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The Anticancer Potential of the Bacterial Protein Azurin and Its Derived Peptide p28
Published in Ananda M. Chakrabarty, Arsénio M. Fialho, Microbial Infections and Cancer Therapy, 2019
Ana Rita Garizo, Nuno Bernardes, Ananda M. Chakrabarty, Arsénio M. Fialho
Currently, the investigation has been directed to segregated soluble factors by bacteria, such as enzymes, secondary metabolites, proteins, or derived peptides and toxins, which may act specifically on cancer cells, being potential anticancer agents [1, 5]. An example of this factor is a small water-soluble protein secreted by P. aeruginosa, called azurin (14 kDa; 128 amino acids), which is composed of one α-helix and eight β-sheets, forming a β-barrel motif. On its surface it contains three distinct binding regions: one face with two charged clusters (one large negative nearby one small positive) and a prominent neutral aromatic-rich hydrophobic patch. This arrangement, centered on Phe114, occupies a region around the copper center. Azurin is part of a group of type I redox proteins, which have an ion copper in their constitution, named cupredoxins (Fig. 9.1; [4, 9–15]). It is known that this protein is involved in the transport of electrons during the denitrification of these organisms [16].
Bacteria and Bioactive Peptides
Published in Prakash Srinivasan Timiri Shanmugam, Understanding Cancer Therapies, 2018
Ameer Khusro, Chirom Aarti, Paul Agastian
Azurin can enter cancer cells much more preferentially than normal cells and interferes in tumor growth by multiple modes of action including complex formation with the tumor suppressor protein p53 (Bizzarri et al., 2011), stabilizing it and enhancing its intracellular level, which then allows induction of apoptosis uniquely in tumors where it entered, leading to cancer cell death and shrinkage in mice (Yamada et al. 2004). In fact, azurin inhibits angiogenesis in tumors through inhibition of the phosphorylation of VEGFR-2, FAK, and AKT22. Azurin also has other tumor growth inhibitory activities that p28 lacks, such as azurin does not enter the cancer cells to form complexes with p53, VEGFR, FAK, and AKT in order to inhibit their functions. Azurin has the property to target cancer cells growing by expressing certain cell signaling receptor tyrosine kinase molecules on the cell surface. For example, a receptor kinase EphB2 is hyperproduced at the surface of cancer cells such as breast, prostate, lung, etc., promoting their rapid growth and proliferation when bound with its cell-membrane-associated ligand ephrin B2. Most importantly, azurin preferentially enters cancer cells and forms complexes with key proteins responsible for tumor formation, finally inhibiting the growth of cancer cells. Azurin has C-terminal four loop regions termed CD loop, EF loop, FG loop, and GH loop as well as its structural similarity with antibody variable domains of various immunoglobulins giving rise to a β-sandwich core and an immunoglobulin fold. This unique structure allows azurin to evade immune action and depicts its anticancer property when present in the bloodstream (Fialho et al. 2007). Azurin's binding domain to EphB2 via its G-H loop region has been used to increase radiation sensitivity of lung tumor cells through conjugation with the radio-sensitizer nicotinamide (Micewicz et al. 2011). Azurin has the ability to inhibit the growth of highly invasive P-cadherin (a member of the type I cadherin family that in certain conditions acts not as a regular cell–cell adhesion molecule, but as a promoter for malignant breast tumor progression) overexpressing breast cancer cells (Bernardes et al. 2013). A sublethal single dose of azurin produced a decrease in the invasion of two P-cadherin expressing breast cancer cell models, the luminal MCF-7/AZ.Pcad and the triple negative basal-like SUM 149 PT through a Matrigel artificial matrix. The decrease in invasion corresponds to a decrease in the total P-cadherin protein levels and a concomitant decrease of its membrane staining, whereas E-cadherin remains unaltered with high expression levels and with normal membrane localization, indicating the very important role of azurin only for invasive cell lines (Bernardes et al. 2013). Despite the potentiality of azurin to inhibit these proteins, the exact mechanism remains elusive.
Protein transduction domain of translationally controlled tumor protein: characterization and application in drug delivery
Published in Drug Delivery, 2022
The lipid raft is a microdomain of the plasma membrane constituted by the interaction of sphingolipid and sterol, which facilitates the internalization of certain macromolecules by clathrin- and caveolae-independent endocytic pathways (Ruseska & Zimmer, 2020). This coat-free pathway can be dynamin-dependent or -independent and is reported in the uptake of PTDs such as azurin fragments and transportan (Ruseska & Zimmer, 2020). Macropinocytosis is a lipid raft-dependent endocytic process, that involves actin-driven plasma membrane protrusion that induces the uptake of extracellular fluids (Ruseska & Zimmer, 2020). Ingestion of extracellular fluids by the membrane ruffles occurs by the action of the actin cytoskeleton (Swanson, 2008; van den Berg & Dowdy, 2011). TAT-protein conjugates and Poly-Arg internalize cells via macropinocytosis (Ruseska & Zimmer, 2020).
A model-based approach for the rational design of the freeze-thawing of a protein-based formulation
Published in Pharmaceutical Development and Technology, 2020
Andrea Arsiccio, Livio Marenco, Roberto Pisano
It was observed that the formation of ice crystals was the primary source of protein denaturation for lactate dehydrogenase (Bhatnagar et al. 2008), and the azurin protein (Strambini and Gabellieri 1996), and this same behavior probably applies to many common pharmaceutical proteins. In line with these considerations, the formation of large ice crystals, and therefore small ice-water surface area was often found to significantly improve protein stability (Chang et al. 1996; Jiang and Nail 1998; Sarciaux et al. 1999). This result could be achieved by adjusting the cooling rate and the nucleation temperature (Eckhardt et al. 1991; Jiang and Nail 1998; Fang et al. 2018). A low cooling rate or a high nucleation temperature promotes the formation of large crystals (Bald 1986; Searles et al. 2001). It was for instance observed that the application of controlled nucleation techniques (Kasper and Friess 2011; Pisano 2019), which make it possible to trigger the formation of ice nuclei at high temperature, resulted in improved protein stability after freeze-thaw (Fang et al. 2018). The use of surfactants may also help in this case, as these molecules mitigate the risk of surface-induced denaturation (Chang et al. 1996; Lee et al. 2011; Arsiccio et al. 2018).
Recent CPP-based applications in medicine
Published in Expert Opinion on Drug Delivery, 2019
Another line of research utilizes a CPP p28 with uptake selectivity and bioactivity toward cancer cells, derived from Azurin, a 128 amino acid cupredoxin, which is secreted by Pseudomonas aeruginosa. It has been shown that the CPP p28 induces p53-mediated cell-cycle arrest in tumor cells [20] and thus has a potential for the treatment of cancers. This CPP showed cytotoxic effects both alone or in combination with DNA-damaging and antimitotic chemotherapeutic agents in human cancer cells [21]. p28 has progressed through 2 phase I clinical trials against adult stage IV solid tumors [22] and pediatric CNS tumors [23], where it proved to be safe and well tolerated, as well as showed treatment efficacy. It is currently on its way to enter phase II clinical trials for pediatric glioblastomas.