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Azurin: Redox Originating from P. aeruginosa with Anti-Cancer Activity

Azurin is a bacterial redox metalloprotein in the family of cupredoxins with the ability to induce apoptosis in mammalian cells.

Sequence and Features

Usage and Biology

First in vitro studies have demonstrated that the cytotoxicity of azurin is due to its ability to raise intracellular levels of p53 by binding and stabilizing it upon internalization.¹ Azurin is potentially a highly versatile and promising anti-cancer agent as additional mechanisms keep being discovered such as inhibition of angiogenesis² and cell invasion (see Table 1).³

CATE transports azurin to tumor cells and releases it via controlled cell lysis. We therefore removed the native signal peptide which is part of the pre-protein form of azurin as no intracellular targeting to the bacterial cell surface is required.

Azurin has a size of 14'000 Daltons and consists of 8 β-strands and an α-helix. As a cupredoxin, azurin contains a type 1 copper center, where a copper ion is coordinated with two nitrogens (His-117 and His-46), two sulfur donors (Cys-112 and Met-121) and an oxygen (Gly-45) arranged in a distorted trigonal bipyramid.

In most studies, azurin is purified after bacterial recombinant expression. This process includes the reconstitution of the copper inn the active site of the protein by growing the cells in media supplemented with copper [Yamada et al].⁴ Investigation of the relationship between the presence of the copper ion in the active site and cytotoxicity showed the following: when two versions of azurin with and without copper were tested for their growth inhibitory effect in vitro on diverse cancer cell lines, the copper impact on the cytotoxicity of azurin was rather low, but detrimental for its oxidoreductase activity.

Characterization

Azurin and sGFP co-expression regulated by a pLux promoter was induced with AHL following which the lysate of each sample was collected and protein concentrations determined with Bradford assays. SDS-PAGE of induced sample lysates resulted in a distinctive band around 14 kD corresponding to the size of azurin. No similar bands can be observed in the negative control and mock-induced (no AHL) sample. For more details, read up on our Anti-cancer Toxin Experiments.

Figure 2: (Left) SDS-PAGE page analysis of pLux-regulated azurin expression in E. coli TOP10. Samples induced with AHL show a distinct band of 14 kD corresponding to the molecular weight of azurin. No similar bands can be observed for negative controls and non-induced samples. (Right) Fluorescent cultures co-expressing azurin and sGFP. - AHL: mock-induced with DMSO

We assessed the cytotoxic potential of azurin-rich bacterial lysates on p53 (+) and p53 (-) cell lines. Literature suggests, that the main mode of action of azurin against tumors the stabilization of p53 protein. For this reason HeLa CCL-2 lc was a suitable candidate for our experiment as it consists a p53 (+) strain and additionally has been well-examined in our project (good lysate tolerance and reliable cell viability assay). Moreover, the HT-29 p53 (-) negative cell line was also tested using the AM-calcein viability assay, in order to examine the effect of azurin against p53 (-) cancer cell - lines.

The cytotoxic effect of our azurin-rich lysate was verified by the decrease in the citrine fluorescence, following an increase in the azurin content. From the following graph (Figure 14) even 3.2% of azurin lysate (~2 nM) is enough to detect a cytotoxic effect.

In collaboration with the Freiburg team, we assessed the cytotoxicity and killing dynamics of azurin in mammalian cells. The Freiburg team observed the effect of incremental doses of purified azurin on HEK239T cell survival throughout one week (triplicates). For more details, visit the collaboration page of iGEM Freiburg 2017. Meanwhile, we applied azurin-rich bacterial lysate onto different mammalian cell lines which enabled us to compare the efficacies of purified azurin and azurin-rich bacterial lysate.

We quantified the azurin content in our bacterial lysates. Freshly prepared bacterial lysate from cultures expressing azurin/gfp after induction with AHL, were analyzed via SDS - PAGE together with a concentration gradient of purified His8-azurin provided by Freiburg iGEM Team. The standard protocol for SDS-PAGE was followed. Quantitative analysis of the SDS-PAGE gel was done with a LI-COR Odyssey CLx scanner.

With this data at hand, we went back to the experiments where we assessed the cytotoxicity of azurin on the HeLa CCL-2 lc cells. The effective azurin concentration was then determined as 2 nM.

Summary

• We expressed mature azurin in E. coli
• Our azurin is cytotoxic towards mammalian cells
• The effective concentration of azurin against p53 + HeLa CCL-2 lc cells is 1.8 - 2 nM

References

1. T. Yamada et al. "Bacterial redox protein azurin, tumor suppressor protein p53, and regression of cancer." Proceedings of the National Academy of Sciences (2002): 14098-14103
2. R. Mehta et al. "A cell penetrating peptide derived from azurin inhibits angiogenesis and tumor growth by inhibiting phosphorylation of VEGFR-2, FAK and Akt." Angiogenesis (2011): 355-369
3. N. Bernardes et al. "The Bacterial Protein Azurin Impairs Invasion and FAK/Src Signaling in P-Cadherin-Overexpressing Breast Cancer Cell Models" PLOS ONE (2013): e69023
4. T. Yamada et al. #FIX#