase. Myrosinase is responsible for conversion of glucosinolates present in cruciferous vegetables to sulphoraphene, a natural metabolite that inhibits tumor growth and promotes apoptosis in cancer cells. A reduced tumor regression and tumor occurrence was observed in murine models of colorectal carcinoma in comparison to the treatment of engineered bacteria or dietary glucosinolates alone [82]. To understand the importance of enzyme prodrug partnership for an effective therapy, [PERSON] et al.