Microbial Azurin Immobilized on Nano-Chitosan as Anticancer and Antibacterial Agent Against Gastrointestinal Cancers and Related Bacteria.

OBJECTIVES: To discover new natural effective anticancer agents and new antibacterial agents against antibiotic-resistant bacteria which are the most serious public health concern. Another important concern is drug delivery which is the transport of pharmaceutical compounds to have a therapeutic effect in organisms having a disease. Azurin is a promising anticancer agent produced from Pseudomonas aeruginosa. This study tried to test the effectiveness of the immobilization of azurin on nano-chitosan to enhance its anticancer and antibacterial activity against gastrointestinal cancer and its related bacteria. METHODS: We purified azurin protein from Pseudomonas aeruginosa and then immobilized it on nano-chitosan. The anticancer activity of the free and nano-azurin is tested against a gastric cancer cell line (CLS-145), pancreatic cancer cell line (AsPC-1), colon cancer cell line (HCT116), esophagus cancer cell line (KYSE-410), and liver cancer cell line (HepG2). The antibacterial activity of both free and immobilized azurin also is tested against bacterial species related to the gastrointestinal cancer biopsies: Helicobacter pylori, Bacteroides fragilis, Salmonella enterica, Fusobacterium nucleatum, and Porphyromonas gingivalis. RESULTS: Both free and nano-azurin showed high anticancer and antibacterial activity. Immobilization significantly increased the anticancer and antibacterial activity of the azurin CONCLUSION: Nano-azurin can be used as an effective anticancer and antibacterial agent against gastrointestinal cancer and bacterial species related to these cancers.

Anticancer Actions of Azurin and Its Derived Peptide p28.

Cancers are a great threat to humans. In cancer therapy, surgical removal of the tumor combined with radiotherapy and chemotherapy is the most routine treatment procedure and usually the most effective. However, radiotherapy and chemotherapy drugs that kill cancer cells efficiently also kill normal cells, thus exhibiting large side effects. Cancer-targeted drugs, which aim to specifically recognize proteins or signaling pathways associated with tumor proliferation and migration, have achieved marked progress in recent years. Azurin is a copper-containing redox protein secreted by Pseudomonas aeruginosa. Azurin and its derived peptide p28 preferentially enter a variety of cancer cells and induce apoptosis or cell cycle arrest. Mechanistic studies revealed that azurin and p28 target the p53 and receptor tyrosine kinase signaling pathways as well as other pathways. Two phase I trials of p28 have been carried out, with findings that p28 is safe and exhibits anticancer activity in both adult and pediatric patients. In this review paper, we provide an up-to-date summary of progress on the anticancer mechanisms and therapeutic strategies for azurin and p28.

Molecular screening and genetic diversity analysis of anticancer Azurin-encoding and Azurin-like genes in human gut microbiome deduced through cultivation-dependent and cultivation-independent studies

Azurin, a bacteriocin produced by a human gut bacterium Pseudomonas aeruginosa, can reveal selectively cytotoxic and induce apoptosis in cancer cells. After overcoming two phase I trials, a functional region of Azurin called p28 has been approved as a drug for the treatment of brain tumor glioma by FDA. The present study aims to improve a screening procedure and assess genetic diversity of Azurin genes in P. aeruginosa and Azurin-like genes in the gut microbiome of a specific population in Vietnam and global populations. Firstly, both cultivation-dependent and cultivation-independent techniques based on genomic and metagenomic DNAs extracted from fecal samples of the healthy specific population were performed and optimized to detect Azurin genes. Secondly, the Azurin gene sequences were analyzed and compared with global populations by using bioinformatics tools. Finally, the screening procedure improved from the first step was applied for screening Azurin-like genes, followed by the protein synthesis and NCI in vitro screening for anticancer activity. As a result, this study has successfully optimized the annealing temperatures to amplify DNAs for screening Azurin genes and applying to Azurin-like genes from human gut microbiota. The novelty of this study is the first of its kind to classify Azurin genes into five different genotypes at a global scale and confirm the potential anticancer activity of three Azurin-like synthetic proteins (Cnazu1, Dlazu11, and Ruazu12). The results contribute to the procedure development applied for screening anticancer proteins from human microbiome and a comprehensive understanding of their therapeutic response at a genetic level

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Phase I trial of p28 (NSC745104), a non-HDM2-mediated peptide inhibitor of p53 ubiquitination in pediatric patients with recurrent or progressive central nervous system tumors: A Pediatric Brain Tumor Consortium Study.

BACKGROUND: p53 is a promising target in human cancer. p28 is a cell-penetrating peptide that preferentially enters cancer cells and binds to both wild-type and mutant p53 protein, inhibiting COP1-mediated ubiquitination and proteasomal degradation. This results in increased levels of p53, which induces cell cycle arrest at G2/M. We conducted a phase I study to determine the maximum-tolerated dose (MTD) and describe the dose-limiting toxicities (DLTs) and pharmacokinetics (PKs) of p28 in children. METHODS: Children aged 3-21 years with recurrent or progressive central nervous system tumors were eligible. Intravenous p28 was administered 3 times weekly for 4 consecutive weeks of a 6-week cycle at 4.16 mg/kg/dose (the adult recommended phase II dose) using a rolling-6 study design. Expression status of p53 was characterized by immunohistochemistry, and serum PK parameters were established on the second dose. RESULTS: Of the 18 eligible patients enrolled in the study, 12 completed the DLT monitoring period and were evaluable for toxicity. p28 was well-tolerated; 7 participants received ≥2 courses, and the most common adverse event attributed to the drug was transient grade 1 infusion-related reaction. PK analysis revealed a profile similar to adults; however, an increased area under the curve was observed in pediatric patients. High p53 expression in tumor cell nuclei was observed in 6 of 12 available tissue samples. There were no objective responses; 2 participants remained stable on the study for >4 cycles. CONCLUSIONS: This phase I study demonstrated that p28 is well-tolerated in children with recurrent CNS malignancies at the adult recommended phase II dose.

Microbial pathogenicity: a new approach to drug development.

Pathogenic microorganisms, particularly pathogenic bacteria that cause debilitating diseases, are considered a threat for human health and well-being. Infectious diseases are rampant, particularly in developing countries. However, there are many other deadly diseases, such as cancer, diabetes, cardiac malfunction, etc., that account for significant loss of life and trauma in our society. In this article, I try to describe the role certain bacterial pathogens with long term residence in the human body can play in providing us with our next generation anticancer and other drugs, demonstrating that a certain amount of good can come out of such bacterial pathogens as well.

A first-in-class, first-in-human, phase I trial of p28, a non-HDM2-mediated peptide inhibitor of p53 ubiquitination in patients with advanced solid tumours.

BACKGROUND: This first-in-human, phase I clinical trial of p28 (NSC745104), a 28-amino-acid fragment of the cupredoxin azurin, investigated the safety, tolerability, pharmacokinetics and preliminary activity of p28 in patients with p53(+) metastatic solid tumours. METHODS: A total of 15 patients were administered p28 i.v. as a short infusion three times per week for 4 weeks followed by a 2-week rest under an accelerated titration 3+3 dose escalation design until either a grade 3-related adverse event occurred or the maximum tolerated dose (MTD) was reached. Single-dose and steady-state serum pharmacokinetics were characterised. Assessments included toxicity, best objective response by RECIST 1.1 Criteria, and overall survival. RESULTS: No patients exhibited any dose-limiting toxicities (DLTs), significant adverse events or exhibited an immune response (IgG) to the peptide. The No Observed Adverse Effect Level (NOAEL) and MTD were not reached. Seven patients demonstrated stable disease for 7-61 weeks, three a partial response for 44-125 weeks, and one a complete response for 139 weeks. Three patients are still alive at 158, 140, and 110 weeks post therapy completion. CONCLUSION: p28 was tolerated with no significant adverse events. An MTD was not reached. Evidence of anti-tumour activity indicates a highly favourable therapeutic index and demonstrates proof of concept for this new class of non-HDM2-mediated peptide inhibitors of p53 ubiquitination.

Escherichia coli Nissle 1917 targets and restrains mouse B16 melanoma and 4T1 breast tumors through expression of azurin protein.

Many studies have demonstrated that intravenously administered bacteria can target and proliferate in solid tumors and then quickly be released from other organs. Here, we employed the tumor-targeting property of Escherichia coli Nissle 1917 to inhibit mouse B16 melanoma and 4T1 breast tumors through the expression of azurin protein. For this purpose, recombinant azurin-expressing E. coli Nissle 1917 was developed. The levels of in vitro and in vivo azurin secretion in the engineered bacterium were determined by immunochemistry. Our results demonstrated that B16 melanoma and orthotopic 4T1 breast tumor growth were remarkably restrained and pulmonary metastasis was prevented in immunocompetent mice. It is worth noting that this therapeutic effect partially resulted from the antitumor activity of neutrophils and lymphocytes due to inflammatory responses caused by bacterial infections. No toxicity was observed in the animal during the experiments. This study indicates that E. coli Nissle 1917 could be a potential carrier to deliver antitumor drugs effectively for cancer therapy.

Induction of apoptosis of azurin synthesized from P. aeruginosa MTCC 2453 against Dalton's lymphoma ascites model.

Azurin derived from P. aeruginosa MTCC 2453 were reported to be an important modulator in cancer regression which leads it to develop as a therapeutic drugs. Earlier studies reported that azurin derived from P. aeruginosa and other organisms, showed in vitro and in vivo antitumor properties by the induction of apoptosis. The present study was premeditated to evaluate the in vivo therapeutic efficacy of azurin from P. aeruginosa MTCC 2453 in Dalton's lymphoma (DL) mice model. The acute toxicity assay of azurin showed the 200 µg/kg as sublethal doses in normal mice. The acute toxicity like body weight, peripheral blood cell count, lympho-hematological and biochemical parameters remained unaffected till 200 µg/kg body weight of azurin. The growth inhibitory properties of azurin against in vivo DL model were vastly significant with its sublethal doses. We found that the DL cells survival rate percentage was 29.69, 64.6 and 88.79% in 50, 100 and 200 µg/kg body weight of azurin, respectively. Investigations of the growth inhibitory mechanism in DL cells were exposed by nuclear fragmentation, and the increased accumulation of DL cells in the sub-G0/G1 phases in cell cycle analysis are indications of apoptosis. Further, the cause of apoptosis was revealed by Western blot which showed the reduction in the ratio of Bcl-2 and Bax protein expression, the activation of caspases-3 through the release of cytochrome c in DL cells. The survival rate of tumor bearing DL mice treated with azurin analyzed by Kaplan Meir method showed an effective antitumor response as with a dose of 200 µg/kg body weight (an 94.19% increase in life span [ILS] %).

Preclinical pharmacokinetics, metabolism, and toxicity of azurin-p28 (NSC745104) a peptide inhibitor of p53 ubiquitination.

PURPOSE: Characterize the preclinical pharmacokinetics, metabolic profile, multi-species toxicology, and antitumor efficacy of azurin-p28 (NSC 745104), an amphipathic, 28 amino acid fragment (aa 50-77) of the copper containing redox protein azurin that preferentially enters cancer cells and is currently under development for treatment of p53-positive solid tumors. METHODS: An LC/MS/MS assay was developed, validated, and applied to liver microsomes, serum, and tumor cells to assess cellular uptake and metabolic stability. Pharmacokinetics was established after administration of a single intravenous dose of p28 in preclinical species undergoing chronic toxicity testing. Antitumor efficacy was assessed on human tumor xenografts. A human therapeutic dose was predicted based on efficacy and pharmacokinetic parameters. RESULTS: p28 is stable, showed tumor penetration consistent with selective entry into tumor cells and significantly inhibited p53-positive tumor growth. Renal clearance, volume of distribution, and metabolic profile of p28 was relatively similar among species. p28 was non-immunogenic and non-toxic in mice and non-human primates (NHP). The no observed adverse effect level (NOAEL) was 120 mg/kg iv in female mice. A NOAEL was not established for male mice due to decreased heart and thymus weights that was reversible and did not result in limiting toxicity. In contrast, the NOAEL for p28 in NHP was defined as the highest dose (120 mg/kg/dose; 1,440 mg/m(2)/dose) studied. The maximum-tolerated dose (MTD) for subchronic administration of p28 to mice is >240 mg/kg/dose (720 mg/m(2)/dose), while the MTD for subchronic administration of p28 to Cynomolgous sp. is >120 mg/kg (1,440 mg/m(2)/dose). The efficacious (murine) dose of p28 was 10 mg/kg ip per day. CONCLUSIONS: p28 does not exhibit preclinical immunogenicity or toxicity, has a similar metabolic profile among species, and is therapeutic in xenograft models.

Bacterial proteins as potential drugs in the treatment of leukemia.

Azurin and Laz are bacterial proteins that have been shown to exert anticancer effects against a variety of solid tumors. Their effects on liquid cancers have never been studied. We now show that they are also effective against liquid-borne cancers such as leukemia. Azurin and Laz can each enter in two leukemia cell lines but Laz exerts a greater cytotoxic effect on both K562 and HL60 cells, while having little effect on peripheral blood mononuclear cells, where they have very limited entry. In addition to Azurin and Laz, we have recently identified another protein, Pa-CARD, from Pseudomonas aeruginosa that carries a caspase recruitment domain (CARD)-like domain. This CARD domain polypeptide, called Pa-CARD, demonstrates cytotoxic activity against leukemia cells. In the leukemia cell lines, HL60 and K562, the anticancer activity of Laz and Pa-CARD is mediated through cell cycle arrest at the G2/M phase involving the Wee1 protein stabilization and the depletion of phosphorylated AKT-Ser-473, the active form of a serine/threonine kinase that is often dysregulated in many cancer types.

Bacterial proteins and CpG-rich extrachromosomal DNA in potential cancer therapy.

Bacterial proteins such as azurin and Laz have recently been shown to enter preferentially to cancer cells and kill them by multiple mechanisms. Historically, bacterial DNA, particularly the unmethylated CpG dinucleotides, have been shown to trigger activation of specific Toll-like receptors (TLRs) in immune cells, leading to various cytokine and chemokine production that allows cancer cell death and their regression. However, the enhanced release of specific protein or extrachromosomal DNA by bacteria in response to exposure to cancer cells has not been previously demonstrated. In this review, we discuss how an opportunistic, extracellular pathogenic bacterium, Pseudomonas aeruginosa, senses the presence of cancer cells and releases a specific protein or extrachromosomal DNA with antitumor activity for inhibition of cancer cell growth.

Bacterial redox protein azurin, tumor suppressor protein p53, and regression of cancer.

The use of live bacteria in the treatment of cancer has a long and interesting history. We report the use of a purified bacterial redox protein, azurin, that enters human cancer (melanoma UISO-Mel-2) cells and induces apoptosis. The induction of apoptosis occurs readily in melanoma cells harboring a functional tumor suppressor protein p53, but much less efficiently in p53-null mutant melanoma (UISO-Mel-6) cells. A redox-negative mutant form of azurin (M44K/M64E) demonstrates much less cytotoxicity to the UISO-Mel-2 cells than the wild-type protein. Azurin has been shown to be internalized in UISO-Mel-2 cells and is localized predominantly in the cytosol and in the nuclear fraction. In the p53-null UISO-Mel-6 cells, azurin is localized only in the cytosol. Thus, intracellular trafficking of azurin to the nucleus is p53-dependent. Azurin forms a complex with p53, thereby stabilizing it and raising its intracellular level in cytosolic, mitochondrial, and nuclear fractions. Corresponding to an increasing level of p53, an inducer of apoptosis, the level of Bax also increases in mitochondria, allowing significant release of mitochondrial cytochrome c into the cytosol, thus initiating the onset of apoptosis. The M44K/M64E mutant form of azurin, deficient in cytotoxicity, is also deficient in forming a complex with p53 and is less efficient in stabilizing p53 than wild-type azurin. Azurin has been shown to allow regression of human UISO-Mel-2 tumors xenotransplanted in nude mice and may potentially be used in cancer treatment.