Engineering a probiotic strain of Escherichia coli to induce the regression of colorectal cancer through production of 5-aminolevulinic acid

Abstract

Bacterial vectors can be engineered to generate microscopic living therapeutics to produce and deliver anticancer agents. Escherichia coli Nissle 1917 (Nissle 1917) is a promising candidate with probiotic properties. Here, we used Nissle 1917 to develop a metabolic strategy to produce 5-aminolevulinic acid (5-ALA) from glucose as 5-ALA plays an important role in the photodynamic therapy of cancers. The coexpression of hemA^M and hemL using a low copy-number plasmid led to remarkable accumulation of 5-ALA. The downstream pathway of 5-ALA biosynthesis was inhibited by levulinic acid (LA). Small-scale cultures of engineered Nissle 1917 produced 300 mg l^-1 of 5-ALA. Recombinant Nissle 1917 was applied to deliver 5-ALA to colorectal cancer cells, in which it induced the accumulation of antineoplastic protoporphyrin X (PpIX) and specific cytotoxicity towards colorectal cancer cells irradiated with a 630 nm laser. Moreover, this novel combination therapy proved effective in a mouse xenograft model and was not cytotoxic to normal tissues. These findings suggest that Nissle 1917 will serve as a potential carrier to effectively deliver 5-ALA for cancer therapy.

Fig. 1

5‐ALA production from glucose in Nissle 1917 (A) and targeted delivery of 5‐ALA into tumour cells (B). 5‐ALA, 5‐aminolevulinic acid; GSA, glutamate‐1‐semialdehyde aminotransferase; Gln^tRNA, glutamyl‐tRNA; LA, levulinic acid; PBG, porphobilinogen; PpIX, protoporphyrin IX; ROS, reactive oxygen species.

Fig. 2

The growth (A), glucose consumption (B), accumulation of 5‐ALA (C) and PBG (D) and colour changes (E) in Nissle 1917 ENAL after addition of LA. 20 g l⁻¹ glucose was added initially as the sole carbon source with 10 g l⁻¹ addition at 40 h. Statistical analysis was conducted using two‐way ANOVA with Tukey’s multiple comparisons test, ***P < 0.001, ****P < 0.0001 (n = 3 per group; each group tested in triplicate; mean ± SEM).

Fig. 3

In vitro analysis of the anticancer effects of 5‐ALA·HCl and Nissle 1917 ENAL. A. Representative histogram (left) and bar graph (right) of PpIX fluorescence measured by flow cytometry in HCT116 cells treated with 5‐ALA·HCl for 6 h and with ENAL for 90 min (n = 3 per group of three replicates, ****P < 0.0001, two‐way ANOVA with Dunnett's multiple comparisons test). B. Cell viability assay of the in vitro anticancer effects of 5‐ALA·HCl and ENAL (n = 3 per group of three replicates, ***P < 0.001, two‐way ANOVA with Dunnett's multiple comparisons test). C. DCFH‐DA assay (representative histogram and bar graph) of intracellular ROS generation using flow cytometry of HCT116 cells treated with 5‐ALA·HCl for 8 h and with ENAL for 90 min (n = 3 per group of three replicates, ****P < 0.0001, two‐way ANOVA with Dunnett's multiple comparisons test). All data are expressed as the mean ± SEM.

Fig. 4

Treatment with 5‐ALA·HCl and Nissle 1917 ENAL reduces the tumour growth rate in a xenograft model. A. The retention of ENAL in different tissues was measured on days 2 (n = 4 mice), 4 (n = 3 mice), 8 (n = 3 mice) and 12 (n = 4 mice) after i.p. administration of 1 × 10⁷ CFU. B–C. Mice transplanted with HCT116 cells were treated daily for 12 days with PBS, ALA 1 (20 mg kg⁻¹ 5‐ALA·HCl), ALA 2 (50 mg kg⁻¹ 5‐ALA·HCl); or treated with 1 × 10⁷ cells 100 μl⁻¹ PBS of EN1920 or ENAL every six days for 12 days (n = 3–4 per group, *P = 0.0227, *P = 0.0457, ****P < 0.0001, two‐way ANOVA with Dunnett's multiple comparisons test). D. Body weight of each group (n = 3–4 per group, n.s. not significant (P > 0.05), two‐way ANOVA with Dunnett's multiple comparisons test). All data are shown as mean ± SEM.