A novel peptide from photosynthetic bacteria that directly targets mitochondria to induce apoptosis in advanced prostate cancer cells

Abstract

We describe the design of a novel non-toxic cell membrane penetrating peptide aurB derived from the bacterial electron transfer protein auracyanin B and show that it induces caspase-mediated apoptotic cell death in prostate cancer cells that is mitochondrial-mediated and independent of the tumor suppressor protein p53. Furthermore, a symbiotic relationship between P. aeruginosa and cancer cells is described.

Description

[Technical field]

[0001] Priority document references This application claims the benefit of U.S. Provisional Patent Application No. 63 / 268,297, filed February 21, 2022, which is incorporated by reference in its entirety.

[0002] Sequence Listing Reference This application contains a Sequence Listing, which has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. Said ASCII copy, created on February 21, 2023, is named 46466-58.xml and is 13054 bytes in size.

[0003] The present invention relates to novel peptides from photosynthetic bacteria that directly target mitochondria to induce apoptosis in advanced prostate cancer cells. [Background technology]

[0004] Mitochondria play a key role in many biological processes and their functions are often altered in cancer cells, making them an attractive target for cancer therapy. Conventional chemotherapy drugs target mitochondria indirectly, via upstream mitochondrial signaling pathways (e.g., p53) that are frequently impaired or inactivated in cancers, particularly cancers such as advanced prostate and ovarian cancer. These cancers are typically fatal and show limited response to immune checkpoint inhibitors. Thus, there is a need to develop novel therapies that directly target the mitochondria of cancer cells.

[0005] Mitochondrial genome sequencing studies have shown a protein with homology to bacterial cupredoxins from the mitochondria of the eukaryotic sunflower plant (Helianthus). Moreover, in some species of bacteria, cupredoxins are involved in electron transport in various biological processes, including nitrogen fixation and ATP production by photosynthesis. For example, the cupredoxin protein azurin is involved in the electron transport chain during the nitrogen fixation process in bacteria such as the opportunistic pathogen Pseudomonas aeruginosa. Azurin secreted by P. aeruginosa induces caspase-mediated apoptotic cell death by complexing with and activating the tumor suppressor protein p53. Furthermore, a cell membrane-permeable peptide derived from azurin (also called p28; NSC745104) also induced p53-mediated tumor growth inhibition in vivo. Summary of the Invention [Problem to be solved by the invention]

[0006] Many advances in cancer treatment have been made through targeting strategies. Mitochondria have been recognized as one of the most important targets for the development of new therapeutic agents. The functions of eukaryotic mitochondria include ATP production and the control of cell proliferation and cell death. For many years, abnormal mitochondrial function has been associated with abnormal cancer cell proliferation. Mitochondrial ATP synthesis is often modulated in cancer cells and plays a critical role in tumor growth and overall survival of patients. Therefore, targeting mitochondria in cancer cells is a promising approach to develop new therapeutic agents. However, currently available drugs target mitochondria indirectly, relying on upstream mitochondrial signaling pathways that are frequently impaired in cancer cells. Therefore, new therapeutic agents that can directly target mitochondria in cancer cells would be ideal.

[0007] Microorganisms, particularly pathogenic bacteria, were used to treat various cancers in humans over 100 years ago, under the premise that toxins produced by a given pathogenic bacterium would inhibit the growth and spread of cancer in humans. With increasing knowledge of the role of the microbiota and the increasing incidence of cancer, treatment options using this approach are being reevaluated.

[0008] Azurin is a 14 kDa periplasmic copper protein containing 128 amino acids (aa) found in several types of bacteria and blue-green algae. One of its known biological functions is as an electron transfer protein in anaerobic energy production by nitrogen fixation. In vitro studies have shown that azurin induces apoptotic cell death in various cancer cells with no or minimal effects on their normal counterparts. After preferentially entering cancer cells, azurin binds to the tumor suppressor protein p53 and induces caspase-mediated apoptosis. Human xenograft studies in athymic mice have shown that systemic administration of azurin inhibits tumor growth without significant adverse effects on the host. In addition, the present inventors have previously identified an active peptide fragment of azurin (i.e., p28 or Azu28), and the efficacy of this 28aa cell membrane-permeable peptide p28 is being actively investigated both experimentally and clinically. [Means for solving the problem]

[0009] Based on the uniqueness of auracyanin among cupredoxins and the known biological functions of cupredoxins, the inventors discovered that these bacterial proteins may regulate human mitochondrial homeostasis by directly targeting mitochondria.

[0010] Here we describe the design of a novel nontoxic cell membrane-permeable peptide, aurB, derived from the bacterial electron transfer protein auracyanin B, and show that it induces caspase-mediated apoptotic cell death in prostate cancer cells that is mitochondrial-mediated and independent of the tumor suppressor protein p53.

[0011] Furthermore, the inventors describe that P. aeruginosa has acquired the ability to both harm (parasitic) and benefit (mutualistic) human health, adjusting its behavior according to physiological / cellular conditions that may change depending on disease states. P. aeruginosa may benefit human health by suppressing and attacking malignant cells in hosts with malignant cells through secretion of the bacterial protein azurin. P. aeruginosa expressing azu (azurin) has been found in tumors of primary melanoma and breast cancer patients who had not received chemotherapy prior to specimen collection.

[0012] Other methods, features and / or advantages will be apparent or will become apparent upon examination of the following figures and detailed description, and it is intended that all such additional methods, features and advantages be included within this description and protected by the accompanying claims.

[0013] Brief Description of the Sequence Listing SEQ ID NO:1 is the auracyanin A peptide (aurA) and corresponds to amino acids 62 to 89. LVK GGE AEA ANI ANA GLS AGP AAN YLPA (SEQ ID NO:1).

[0014] SEQ ID NO:2 is the auracyanin B peptide (aurB) and corresponds to amino acids 61 to 88. LVN GGD DVA AAV NTA AQN NAD ALF VPPP (SEQ ID NO:2).

[0015] SEQ ID NO:3 is the p28 peptide, which corresponds to amino acids 50-77 of azurin: LST AAD MQG VVT DGM ASG LDK DYL KPDD (SEQ ID NO:3).

[0016] SEQ ID NO: 4 is the ATPG human ATP synthase subunit gamma, mitochondrial sequence. Met Phe Ser Arg Ala Gly Val Ala Gly Leu Ser Ala Trp Thr Leu Gln Pro Gln Trp Ile Gln Val Arg Asn Met Ala Thr Leu Lys Asp Ile Thr Arg Arg Leu Lys Ser Ile Lys Asn Ile Gln Lys Ile Thr Lys Ser Met Lys Met Val Ala Ala Ala Lys Tyr Ala Arg Ala Glu Arg Glu Leu Lys Pro Ala Arg Ile Tyr Gly Leu Gly Ser Leu Ala Leu Tyr Glu Lys Ala Asp Ile Lys Gly Pro Glu Asp Lys Lys His Leu Leu Ile Gly Val Ser Ser Asp Arg Gly Leu Cys Gly Ala Ile His Ser Ser Ile Ala Lys Gln Met Lys Ser Glu Val Ala Thr Leu Thr Ala Ala Gly Lys Glu Val Met Leu Val Gly Ile Gly Asp Lys Ile Arg Gly Ile Leu Tyr Arg Thr His Ser Asp Gln Phe Leu Val Ala Phe Lys Glu Val Gly Arg Lys Pro Pro Thr Phe Gly Asp Ala Ser Val Ile Ala Leu Glu Leu Asn Ser Gly Tyr Glu Phe Asp Glu Gly Ser Ile Ile Phe Asn Lys Phe Arg Ser Val Ile Ser Tyr Lys Thr Glu Glu Lys Pro Ile Phe Ser Leu Asn Thr Val Ala Ser Ala Asp Ser Met Ser Ile Tyr Asp Asp Ile Asp Ala Asp Val Leu Gln Asn Tyr Gln Glu Tyr Asn Leu Ala Asn Ile Ile TyrTyr Ser Leu Lys Glu Ser Thr Thr Ser Glu Gln Ser Ala Arg Met Thr Ala Met Asp Asn Ala Ser Lys Asn Ala Ser Glu Met Ile Asp Lys Leu Thr Leu Thr Phe Asn Arg Thr Arg Gln Ala Val Ile Thr Lys Glu Leu Ile Glu Ile Ile Ser Gly Ala Ala Ala Leu Asp (SEQ ID NO: 4).

[0017] SEQ ID NO:5 is an azu-specific primer; 5'-CAGTTCACCGTCAACCTGTCC-3' (SEQ ID NO:5).

[0018] SEQ ID NO:6 is an azu-specific primer, 5'-TGGTGTGGGCGATGACACG-3' (SEQ ID NO:6).

[0019] SEQ ID NO:7 is the human GAPDH primer, which was also amplified as a loading control; 5'-AACGGGAAG CTTGTCATCAA-3' (SEQ ID NO:7).

[0020] SEQ ID NO:8 is a human GAPDH primer, which was also amplified as a loading control 5'-TGGACTCCACGACGTACTCA-3' (SEQ ID NO:8).

[0021] SEQ ID NO: 9 is the primer sequence Aldolase A: forward 5'-CGG GAA GGA GAA CCT G-3' (SEQ ID NO: 9).

[0022] SEQ ID NO:10 is the primer sequence aldolase A reverse 5'-GAC CGC TCG GAG TGT ACT TT-3' (SEQ ID NO:10).

[0023] SEQ ID NO:11 is the primer sequence β-actin: forward 5'-ACT GGA ACG GTG AAG GTG AC-3' (SEQ ID NO:11).

[0024] SEQ ID NO:12 is the primer sequence β-actin: reverse 5'-AGA GAA GTG GGG TGG CTT TT-3' (SEQ ID NO:12).

[0025] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings. [Brief description of the drawings]

[0026] [Figure 1A] ~ [Figure 1I] Overall structure of auracyanin A and B from C. aurantiacus and azurin from P. aeruginosa. Ribbon diagrams and molecular surfaces of each protein (1A: auracyanin A, 1B: auracyanin B, 1C: azurin) were imaged by QuteMol and DeepView (Swiss Institute of Bioinformatics). Dotted boxes indicate the location of helical (red) peptides aurA, aurB, and p28. Blue: copper. Hydrophobicity plots of aurA (1D), aurB (1E), and p28 (1F) were generated according to the Kyte and Doolittle hydrophobicity scale. Polarity scores of aurA (1G), aurB (1H), and p28 (1I) were calculated according to the polarity propensity scale. [Figure 2A] ~ [Figure 2B]Characterization of the three proteins and their peptides. The primary sequence of the mature protein was used. The peptide sequences are described in Methods (2A). Multiple sequence alignment by ClustalO showed that none of the amino acids were conserved among all three peptides (2B). aurA (aa 62-89 of auracyanin A): LVK GGE AEA ANI ANA GLS AGP AAN YLPA (SEQ ID NO:1), aurB (aa 61-88 of auracyanin B): LVN GGD DVA AAV NTA AQN NAD ALF VPPP (SEQ ID NO:2), and p28 (aa 50-77 of azurin): LST AAD MQG VVT DGM ASG LDK DYL KPDD (SEQ ID NO:3). ":" and "." indicate conservative substitutions (between two of the three peptides) and semi-conservative substitutions (between all three peptides), respectively. [Figure 3A] ~ [Figure 3B] Antiproliferative effects of aurA and aurB. Human cancer cells (2,000 cells / well) were incubated with different concentrations (5-100 μM) of aurA (3A) and aurB (3B) for 24 h at 37°C. Cell viability was detected by MTT assay, and control (PBS-treated) cells were considered as 100% viable. Mean + SE (N = 3). *P < 0.05, **P < 0.01, ***P < 0.001. [Figure 4A] ~ [Figure 4D] Effect of peptides on prostate cancer cell lines differing in p53 and AR expression status. MTT cell viability assays of prostate cancer cell lines (4A: LNCaP, 4B: DU145, 4C: PC3) and normal prostate cells (4D: CRL11611) were performed in the presence of aurA, aurB, p28, or Ptxl for 24-72 h. Control (PBS) treated cells were considered to have 100% viability. Mean + SE (N=3). [Diagram 5]Effects of aurB on p53-null human cancer cells. The dose-dependent effects of aurB on PC3 prostate cancer cells, MDD2 breast cancer cells, and SKOV3 ovarian cancer cells were measured by MTT assay. Mean + SE (N = 3). Each cancer cell line was exposed to aurB at 0.1-100 μM for 24 hours. [Figure 6A] ~ [Figure 6B] Cell membrane toxicity assay. Lactate dehydrogenase (LDH) leakage assay was performed on PC3 cells exposed to different concentrations (0.2-200 μM) of aurA (6A) or aurB (6B) at 37 °C. Cells treated with lysis buffer were defined as having 100% LDH release. Mean + SE (N = 3). [Figure 7A] ~ [Figure 7D] Measurement of apoptotic cell death. Prostate cancer cell lines (7A: LNCaP, 7B: DU145, and 7C: PC3) and normal prostate CRL11611 cells (7D) were exposed to the indicated drugs for 48 h and stained using an apoptosis assay kit according to the manufacturer's instructions. Samples were analyzed by flow cytometry. Mean + SE (N=3). [Figure 8A] ~ [Figure 8D] AurB localizes to mitochondria and targets ATP synthase. 8A, TEM image of PC3 cells. Representative TEM micrographs of aurB-treated cells illustrating the intramitochondrial localization of aurB-GNRs (arrowheads) and mitochondrial (Mt) swelling. Scale bar: 600 nm. Mt: mitochondria. 8B-8D, Identification of aurB-binding proteins. Mitochondrial fractions of PC3 cells were incubated with biotin-labeled p28, aurA, or aurB. A Coomassie-stained SDS-PAGE gel is shown (8B). A protein band of approximately 35 kDa (arrowheads) was identified as ATP5C using mass spectrometry. The identified fragment is shown with a green underlined sequence (SEQ ID NO: 4) (8C). IB with anti-ATP5C antibody confirmed that aurB physically binds to ATP5C (8D). [Figure 9A] ~ [Figure 9B] Effects of aurB on mitochondrial apoptosis signaling pathway. 9A, PC3 cancer cells were exposed to aurA or aurB at the indicated concentrations. Cells were stained using JC-1 mitochondrial membrane potential assay kit according to the manufacturer's instructions to measure mitochondrial membrane potential, and fluorescence was measured by flow cytometry. 9B, Caspase-3 is involved in aurB-induced apoptosis. The effects of caspase-3 inhibitor Z-DEVD-FMK on PC3 cells exposed to 100 μM aurB were measured by MTT assay at 24 h (circles) and 48 h (squares). Inhibitors were used at the indicated concentrations. Mean ± SE (N=3). [Figure 10A] ~ [Figure 10D] Inhibition of PC3 xenograft tumor growth. 10A, Athymic mice were randomized into control (PBS) and intraperitoneal treatment groups when subcutaneous tumors reached approximately 5 mm. Mean + SE (N = 5). *P < 0.01. 10B, None of the treatments significantly modulated the body weight of the animals. 10C, At the end of treatment, tumors were dissected and weighed. *P < 0.01. 10D, In tumor sections from the control group, there was a significant increase in Ki-67-positive proliferating cells (stained brown and counterstained with hematoxylin, × 40), and only a small number of apoptotic cells were observed as measured by TUNEL and caspase-3 (Casp-3) staining. There was a clear sharp decrease in Ki-67-positive proliferating cells and a greater number of TUNEL-positive apoptotic cells in the aurB-treated group compared to the control group. Similarly, Casp-3 expression in aurB-treated tumors was significantly increased compared to that in untreated controls. [Figure 11A] ~ [Figure 11B]Model for direct targeting of the mitochondrial energy production system. 11A: The electron transport complex and subunits of ATP synthase are localized to the inner mitochondrial membrane and generate energy in eukaryotic cells. In the bacterium Chloroflexus, both auracyanin A and B are membrane-anchored electron transport proteins, unlike azurin in P. aeruginosa. Auracyanin A is proposed to be transported to the outer membrane, while auracyanin B is anchored to the inner membrane. 11B: Phylogeny tree constructed based on the DNA sequences of azurin, auracyanin A, auracyanin B, and ATP5C using the program NGPhylogeny.fr. [Figure 12] TEM images of PC3 cells exposed to aurA. Representative TEM micrographs of aurA-treated PC3 cells showed that aurA-GNRs (arrowheads) were prominently found in large intracellular vacuoles but not in mitochondria. Scale bar: 600 nm. *: mitochondria. [Figure 13A] ~ [Figure 13C] Serum azurin levels are elevated in cystic fibrosis patients: 13A. Patient demographics. Serum azurin levels were measured using sera from cystic fibrosis (CF) patients and controls (N=50 each) of similar age range / age median. 13B. Serum levels of azurin were measured in CF patients (N=50) and controls (N=50) by ELISA using rabbit polyclonal anti-azurin antibodies. Purified azurin was used as an internal standard. 13C. A linear regression line of azurin levels against CF patient age was plotted. R=0.596, P=0.0038. Statistical analysis (t-test) showed significant differences in azurin levels between CF patients and healthy controls. ****: P<0.0001. [Figure 14A] ~ [Figure 14D]Azurin secretion is stimulated by human cancer cells. 14A induction of azurin secretion by P. aeruginosa in the presence of host cells. Human cells and P. aeruginosa were co-incubated for 30 min with P. aeruginosa (Pa) at a concentration of 500,000 human cells / ml and optical density (OD) = 0.3. Human cell lines derived from different tissues and tumors were used: melanoma Mel-2, congenital melanocytic nevus CMN, prostate cancer DU-145, normal prostate CRL-11611, ovarian cancer SK-OV3, normal ovarian HOSE6-3, breast cancer MDA-MB-231, and normal breast MCF-10A. Secretion of azurin by P. aeruginosa into the culture supernatant was assessed by Western blot analysis. The graph shows the observed band intensities measured by densitometer UN-SCAN-IT Gel version 5.1. Mean + SE values ​​were calculated for skin, prostate, ovarian, and breast cell pairs. 14B-14C Azurin secretion is cancer cell dose dependent. P. aeruginosa secretes higher levels of azurin in the presence of human breast cancer MDA-MB-231 (14B) and melanoma Mel-2 cells (14C) than in the presence of melanocytes (CMN) or non-malignant MCF-10A breast cells in a dose-dependent manner. Cancer cells and normal / non-malignant cells were co-incubated with P. aeruginosa for 30 min at concentrations ranging from 0-2,000,000 cells / ml and OD=0.3. Secretion of azurin by P. aeruginosa into the culture supernatant was assessed by Western blot analysis. The graph shows the observed band intensities. Mean + SE values ​​were calculated. 14D Soluble extracellular factors stimulate azurin secretion in a distance-dependent manner. Mel-2 and P. aeruginosa cells were separated by a permeable Transwell® insert membrane with a pore size of 0.4 μm.In this co-culture system, P. aeruginosa (OD = 0.3) and Mel-2 cells (3,000,000 cells / ml) were separated by a distance ranging from 2 mm to 12 mm and incubated at 37°C for 30 min (Inset: Schematic of the co-culture system. Top: P. aeruginosa, bottom: Mel-2). Secretion of azurin by P. aeruginosa into the culture supernatant was assessed by Western blot analysis. The graph shows the observed band intensity. [Figure 15] Azurin transcription is induced by co-incubation of P. aeruginosa with Mel-2 cells. P. aeruginosa and Mel-2 cells were indirectly co-incubated for 0-120 min and azu transcription was assessed by real-time PCR. Analysis of ΔΔCt values ​​of azu transcripts was performed and normalized to rpoD transcripts. azu and rpoD mRNAs were isolated at various time points of co-incubation of P. aeruginosa cells with Mel-2 cells. Data revealed a 2-fold increase in azurin transcript levels at 30 min. Mean + SD, *P<0.05, ***P<0.001 (ANOVA vs. control (rpoD). [Figure 16] P. aeruginosa does not pass through 0.4 μm filter membranes. Corning Transwell® polyester membrane cell culture plates and inserts (0.4 μm pore TC-treated sterile 24 mm Transwell) were used for the assay. Various concentrations of P. aeruginosa in 0.5% MGM were incubated in the upper compartment (insert) at 37° C. After 30 min of incubation, culture media from the upper and lower (well) chambers were plated on LB agar plates to measure colony forming units (CFU / ml). [Figure 17A] ~ [Figure 17C]Aldolase A secretion by melanoma cells is induced by exposure to azurin. Aldolase A is secreted by Mel-2 cells in the presence of P. aeruginosa. 17A. Mel-2 or CMN cells (3,000,000 cells / ml) and P. aeruginosa (OD=0.3) were co-incubated in a Transwell system, and culture supernatants were collected and subjected to SDS-PAGE followed by Coomassie staining. Mel-2+P. aeruginosa samples showed one additional band (40 kDa) compared to Mel-2 samples. 17B. This protein band was analyzed by mass spectrometry, and the protein was identified as human aldolase A. 17C. The numbers in the red circles indicate peptides that matched the aldolase A sequence. Based on the mass spectrometry data, the protein / peptide sequences were identified using Mascot software. Peptide sequences consistent with human aldolase A from mass spectrometry data are shown in red. [Figure 18A] ~ [Figure 18G]Induction of aldolase A secretion by host cells in the presence of P. aeruginosa. 18A. Human host cells (cancer and normal) and P. aeruginosa were co-incubated with P. aeruginosa at OD=0.3 at a concentration of 500,000 human cells / ml for 30 min. Aldolase secretion differed between co-cultures with cancer and normal cells. Secretion of azurin and aldolase into culture supernatants was assessed by Western blot analysis. Graph shows observed band intensity. 18B Correlation between aldolase A and azurin levels in co-cultures of P. aeruginosa with cancer or normal cells. 18C cancer cells (Mel-2 and MDA-MB-231) and P. aeruginosa (WT and azu null mutant) were co-incubated with P. aeruginosa at OD = 0.3 at a concentration of 500,000 human cells / ml for 30 min. Secretion of aldolase into the culture supernatant was assessed by Western blot analysis. 18D P. aeruginosa (OD = 0.3) was treated with purified aldolase A protein at concentrations of 1 nM, 10 nM, 100 nM, and 1 μM for 30 min. Treatment with 1 μM aldolase A stimulated azurin secretion from P. aeruginosa, suggesting that aldolase A is a stimulatory factor for azurin secretion. Secretion of azurin by P. aeruginosa into the culture supernatant was evaluated by Western blot analysis. The graph shows the observed band intensity. MDA-MB-231 (18E) and Mel-2 (18F) cells were treated with purified azurin protein at concentrations of 100 nM, 10 μM, and 1 mM for 30 min. Secretion of aldolase A by cancer cells into the culture supernatant was evaluated by Western blot analysis. The graph shows the observed band intensity. P. aeruginosa (only Pa) did not show any signal because it does not secrete any protein that cross-reacts with anti-aldolase A antibody.18G aldolase A secretion in the presence of P. aeruginosa is distance dependent. Mel-2 cells and P. aeruginosa were separated by permeable Transwell® inserts. In this co-culture system, P. aeruginosa (OD=0.3) and Mel-2 cells (3,000,000 cells / ml) were separated by distances ranging from 0 mm to 12 mm and incubated at 37°C for 30 min. Aldolase A secretion decreased as the distance between the two cell populations increased. Secretion of aldolase by Mel-2 cells into the culture supernatant was assessed by Western blot analysis. The graph shows the observed band intensities. [Figure 19A] ~ [Figure 19B] P. aeruginosa does not induce cytotoxicity in Mel-2 cells during 30 min of co-incubation. To confirm that factors found in the extracellular environment of Mel-2 cells are released due to active secretion and not cytotoxicity, cell viability assays were performed on Mel-2 cells co-incubated with P. aeruginosa. Mean + SEM values ​​were calculated from (19A) cell count and (19B) MTT cell proliferation assay data, and no significant differences were observed between the viability of Mel-2 cells cultured alone or co-cultured with P. aeruginosa. [Figure 20] Western blot analysis of azurin in WT and mutant P. aeruginosa. Cell lysates from wild-type (WT) and azu gene null P. aeruginosa PAO1 were loaded onto 4-12% NuPAGE gels. Bacterial proteins on the gels were transferred to nitrocellulose membranes. Rabbit anti-azurin antibody (1:5,000) and anti-GAPDH (1:5,000) antibodies were added, and HRP-conjugated secondary antibody was used for band visualization. [Figure 21]E. coli carrying the P. aeruginosa azu gene (ECAzu) induces aldolase secretion from Mel-2 cells. Both P. aeruginosa (Pa) and ECAzu showed azurin secretion in the presence of Mel-2 cells. However, compared with ECAzu, Pa induced more than a four-fold increase in azurin secretion. E. coli (EC), ECAzu, and Pa stimulated aldolase secretion from Mel-2 cells. Compared with EC, ECAzu showed a two-fold increase in aldolase secretion and Pa showed a six-fold increase. Secretion of azurin and aldolase into the culture supernatant was assessed by Western blot analysis. Mean + SEM. [Figure 22A] ~ [Figure 22B] Azurin does not regulate gene expression or intracellular levels of aldolase A. MDA-MB-231 and Mel-2 cells were treated with purified azurin protein at concentrations of 100 nM, 10 μM, and 1 mM for 30 min. Aldolase A gene expression in MDA-MB-231 (black bars) and Mel-2 (white bars) cells was measured by RT-PCR (22A). NS: not significant. Mean + SEM. 22B: Intracellular levels of aldolase A were assessed by Western blot analysis with anti-aldolase A antibody. GAPDH was used as a loading control. [Figure 23A] ~ [Figure 23B]siRNA-induced silencing of aldolase A (23A) and MUC1 (23B) genes in MDA-MB-231 (left) and Mel2 (right) cells. SMARTpool human ALDOA, MUC1, and non-targeting siRNA pool (Ctrl) were used as siRNAs targeting aldolase A, MUC1, and control (Ctrl), respectively. After 48 h of transfection, whole cell lysates (30 μg / lane with circumflex) were loaded onto a 4-12% NuPAGE gel. Proteins on the gel were transferred to a nitrocellulose membrane. Anti-aldolase, MUC1, and anti-GAPDH antibodies were added, and HRP-conjugated secondary antibodies were used for band visualization. [Figure 24A] ~ [Figure 24E]24A-Adhesion assays were performed essentially as described previously38. They were compared to siRNA-induced silencing of the aldolase gene in cancer cell lines (+) in monolayers of MDA-MB-231 (red) and Mel-2 (blue) cells when co-incubated with P. aeruginosa for 2 h. To assess total cell attachment, monolayers were washed to remove unbound P. aeruginosa and then disrupted and homogenized with 0.1% saponin / PBS. P. aeruginosa cells were counted by serial dilution of the homogenized suspension followed by plating on LB agar to determine colony forming units (CFU). Control (-) was expressed as 100%. Mean + SE, *P<0.05. 24B-monolayer MDA-MB-231 (red) and Mel-2 (blue) cells were co-incubated with P. aeruginosa in the presence or absence of exogenous aldolase A for 2 h. As above, the total binding of P. aeruginosa to cancer cells was counted by plating on LB agar. The control (0 μM aldolase A) was expressed as 100%. 24C-monolayer MDA-MB-231 (red) and Mel-2 (blue) cells were co-incubated with P. aeruginosa in the presence or absence of 200 nM exogenous aldolase A for 2 h, and compared with siRNA-induced silencing (+) of the MUC1 gene in cancer cell lines. Total binding of P. aeruginosa to cancer cells was counted by plating on LB agar. *P<0.05, NS: not significant. Azurin secretion is P. aeruginosa dose-dependent. MDA-MB-231 (24D) and Mel-2 (24E) cells (500,000 cells / ml) were co-incubated with P. aeruginosa at concentrations corresponding to OD ranging from 0.0 to 0.6. Secretion of azurin by P. aeruginosa into the culture supernatant was assessed by Western blot analysis. The graph shows the observed band intensities.Mean + SE values ​​were calculated. [Diagram 25] Melanoma and Breast Cancer Patient Demographics. Primary and metastatic tumors from patients with similar age ranges and age medians were used. [Figure 26A] ~ [Fig. 26H] Azurin-producing P. aeruginosa in human tumors. The gene encoding azurin (azu) was detected in tumors from patients with breast cancer and melanoma. It was amplified by PCR with P. aeruginosa azu-specific primers and confirmed by DNA sequencing. The amplified PCR product, as a single band, was sequenced and showed 100% identity to P. aeruginosa azu. In melanoma (26A), 27.6% (8 / 29) of primary tumors and 5.9% (2 / 34) of metastatic tumors were azu-positive (P<0.05). In breast cancer (26B), 22.7% (5 / 22) of primary tumors and 14.3% (2 / 14) of metastatic tumors were azu-positive (P>0.05). In addition, frozen tumor samples were processed for immunogold transmission electron microscopy (TEM) using anti-P. aeruginosa and anti-azurin antibodies. TEM images of uranyl acetate stained sections (arrowheads) showed intracellular localization of P. aeruginosa (26C) and its product azurin (26D) in human melanoma sections that were azu-positive by PCR. Magnification: ×3000. Patients with 26E, 26F-azu-positive tumors showed increased survival. Survival analysis of patients with azu-positive versus azu-negative primary melanoma (26E, P<0.01) and primary breast cancer (26F, P<0.05) tumors. 26G-hemizygous MMTV-PyMT transgenic mice were injected with either PBS control or 2.5 mg / kg azurin (i.p., 3 times weekly) for 2 months. The mean + SE values ​​of tumor volumes were calculated. Tumor-free survival curves were generated for the PBS control and 2.5 mg / kg azurin groups of MMTV-PyMT transgenic mice. Median tumor-free survival for the PBS control and azurin-treated groups was 67 and 72 days, respectively. P=0.015. [Figure 27A] ~ [Figure 27C] Melanoma specimen sections contain P. aeruginosa and azurin. 27A-H&E staining of human melanoma imaged by TEM in Figure 26. H&E stained sections (20x magnification) confirmed a malignant tumor with a high degree of cellular atypia. In approximately 20% of the tumor cells, the cytoplasm was stained brown due to the expression of melanin. TEM images showed the intracellular localization of azurin (27B, 6,300x; 27C, 1,000x) in human melanoma sections (arrowheads). [Figure 28] Invasive estrogen receptor- and progesterone receptor-negative breast cancer cells induce the highest levels of azurin secretion by P. aeruginosa. P. aeruginosa secretes higher levels of azurin in the presence of highly invasive MDA-MB-231 (ER-, PR-, Her2-) human breast cancer cells than in the presence of noninvasive T-47D (ER+, PR+, Her2-) human breast cancer cells or the MCF-10A normal breast cell line. Cancer (highly invasive MDA-MB-231 (ER-, PR-, Her2-) human breast cancer cells and non-invasive T-47D (ER+, PR+, Her2-) human breast cancer cells) and normal / non-malignant cells (MCF-10A cells) were co-incubated with P. aeruginosa for 30 min. Azurin secretion by P. aeruginosa into the culture supernatant was assessed by Western blot analysis. Graph shows observed band intensity. Mean + SEM. [Figure 29A] ~ [Figure 29C]Effects of azurin from P. aeruginosa in a transgenic animal model. 29A. Near infrared red (NIR) dye-conjugated azurin or PBS was injected intravenously into transgenic mice spontaneously developing mammary tumors. 24 hours after azurin injection, the mice were imaged by a photodynamic eye (PDE) system (left: photographic image, right: NIR fluorescence image at 800 nm). 29B. At the end of the study, the tumors were excised. The tumor weights of azurin-treated mice were significantly lower than those of control mice (**P<0.01), but body weights were not different (29C). [Diagram 30] A model for P. aeruginosa-cancer interactions. Secretion of aldolase A in response to the bacterial protein azurin, a beneficial anticancer activity of the bacteria. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] definition As used in this specification and the claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0028] The term "cell" as used herein includes both the singular and plural forms of the term. The terms "isolated," "purified," or "biologically pure" refer to material that is substantially or essentially free from components that normally accompany the material when found in its natural state. The terms "heterologous DNA," "heterologous nucleic acid sequence," "exogenous," and the like, as used herein, refer to a nucleic acid sequence in which at least one of the following is true: (a) the nucleic acid sequence is foreign to (i.e., not found in nature) a given host microorganism, (b) the sequence may be found naturally in a given host microorganism, but in a non-natural (e.g., greater than expected) amount, or (c) the nucleic acid sequence includes two or more subsequences that are not found in the same relationship to each other in nature.

[0029] "Peptide" and "polypeptide" are used interchangeably herein to refer to a compound comprised of a chain of amino acid residues linked by peptide bonds. An "active portion" of a polypeptide means a peptide that is smaller than the full-length polypeptide but that retains measurable biological activity and retains biological detection.

[0030] The term "tumor" as used herein refers to any neoplastic growth, proliferation, or mass of cells, whether benign or malignant (cancerous), whether as a primary site lesion or a metastasis.

[0031] As used herein, a "therapeutically effective amount" refers to an amount of a composition that relieves one or more symptoms of a disease or condition in a mammal (to any extent as determined by a physician of ordinary skill in the art). In addition, a "therapeutically effective amount" of a composition refers to an amount that restores, either partially or completely, normal physiological or biochemical parameters associated with or causing a disease or condition. A skilled clinician can determine the therapeutically effective amount of a composition to treat or prevent a specific disease, condition, or disorder when it is administered, for example, intravenously, subcutaneously, intraperitoneally, orally, or by inhalation. The exact amount of a composition required to be therapeutically effective will depend on a number of factors, such as the specific activity of the active agent, the delivery device employed, the physical properties of the agent, the purpose of administration, and the like, in addition to many patient-specific considerations. However, determining a therapeutically effective amount is within the capabilities of a skilled clinician armed with the disclosure set forth herein.

[0032] As used herein, "treat," "treating," and "treatment" refer to any action that provides a benefit to a patient at risk for or affected by a disease, including amelioration of the condition by alleviating or suppressing at least one symptom, slowing the progression of the disease, preventing or delaying the onset of the disease, and the like. Treatment also includes the partial or complete destruction of undesirable proliferating cells with minimal destructive effects on normal cells. An at-risk subject is one in which it has been determined that the subject has an above average risk that he or she will develop cancer, which may be determined, for example, by family history or detection of genes that cause a predisposition to developing cancer.

[0033] As used herein, the term "subject" refers to mammalian species, including, but not limited to, primates, including monkeys and humans, equines (e.g., horses), canines (e.g., dogs), felines, various domesticated farm animals (e.g., ungulates, e.g., swine, domestic pigs, goats, sheep, etc.), as well as domestic pets and animals kept in zoos.

[0034] Where methods and steps described herein indicate that some events occur in a certain order, those skilled in the art will recognize that the order of some steps may be modified and that such modifications are in accordance with variations of the invention. In addition, some steps may be performed not only sequentially, but also simultaneously in a parallel process when possible.

[0035] The meanings of the abbreviations are as follows: As is clear from the wording, "C" stands for Celsius or degrees Celsius, "s" stands for seconds, "min" stands for minutes, "h", "hr", or "hrs" stands for hours, "psi" stands for pounds per square inch, "nm" stands for nanometers, "d" stands for days, "μL" or "uL" or "ul" stands for microliters, "mL" stands for milliliters, "L" stands for liters, "mm" stands for millimeters, "nm" stands for nanometers, "mM" stands for millimolar, "μM" or "uM" stands for micromolar, and "M" stands for molar. "mmol" means millimole, "μmol" or "uMol" means micromole, "g" means gram, "μg" or "ug" means microgram, "ng" means nanogram, "PCR" means polymerase chain reaction, "kDa" means kilodalton, "g" means the gravitational constant, "bp" means base pair, "kbp" means kilobase pair, "%w / v" means weight / volume percent, "%v / v" means volume / volume percent, "rpm" means revolutions per minute, "HPLC" means high performance liquid chromatography, and "GC" means gas chromatography.

[0036] It is understood that "aurA" and "aurB" may refer to any molecule having a peptide sequence having substantial similarity to SEQ ID NO: 1 or 2. It is understood that any polypeptide that partially or completely comprises SEQ ID NO: 1 or 2 or is at least 96% identical to p28 can be used as a probe in the methods described herein. That is, if one amino acid of aurA, aurB, or p28 is modified by substitution with a different amino acid, the new sequence will be 96% identical to SEQ ID NO: 1 or 2. Similarly, one amino acid will be added to the p28 sequence, resulting in a sequence that is 96% identical to SEQ ID NO: 1 or 2.

[0037] overview Because mitochondria evolved from bacterial endosymbionts, several protein features are shared between mitochondria and extant bacteria that allow bacterial proteins to modulate mitochondrial function. Consistent with this model, we observed that aurB induces apoptosis by targeting mitochondrial ATP synthase and regulating the mitochondrial apoptotic pathway in cancer cells but not in healthy cells. In this study, we developed new drugs from such bacterial proteins that can directly target and regulate mitochondrial signaling pathways that are aberrantly expressed in cancer. Together, our findings indicate that bacterial electron transfer proteins are a significant source of novel therapeutic agents for directly targeting the mitochondrial energy production system of cancer cells.

[0038] Here we describe the design of a novel nontoxic cell membrane-permeable peptide, aurB, derived from the bacterial electron transfer protein auracyanin B, and show that it induces caspase-mediated apoptotic cell death in prostate cancer cells in a mitochondrial-mediated manner and independent of the tumor suppressor protein p53.

[0039] We have identified a novel non-toxic aurB peptide derived from bacterial photosynthetic protein that targets mitochondria and induces caspase-mediated apoptotic cancer cell death in a p53-independent manner, and demonstrated its potential therapeutic value in directly targeting the mitochondria of cancer cells. The mitochondria of cancer cells are structurally and functionally different from those of normal cells. Many conventional drugs induce cell death by targeting signaling pathways that are upstream of mitochondria and converge on these organelles. It is suggested that the development of potential anticancer drugs that directly target the mitochondrial energy production machinery is a logical approach. To the best of our knowledge, this is the first study to show that bacterial photosynthetic protein could be a significant source for the development of immune-independent drugs that directly target the mitochondrial energy production system. This study provides an important reference for the development of novel therapeutic agents by regulating mitochondrial homeostasis.

[0040] We have investigated the therapeutic role of microbes such as the opportunistic pathogen Pseudomonas aeruginosa in the management of human cancer. P. aeruginosa is the major pathogen of cystic fibrosis (CF) and causes significant morbidity and mortality, but CF patients have a lower incidence of melanoma and breast cancer than non-CF patients. When P. aeruginosa culture medium supernatants were co-incubated with tumor-derived J774A.1 macrophages, they induced apoptosis of J774A.1 in a dose-dependent manner. Culture medium supernatants contained high concentrations of azurin, a redox protein secreted by P. aeruginosa.

[0041] Here, we further show and describe that P. aeruginosa upregulated azurin secretion in response to increasing numbers and proximity of cancer cells. Conversely, cancer cells upregulated aldolase A secretion in response to increasing proximity of P. aeruginosa, which also correlated with enhanced P. aeruginosa adhesion to cancer cells. In addition, we show that cancer patients had detectable P. aeruginosa and azurin in their tumors and showed increased overall survival when they did, and that azurin administration reduced tumor growth in transgenic mice. Our results demonstrate a host-bacteria symbiotic mutualism that serves as a diverse complement of the host defense system through interkingdom communication mediated by the evolutionarily conserved proteins azurin and human aldolase A. This improved understanding of bacterial symbiotic relationships with humans indicates a potential contribution to tumor homeostasis.

[0042] We have demonstrated the interaction of P. aeruginosa with human cancer cells and its role in tumor homeostasis. In addition to highlighting the importance of this specific bacteria-cancer interaction, we show for the first time a bidirectional regulation of bacteria-cancer communication in relation to the potential of azurin secreted by P. aeruginosa to inhibit tumor growth.

[0043] In some embodiments, the present invention describes auracyanin peptides that modulate apoptotic pathways mediated by mitochondria in eukaryotic cells. Modulation can include increasing or decreasing any pathway typically found in mitochondria. Of particular interest are pathways that result in stimulation of pathways leading to cell death. Of particular interest is activation of the capase-dependent apoptotic pathway in the mictochondria of eukaryotic cells. In some embodiments, the auracyanin peptide is 20-35 amino acids in length. In some embodiments, the peptide is 28 amino acids in length. In some embodiments, the peptide forms an alpha helix and a beta sheet motif in solution. In some embodiments, the peptide is auracyanin obtained from the microorganism C. aurantiacus. In some embodiments, the peptide is amino acids 62-89 of the auracyanin A protein. In some embodiments, the peptide is amino acids aa 61-81 of the auracyanin B protein. In some embodiments, the peptide localizes to the mitochondrial membrane of eukaryotic cells and binding of the peptide to the gamma subunit of human mitochondrial ATP synthase protein occurs. In some embodiments, the peptide targets apoptotic pathways mediated by mitochondria in cancer cells but not in healthy cells. In some embodiments, the peptide has a sequence that is 96% identical to SEQ ID NO: 1 or 2. In some embodiments, the peptide has a sequence that is SEQ ID NO: 1 or 2. In some embodiments, the peptide has a pI of about 3.42 and a molecular weight of about 2400-2700 Da. In some embodiments, the auracyanin peptide is non-toxic to and permeates eukaryotic cells. In some embodiments, the peptide causes a decrease in mitochondrial membrane potential in a dose-dependent manner.

[0044] In some embodiments, methods of modulating the mitochondrial mediated apoptotic pathway in eukaryotic cells are described. The methods can include administering a therapeutically effective amount of an auracyanin peptide to a eukaryotic cell, such that the auracyanin peptide induces and results in a decrease in mitochondrial membrane potential, resulting in induction of the caspase mediated apoptotic pathway. In some embodiments, the auracyanin peptide induces caspase mediated apoptotic cell death in cancer cells but not in healthy cells. In some embodiments, the cell death induced by the auracyanin peptide is independent of the tumor suppressor protein p53 pathway. In some embodiments, the auracyanin peptide is amino acids aa61-81 of the auracyanin B protein. In some embodiments, methods of administering a therapeutically effective amount of an auracyanin peptide to a subject in need of cancer treatment are described. The method may include providing an effective amount of an auracyanin peptide in a pharma- ceutically acceptable form to enhance the mitochondrial-mediated apoptosis pathway in cancer cells, and administering an effective amount of the auracyanin peptide to a subject. By practicing the method, the auracyanin peptide will induce the caspase-mediated apoptosis pathway in the cancer cells. In some embodiments, the auracyanin peptide applied in the method may be amino acids aa61-81 of the auracyanin B protein.

[0045] In some aspects, the present invention describes non-toxic, cell membrane permeable peptides and their application to cells or subjects, however, it will be understood that administration of the auracyanin peptide may be by other means, i.e., for example, a plasmid or other nucleic acid delivery vehicle containing a nucleic acid encoding the peptide may be administered to a cell or subject to provide for expression of the auracyanin peptide in the cell or subject. EXAMPLES

[0046] Example 1: Characterization of peptide-based drugs from C. aurantiacus proteins C. aurantiacus produces at least two distinct forms of auracyanin. Both auracyanin A protein (13.9 kDa, 139 aa) and auracyanin B protein (14.4 kDa, 140 aa) have a core molecular structure of two β-sandwich domains formed by eight polypeptide strands in a typical cupredoxin fold (Figures 1A-1C). In a previous study, we designed and identified a cell membrane penetrating peptide p28 from P. aeruginosa azurin, which forms α-helical and β-sheet motifs together with random coils in solution. Here, by a similar approach, we designed peptide fragments from the predominantly helical motifs of auracyanin A and B. These fragments, aurA (aa62-89 of auracyanin A) and aurB (aa61-88 of auracyanin B), are highly water-soluble linear peptides of 28 aa with molecular weights of 2,610 and 2,721 Da, respectively. These two peptides and p28 are all anionic peptides, but the pI value of aurB is close to that of p28 (Fig. 2A). Multiple sequence alignment showed that there was very little amino acid conservation among the three peptides (Fig. 2B), and hydrophobicity / polarity plots (Figs. 1D-1I) showed that the polarity of aurA in the central to C-terminal regions was very low compared to that of p28, suggesting that the biological functions of these three peptides are probably different due to their different chemical properties, despite their similar secondary structures (Fig. 1).

[0047] Example 2: Antiproliferative effects of aurA and aurB on cancer cell lines Given that p28 acts in cancer cells through a p53-dependent mechanism, we examined the antiproliferative effects of aurA and aurB on various cancer cell lines differing in p53 status in vitro. aurA treatment had little effect on human cancer cells at any concentration tested (Fig. 3A). In contrast, aurB exposure showed clear dose-dependent effects on ovarian (SKOV-3), breast (MCF-7), and prostate (DU145) cancer cell lines, regardless of p53 expression status (Fig. 3B).

[0048] Although targeting the androgen receptor (AR) axis is one of the most successful therapeutic approaches, approximately 20% of prostate cancer patients develop castration-resistant prostate cancer (CRPC) stage within 5 years of follow-up during androgen deprivation therapy. CRPC has a poor prognosis and impairs quality of life. In addition, current immunotherapies have shown limited efficacy against advanced prostate cancer, unlike melanoma or lung cancer. Therefore, our observation that aurB significantly reduces the proliferation of AR-negative DU145 prostate cancer cell line motivated us to evaluate the effects of aurB, aurA, p28, and paclitaxel (Ptxl; one of the current standard drugs for prostate cancer) on additional prostate cancer cell lines with different molecular signatures. As expected, exposure to 1 nM paclitaxel significantly induced time-dependent cytotoxicity in the prostate cancer metastases LNCaP (derived from left supraclavicular lymph node metastasis, p53 wild-type, AR+), DU145 (derived from central nervous system metastasis, heterozygous p53 mutants P223L and V274F, AR-), and PC3 (derived from bone metastasis, p53 null, AR-), as well as the normal prostate cell line CRL11611 (p53+, AR+) (Figures 4A-4D). p28 also showed a dose-dependent effect on p53-expressing prostate cancer cells (LNCaP and DU145), but not on p53 null PC3 cells or normal CRL11611 cells, confirming our previous report. Consistent with the results shown in Figure 5, aurA was not active against these prostate cancer cells. In stark contrast, aurB significantly induced dose-dependent cytotoxic effects against all three prostate cancer cell lines for 72 h, with an inhibition rate of about 50% at 100 μM (Figures 4A-4C). However, this effect of aurB was not evident in normal prostate cells (Figure 4D). Moreover, the effects induced by aurB against p53-inactive cancer cell lines, including MDD2 breast cancer (p53 dominant negative) and p53-null SKOV3 ovarian and PC3 prostate cancer cells, were dose-dependent (Figure 5). Taken together, these data indicate that the cytotoxic effects of aurB against cancer cells were independent of p53 status, AR expression, and cell type.

[0049] We have demonstrated the antiproliferative activity of aurB in human cancer cells of various histological types, including a set of prostate cancer cell lines that are widely used in therapeutic research. Importantly, aurB was effective against p53WT, p53mutant, and p53null lines. The fact that the tumor suppressor p53 is the most frequently mutated gene in cancer53 and that these mutations typically modulate p53 activity, depending on the mutation site on the gene, indicates that p53 is a valid therapeutic target for cancer drug development. There have been intensive efforts to target mutant p53 reactivation in cancer, and several compounds have been identified. However, this promising approach does not apply to p53null cancer cells, suggesting that a different therapeutic strategy is needed to treat p53null cancer cells. Moreover, p53-null PC3 cells, but not LNCaP cells, are considered small cell neuroendocrine carcinomas, which do not form glands and are AR and prostate-specific antigen (PSA) negative. Neuroendocrine prostate cancer is extremely aggressive, does not respond to hormonal therapy, and, like other metastatic prostate cancers, is characterized by a relatively "cold" tumor immune microenvironment. Because the 5-year survival rate for metastatic and advanced prostate cancer in the United States is 31%, as opposed to nearly 100% for localized prostate cancer, new treatment options need to be developed to improve survival.

[0050] Example 3: The cytotoxic effect of aurB is due to cell membrane damage. Many antimicrobial peptides induce their cytotoxic effects by disrupting cell membranes. To examine whether the cytotoxic effect of aurB is due to cell membrane damage, a membrane toxicity assay was performed. Neither aurA nor aurB induced cell membrane toxicity at any concentration (Figure 6). Therefore, we examined apoptotic cell death in prostate cancer cell lines exposed to p28, aurA, aurB, and Ptxl (Paxitel) by flow cytometry analysis. In all cell lines, including normal cells, 1 nM Ptxl induced apoptotic cell death, while aurA had very little effect (Figures 7A-7D). In addition, p28 induced apoptotic cell death in LNCaP and DU145 cells, but not in PC3 and CRL11611 cells. Treatment with aurB induced significant dose-dependent apoptosis in all prostate cancer cell lines, but to a lesser extent in normal prostate cells (Figures 7A-7D). Importantly, although p53 and AR play significant roles in inducing apoptosis, aurB could induce apoptosis in both p53-null and AR-negative cancer cells, suggesting that its mechanism of action is p53 / AR-independent.

[0051] Apoptotic programmed cell death occurs in multicellular organisms, and mitochondria are particularly distinct from other intracellular organelles because they play critical roles in the p53-dependent and p53-independent regulation of cellular energy metabolism and programmed cell death. Given that aurB is derived from a bacterial electron transport chain protein, the present inventors hypothesized that aurB may have affinity for mitochondria because aurB was identified from auracyanin B, which is involved in the electron transport chain process for energy production in C. aurantiacus.

[0052] In this study, we compared the effects of aurA and aurB with those of Ptxl, one of the current standard chemotherapy drugs for patients with prostate, breast, and ovarian cancer. Ptxl was most toxic in normal prostate cell lines (CRL11611), and p53 mutant cell lines (DU145 and PC3) were less sensitive to Ptxl treatment than p53 wild-type and androgen-sensitive LNCaP cells. In contrast, all three tested prostate cancer cell lines were more sensitive to aurB than normal prostate cell lines. These results also confirmed that aurB significantly induced apoptotic cell death in prostate cancer cells compared to their normal counterparts (Figure 7). It has been reported that the overall mitochondrial mass is increased in cancer tissues of all Gleason grades compared to benign prostate tissues. This may be one reason why the effect of aurB on prostate cancer cells is significantly greater than that on normal prostate cells (Figures 4, 7). Moreover, in cancer, high levels of mitochondrial ATP synthase complex V components, including ATP5C, are significant risk factors (e.g., in terms of overall survival), suggesting that this complex is a promising druggable target.

[0053] Example 4: Localization of aurB in PC3 prostate cancer cells by transmission electron microscopy (TEM) We first observed the localization of aurB in PC3 prostate cancer cells by transmission electron microscopy (TEM). AurB was conjugated to non-spherical gold nanorods (GNRs, 25 nm diameter × 73 nm length) for labeling. Relatively large GNRs were used because GNRs with diameters smaller than 15 nm can enter tumor tissues by themselves. GNR-conjugated aurB was clearly internalized by PC3 cells and localized to mitochondria (Figure 8A). In contrast, GNR-conjugated aurA was internalized by PC3 cells and was prominently found in large intracellular vacuoles rather than mitochondria (Figure 9). Pull-down assays with biotin-labeled p28, aurA, or aurB revealed that aurB pulled down an endogenous protein with a molecular weight of approximately 35 kDa, but p28 and aurA did not (Figure 8B). Mass spectrometry and immunoblot (IB) of the 35 kDa protein band identified the binding partner of aurB as the mitochondrial ATP synthase gamma subunit (ATP5C) (Figures 8C-8D). Furthermore, phylogenetic analysis to determine the evolutionary relationship between these three cupredoxins and ATP5C showed that auracyanin B is closely related to ATP5C (Figure 8E), at least in our phylogenetic analysis based on their gene sequences. Taken together, these findings suggest that the intramitochondrial localization of aurB occurs through binding to mitochondrial ATP5C, leading to mitochondrial swelling (Figure 8A).

[0054] Example 5: Mitochondrial membrane potential of PC3 cells treated with different compounds Modulation of mitochondrial morphology is generally associated with mitochondrial membrane potential (Δ Ψm Since aurA is associated with a decrease in mitochondrial membrane potential and caspase activation, we used a specific fluorescent probe (JC-1) to examine the mitochondrial membrane potential of PC3 cells treated with different compounds. Ψm Although aurB exposure did not affect the Δ ΨmIn addition, the mitochondrial localization of apoptosis regulators was increased by Δ Ψm It has been reported that aurB induces a decrease in mitochondrial function, which induces activation of caspases, critical mediators of apoptotic cell death. Because caspase-3 is a key mediator of mitochondrial apoptosis, we assessed the effect of Z-DEVD-FMK, a cell membrane-permeable inhibitor that irreversibly binds to the catalytic site of caspase-3, to determine whether caspases mediate aurB-induced apoptosis. The caspase-3 inhibitor significantly blocked the cytotoxic / apoptotic effects induced by aurB in PC3 cells in a dose-dependent manner (Figure 9B). These in vitro results indicate that aurB localizes to mitochondria and mediates ΔAurB activation in a p53- and AR-independent manner. Ψm We demonstrated that IL-1 induces apoptotic prostate cancer cell death by downregulating IL-1 and IL-2 activation.

[0055] Example 6: Effects of aurB observed in vivo. To examine whether the effects of aurB observed in vitro could be translated in vivo, we used an athymic mouse xenograft model. In mice bearing PC3 xenograft tumors, aurB significantly inhibited PC3 tumor growth over the course of 4 weeks of intraperitoneal treatment (P<0.01) (Fig. 10A) without inducing any behavioral changes or weight loss (Fig. 10B). At the end of treatment, aurB and Ptxl treatment inhibited tumor growth by approximately 65% ​​and 52%, respectively (relative to PBS control). In contrast, consistent with the in vitro data, p28 showed no antitumor effect against p53-null PC3 xenografts (Figs. 4, 5, 7). Consistent with the tumor growth data, aurB significantly reduced tumor weight (Figs. 10A, 10C). Histological analysis of tumors showed that Ki-67 stained proliferating cells were randomly distributed in the tumor parenchyma of control animals (Fig. 10D). However, treatment with aurB substantially reduced the number of proliferating Ki-67 positive cells (Fig. 10D). To determine the apoptotic effect of aurB in vivo, we employed a TUNEL assay to detect caspase-3 staining and DNA fragmentation. Tumors from control animals contained a small number of TUNEL positive apoptotic cells, whereas the number of apoptotic cells in aurB-treated animals was increased (Fig. 10D). A substantial increase in caspase-3 positive cancer cells was found in tumors from aurB-treated animals compared with tumors from control animals (Fig. 10D). These data confirm the in vitro data above and suggest that aurB suppresses tumor growth by inducing caspase-mediated apoptosis.

[0056] Example 7: Bacteria and Auracyanin C. aurantiacus can be commonly found in bacterial mats in hot springs at temperatures ranging from 45 to 70 °C and pH ranges from 6.5 to 9.0, and grows phototrophically under anaerobic conditions or chemosynthetically under aerobic and dark conditions. The presence of multiple metabolic pathways and the ability to grow under such extreme conditions suggests that Chloroflexus evolved in a variety of environments. Based on 16S rRNA analysis, Chloroflexus species are the earliest divergent bacteria capable of photosynthesis, and C. aurantiacus has long been regarded as a key organism for elucidating the mysteries of the origin and early evolution of photosynthesis. In general, prokaryotes, including C. aurantiacus, use a variety of electron donors and acceptors during energy production and may have alternative complexes that perform the same catalytic reactions as mitochondrial complexes. Mitochondria play important roles in energy production and apoptosis in eukaryotic cells and are also considered to be the descendants of endosymbiotic bacteria. In the bacterium Chloroflexus, both auracyanin A and B are membrane-anchored electron transfer proteins involved in energy production, as are many mitochondrial proteins, but they are localized to different membrane sites and their functions do not overlap. Thus, each protein has a separate biological function (see model in Figure 11). Auracyanin A is proposed to be transported to the outer membrane to facilitate aerobic respiration, while auracyanin B is anchored to the inner membrane and has functions in photosynthesis and aerobic respiration. Unlike auracyanin A and B, azurin is found only in non-photosynthetic proteobacteria and is transported to the periplasmic space without membrane anchoring. In contrast, the electron transfer complex and subunits of ATP synthase in eukaryotic cells are localized to the inner mitochondrial membrane, as is auracyanin B, which is bound to the bacterial inner membrane (Figure 11). Our results suggest that shared properties of some bacterial and mitochondrial proteins inherently enable bacterial proteins to target mitochondria (Fig. 8, Fig. 12). Despite their structural similarities, the differences in the subcellular localization and mechanism of action of these proteins may be explained by differences in their hydrophobicity / polarity.These differences may be the reason why aurB, unlike aurA and p28, can localize to mitochondria, bind to mitochondrial proteins, and subsequently induce caspase-mediated apoptotic cancer cell death in a p53-independent manner. Based on our ideas and results above, other mitochondria-targeting peptides can be identified from cupredoxins by larger-scale screening studies.

[0057] Example 8: High levels of P. aeruginosa azurin in serum of CF patients Based on the high frequency of colonization by P. aeruginosa in CF patients, the lower incidence of melanoma and breast cancer in CF compared to non-CF patients, and previous descriptions of the cancer-specific effects of azurin, we hypothesized that azurin from P. aeruginosa plays a role in tumorigenesis. To test our hypothesis that the bacterial protein azurin is detectable in human serum and that there is a significant increase in azurin levels in CF patients, we first compared the serum levels of azurin in CF patients with chronic Pseudomonas infection with those of disease-free healthy volunteers (controls; see Figure 13A) and found that the azurin levels in CF patients were significantly higher than those in controls (Figure 13B). The serum levels of azurin correlated with the age of the CF patients (Figure 13C). This clinical evidence demonstrated that CF patients have significantly elevated levels of azurin in their circulation.

[0058] Example 9: P. aeruginosa azurin secretion is stimulated by cancer cells To examine how host cells, either human cancer cells or their corresponding normal counterparts, affect P. aeruginosa azurin secretion, azurin secretion levels were measured by P. aeruginosa-host cell culture. Azurin secretion was significantly higher in the presence of various human cancer cell lines than in the presence of their normal counterparts, as were the transcript levels of the azurin-encoding gene, azu (Fig. 14A, Fig. 15). The evidence that the incidence of melanoma and breast cancer is low in CF patients and that these two cancer types showed large differences in azurin secretion in our experimental system led us to focus on these cancers in the remainder of this study. Azurin secretion by P. aeruginosa correlated positively with the number of human breast cancer (Fig. S4B) or melanoma (Fig. S4C) cells, and azurin secretion was significantly lower when P. aeruginosa was incubated with normal breast cells or benign nevus (congenital melanocytic nevus, CMN) cells (Fig. S4A-S4C). These results suggest that P. aeruginosa preferentially secretes azurin in the presence of cancer cells and that this effect is dose-dependently related to the population of cancer cells.

[0059] We next determined whether the regulation of azurin secretion mediated by bacteria-cancer interactions required direct cell-cell contact between P. aeruginosa and cancer cells. Secreted azurin levels were highest at the shortest distance (2 mm) between bacteria and cancer cells and extremely low at distances greater than 12 mm, showing an inverse correlation (Figure 14D). Despite the lack of physical contact, azurin secretion was induced, thereby indicating that a soluble factor secreted by Mel-2 cells could act as a stimulus. The change in azurin levels was not due to contamination of the top or bottom wells with P. aeruginosa (Figure 16). These results suggest the presence of at least one soluble agent originating from cancer cells that exerts a concentration-dependent stimulatory effect on azurin secretion.

[0060] Example 10: Cancer cells secrete aldolase A in response to azurin The significant stimulation of azurin secretion by malignant host cells compared to that by their normal counterparts suggested that these cell populations differ in their secretion of host factors. To identify any proteins that are differentially secreted in the presence of P. aeruginosa, we used a mass spectrometry-based proteomic approach. We found that human aldolase A was extracellularly released into the culture medium when human cancer cells or their normal counterparts were co-incubated with P. aeruginosa (Figures 17A-17C).

[0061] Aldolase, also called fructose-bisphosphate aldolase (FBA), is a glycolytic enzyme involved in the Embden-Meyerhof-Parnas glycolysis pathway and gluconeogenesis, and is highly conserved in bacteria, archaea, and eukaryotes. Human aldolase A secretion by human cancer cells in the presence of P. aeruginosa was significantly higher than that by their normal counterparts (Figure 18A). The change in aldolase A secretion was not due to modulation of cell growth rate or induction of toxicity in cancer cells (Figures 19A-19B). When human malignant and benign cells derived from various tumors and tissues were co-incubated with P. aeruginosa, the secretion level of aldolase A in the culture medium was related to the secretion level of azurin (Figure 18B). To address the role of azurin in aldolase A secretion, we took three experimental approaches: (1) aldolase A secretion was induced by wild-type (WT) and azu gene deletion mutant P. aeruginosa, (2) nonpathogenic E. coli expressing the azurin gene, and (3) recombinant azurin protein. ヌルAldolase A secretion was measured when co-incubated with mutant P. aeruginosa. Aldolase A secretion induced by WT P. aeruginosa was azure. ヌル The azu mutant was significantly higher than that of the ヌル The mutants still induced aldolase A secretion from cancer cells (Figure 18C, Figure 20). Next, the interaction of the nonpathogenic E. coli laboratory strain JM109 (a K-12 derivative) transformed with the gene encoding azurin with cancer cells was evaluated to determine whether the secretion of azurin and aldolase A was specific to the P. aeruginosa strain we used. E. coli expressing the azu gene secreted significant amounts of azurin in the presence of Mel-2 cells. In addition, aldolase A secretion from Mel-2 cells was induced by azu-expressing E. coli (Figure 21), suggesting that (1) azurin is the major inducer of aldolase A secretion but not the only inducer from P. aeruginosa and (2) E. coli has a secretion mechanism similar to that of P. aeruginosa.

[0062] To further explore the effect of human aldolase A on azurin secretion during the interaction of bacteria and human cancer cells, P. aeruginosa cells were cultured in the presence of purified human aldolase A (purity >95%), and azurin secretion was measured by Western blotting. Exposure of P. aeruginosa to aldolase A induced azurin secretion (Fig. 18D). Conversely, when breast cancer and melanoma cells were cultured in the presence of purified azurin, these cancer cells secreted significant amounts of human aldolase A into the culture medium without increasing the gene expression or intracellular levels of aldolase A (Fig. 18E, 18F), suggesting that azurin-induced aldolase A secretion occurs in a transcript-independent manner (Fig. 22A-22B). Similar to the azurin secretion pattern shown in Figure 14D, the level of secreted aldolase A was highest at the shortest distance (2 mm) between P. aeruginosa and cancer cells (Figure 18G). The secreted azurin played a major role in inducing aldolase A secretion, and their interaction is a bidirectional and concentration-dependent mutual communication between P. aeruginosa and cancer cells via azurin and aldolase A (Figure 18).

[0063] Example 11: Aldolase A promotes P. aeruginosa localization on cancer cells Although the cytosolic role of aldolases in glycolysis and gluconeogenesis has been recognized for many years, aldolases are also reportedly involved in host cell adhesion and biofilm formation in bacteria and parasites, such as Streptococcus, Neisseria, Toxoplasma, and Plasmodium. This motivated us to investigate whether aldolase A secreted by human cancer cells plays a similar biological role in the adhesion of P. aeruginosa to cancer cells. First, we tested whether silencing the aldolase A gene would modulate the adhesion of P. aeruginosa to cancer cells. P. aeruginosa adhesion assays showed that siRNA-induced silencing of aldolase A in MDA-MB-231 and Mel-2 cells (Figure 23A) significantly reduced P. aeruginosa adhesion (Figure 24A). Conversely, in the presence of purified human aldolase A, P. aeruginosa showed a significant increase in adhesion to cancer cells, which was dose-dependent and saturable (>1 μM) (Figure 24B). It has been reported that the O-glycosylated membrane-anchored mucin MUC1 of cancer cells interacts with P. aeruginosa via flagellin44,45. Muc1- / - animals showed approximately 50% less adhesion of P. aeruginosa in the lungs compared to Muc1+ / + mice46. Therefore, we tested the effect of siRNA-induced MUC1 silencing in cancer cells (Fig. S4B) and in our P. aeruginosa adhesion assay. Silencing of MUC1 in MDA-MB-231 and Mel-2 cells significantly reduced P. aeruginosa adhesion (Fig. S4C).Furthermore, when recombinant aldolase A was added to MUC1-silenced cancer cells, the adhesion rate of P. aeruginosa was similar to that of the control, suggesting that aldolase-mediated adhesion of P. aeruginosa is, at least in part, independent of MUC1-mediated adhesion.

[0064] These results suggest that aldolase A induces P. aeruginosa adhesion and colonization on cancer cells. Although mucins have been suggested as the preferred attachment and colonization site of P. aeruginosa on host cells, P. aeruginosa can also bind to several other host proteins. These proteins include aldolases, and this binding is mediated by hydrophobic interactions.

[0065] Example 12: Azurin secretion is P. aeruginosa density dependent The complexity of the P. aeruginosa genome reflects evolutionary adaptations, allowing it to thrive in diverse environments, including eukaryotic hosts with which it has coexisted for millions of years. Among prokaryotes, Pseudomonas spp. are known for their complex quorum sensing (QS) systems that control biofilm formation. In general, bacterial QS of stimuli and responses correlates with population density. Based on our data, P. aeruginosa secretes low levels of azurin due to the presence of only small populations of either cancer cells (Figures 14B, 14C) or P. aeruginosa (Figures 24D, 24E). This finding suggests that high concentrations of aldolase A due to large populations of cancer cells enhance azurin secretion by increasing bacterial adhesion and bacterial population density. However, this quorum-sensing mechanism in P. aeruginosa is probably independent of the cell-cell QS communication system, because it has been previously shown that azurin is actively expressed in Pseudomonas mutants with mutations in the Gac / Rsm system, which activates the QS mechanism mainly by stimulating N-butanoyl-L-homoserine lactone (C4-HSL) production. In addition, azurin expression is not regulated as a virulence factor in P. aeruginosa, because GacA is the major positive regulator of virulence in P. aeruginosa. Similar to the relationship between Burkholderia (previously considered to be members of the genus Pseudomonas) and plants, this QS-independent mechanism appears to play an important role in P. aeruginosa-cancer interactions.

[0066] Example 13: P. aeruginosa is abundant in primary tumors and its product azurin inhibits tumor growth Our in vitro results showed that P. aeruginosa and cancer cells communicate with each other through the secreted proteins azurin and aldolase A, leading to the localization of P. aeruginosa on cancer cells (Figs. 14, 18, 24). This effect may be applicable in clinical settings. To measure P. aeruginosa localization in human melanoma and breast tumors, we performed PCR with azu-specific primers. We included both primary and metastatic tumors because their different characteristics may affect P. aeruginosa localization. In tumors from patients with melanoma (primary: N = 29, age range = 21-84 years; metastasis: N = 34, age range = 24-85 years) and breast cancer (primary: N = 22, age range = 30-81 years; metastasis: N = 14, age range = 28-79 years) (Figure 25), the P. aeruginosa azu gene was detectable in 27.6% of primary melanomas and 5.9% of metastatic melanomas (Figure 26A). Primary breast cancers showed a higher azu gene positivity rate than metastatic breast tumors (22.7% vs. 14.3%, Figure 26B). The presence of P. aeruginosa and its product azurin was further confirmed by transmission electron microscopy (TEM) in azu-positive melanoma cells stained with anti-P. aeruginosa or anti-azurin antibodies (Fig. 26C, 26D, Fig. 27A-27C). P. aeruginosa cells were found in the cytoplasm, and azurin was localized in both the cytoplasm and nucleus. This finding is consistent with our preclinical study showing that azurin is localized in the nucleus. These results indicate that P. aeruginosa was detectable in human tumors, raising the possibility that in some individuals, azurin-producing P. aeruginosa may affect tumor biological activity via bacteria-cancer interactions. To test this possibility, we compared survival between patients with azu-positive and azu-negative primary melanoma and breast cancer tumors.In both melanoma (Figure 26E) and breast cancer (Figure 26F), patients with azu-positive tumors had better overall survival times than patients with azu-negative tumors, suggesting that P. aeruginosa azurin positively affects patient prognosis, at least in melanoma or breast cancer patients.

[0067] In this study, we investigated the effect of P. aeruginosa azurin on cancer in vivo using a transgenic mouse model that spontaneously develops mammary tumors. Based on the critical role of azurin in P. aeruginosa-cancer interactions (Figures 14, 18, 24), purified P. aeruginosa azurin was injected into transgenic mice that spontaneously develop mammary tumors. In vitro experiments showed that P. aeruginosa azurin secretion was significantly higher in the presence of triple-negative MDA-MB-231 cells than in the presence of T-47D (ER+, PR+, Her2-) cells (Figure 28). Therefore, triple-negative p53wt MMTV-PyMT transgenic mice were used. When these mice were exposed to azurin, NIR dye-conjugated azurin preferentially localized in spontaneous mammary tumors (Figure 29A). In the azurin-treated group, tumor growth and tumor weight were significantly inhibited without affecting body weight (Figure 26G, Figure 29B, 29C), and tumor-free survival was significantly extended (Figure 26H). These findings suggest a novel role for the bacterium P. aeruginosa in host defense through azurin production.

[0068] We demonstrate bidirectional control of bacteria-cancer communication in relation to the potential of azurin secreted by P. aeruginosa to inhibit tumor growth. Human cancer cells upregulated aldolase A secretion in response to increased P. aeruginosa / azurin proximity, which correlated with enhanced P. aeruginosa adhesion to cancer cells. Our results also show that cancer patients had detectable P. aeruginosa and azurin in their tumors and showed increased overall survival when they did. Finally, our results suggest that host-bacteria symbiotic mutualism serves as a versatile adjunct to the host defense system for tumor homeostasis.

[0069] This evidence suggests that some bacteria may be dual-purpose microorganisms with symbiotic relationships that can both harm (parasitic) and benefit (mutualistic) the host. During evolution, P. aeruginosa has acquired the ability to both harm (parasitic) and benefit (mutualistic) human health, adjusting its behavior according to physiological / cellular conditions that may change depending on disease states. Our results suggest that P. aeruginosa may have a symbiotic relationship with humans that is characterized by both mutualism and parasitism. In its parasitic relationship, the opportunistic pathogen P. aeruginosa increases morbidity and mortality in CF patients, whereas in its mutualistic relationship, P. aeruginosa can benefit human health by suppressing and attacking malignant cells in the host through the secretion of the bacterial protein azurin (Figure 30). azu-expressing P. aeruginosa was found in tumors of primary melanoma and breast cancer patients who had not received chemotherapy prior to specimen collection. Furthermore, our preliminary data on human tumors showed that azu-positive patients had longer overall survival times than azu-negative patients, suggesting that tumor-localized P. aeruginosa may positively affect cancer prognosis. We demonstrate that molecular determinants of host-bacteria mutualism act as diverse adjuncts of the host defense system through inter-kingdom signaling by evolutionarily conserved proteins, the bacterial cupredoxin azurin and human aldolase A. These data on host-microbe interactions provide novel insight into symbiotic strategies based on the microbial cupredoxin azurin in regulating host defense mechanisms that occur independent of immune system stimulation.

[0070] method: Peptide synthesis: Peptides were chemically synthesized with >95% purity and mass balance (CS Bio, Menlo Park, CA). The sequences were as follows: aurA (aa 62-89 of auracyanin A): LVK GGE AEA ANI ANA GLS AGP AAN YLPA (SEQ ID NO:1), aurB (aa 61-88 of auracyanin B): LVN GGD DVA AAV NTA AQN NAD ALF VPPP (SEQ ID NO:2), and p28 (aa 50-77 of azurin): LST AAD MQG VVT DGM ASG LDK DYL KPDD (SEQ ID NO:3).

[0071] Cell lines and culture: Human cancer and non-cancer (immortalized and non-immortalized) cell lines were obtained from the American Type Culture Collection [prostate cancer (PC3, DU145, and LNCaP), normal prostate (CRL11611), breast cancer (MCF-7), ovarian cancer (SK-OV3 adenocarcinoma), and lung cancer (A549)]. MDD2 breast cancer cells (p53 dominant negative) were kindly provided by Dr. Andrei V. Gudkov at Roswell Park Cancer Institute. All 20 cell lines were cultured in MEM-E (Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum (Atlanta Biological, Inc.), 100 units / mL penicillin, and 100 μg / mL streptomycin at 37°C with 5% CO2.

[0072] Cytotoxicity assay: MTT assays were performed as previously described by the inventors (Cha et al., Cancer Research 2020, Vol. 80: p. 1615-1623).

[0073] Cell membrane toxicity: LDH assays were performed as previously described by the inventors (Cha et al., Cancer Research 2020, Vol. 80: p. 1615-1623).

[0074] TEM: GNRs (Nanopartz Inc., CO) were conjugated in vitro with aurA and aurB according to the manufacturer's instructions. Briefly, NHS-functionalized non-spherical GNRs with diameter 25 nm × length 73 nm were conjugated with 500 molar excess of aurA or aurB in 0.1 M borate buffer (pH 8.0) at room temperature for 4 h. The labeled peptides were washed with 1% PBS / 0.1% Tween 20 at 9,000 rcf for 10 min and resuspended in PBS. After exposing PC3 cells to aurA-GNRs or aurB-GNRs (300 μg / ml) for 16 h, approximately 1 mm3 cell culture samples were fixed with 4% phosphate-buffered glutaraldehyde, and then the samples were washed with 2% sucrose in 0.1 M Sorensen's phosphate buffer at room temperature. The samples were then treated with 1% osmium tetroxide in 0.1 M Sorensen's phosphate buffer for 1 h at room temperature. After washing with dH2O and dehydration with acetone, resin infiltration (EMBed 812 resin [EMS]) was performed as follows: 30 min, 2:1 mixture of 100% acetone:resin; 30 min, 1:1 mixture of 100% acetone:resin; 1 h, 1:2 mixture of 100% acetone:resin; and 1.5 h, 100% resin. The samples were then placed in embedding molds and polymerized overnight at 60°C. Sections (60 nm thick) were made using an Ultratome and mounted on 200 mesh copper grids. TEM images of uranyl acetate and lead citrate stained samples were taken with a JEOL 1220 TEM.

[0075] Pull-down assay and protein identification: PC3 cells were washed twice with PBS, and mitochondrial fractions were prepared with Qproteome mitochondrial isolation kit (Qiagen). Mitochondrial proteins were extracted with 10% n-dodecyl-β-D-maltoside (Mitochondrial Protein Immunoprecipitation Kit, Sigma) and incubated with biotin-labeled p28, aurA, or aurB for 16 h at 4°C. The peptides were then preincubated with streptavidin-agarose beads for 4 h. After washing, the beads were boiled, and the released proteins were subjected to SDS-PAGE analysis. The approximately 35 kDa protein band was subjected to microcapillary LC / MS / MS for protein identification (Taplin Mass Spectrometry Facility, Harvard Medical School). For IB, proteins were transferred to nitrocellulose membranes. After blocking with 5% BSA in TBST, the membrane was incubated with anti-ATP5C antibody (Abcam) at 1:25,000 in 5% BSA / TBST for 16 h at 4°C. Secondary antibody was added (anti-goat IgG-HRP; Santa Cruz Biotechnology). Signals were detected using enhanced chemiluminescence (ECL).

[0076] Flow cytometry analysis: Three prostate cancer cell lines and normal cell lines were treated with 1 nM Ptxl, 50 μM p28, 0.5-50 μM aurA, or 0.5-50 μM aurB. After 48 hours, Annexin-V apoptosis assay (Thermo Fisher Scientific) was performed to detect apoptotic cells. At least 10,000 cells were analyzed by FACS each (RRC, UIC).

[0077] Mitochondrial membrane potential was measured using JC-1 dye (Invitrogen). PC3 cells were exposed to 1, 10, and 100 μM aurA or aurB. After washing with PBS, trypsinized PC3 cells were incubated with JC-1 dye and analyzed by FACS.

[0078] Caspase assay: Similar to the procedure for the MTT assay, PC3 cells were exposed to aurB in the presence or absence of the specific caspase inhibitor Z-DEVD-FMK.

[0079] Xenograft animal model: Human prostate cancer cells (PC3) were injected subcutaneously into the right flank of 5-6 week old male athymic mice78. When tumors reached 5 mm in diameter, animals were randomized into control and treatment groups. Doses were as follows: Ptxl (10 mg / kg once weekly), p28 (10 mg / kg daily), aurA (10 mg / kg daily), and aurB (5 mg / kg daily), intraperitoneally. Results are presented as mean + SE (N = 5). All animals were weighed twice weekly. Tumors were dissected and weighed at necropsy. Statistical comparisons were performed by one-way analysis of variance (ANOVA) (control vs. treatment). Tumor samples were fixed overnight in 10% buffered formalin. Fixation was followed by dehydration, clearing, and infiltration. Samples were then embedded in paraffin and 4 μm sections were cut on a microtome. Ki67 (LabVision) staining was performed on tissue sections using the Vector Vectastain Elite ABC kit. TUNEL assays were performed on sections using the Millipore Sigma ApopTag® Peroxidase In Situ Apoptosis Detection Kit.

[0080] P. aeruginosa and human cell lines: P. aeruginosa strain 8822, isolated from sputum of a CF patient, was a gift from Dr. Ananda M. Chakrabarty. Human cell lines of prostate cancer (DU-145), normal prostate (CRL-11611), ovarian cancer (SK-OV3), normal ovary (HOSE6-3), breast cancer (MCF-7, T-47D, MDA-MB-231), and normal breast (MCF-10A) were purchased from the American Type Culture Collection (ATCC, Virginia (VA)). Human melanoma (UISO-Mel-2) and CMN cell lines were developed in our laboratory as described.

[0081] ELISA: CF subjects were patients followed at the Adult CF Clinic at Emory University. Patients had signed informed consent to provide blood samples in accordance with the Emory University IRB (Emory #00042577). Control serum from non-diseased volunteers was obtained from Discovery Life Sciences (Los Osos, CA). Serum samples (N=50 in each group) in carbonate-bicarbonate buffer (pH 9.4) were used to coat 96-well plates (MaxiSorb, Thermo Fisher) in triplicate. A standard curve was generated using purified azurin 75 to coat the 96-well plates as an internal control. Azurin was detected using polyclonal rabbit anti-azurin antibodies 1 and 2 and alkaline phosphatase-conjugated secondary anti-rabbit antibody (Sigma Millipore).

[0082] Growth medium: All human cell lines were maintained in DMEM with 10% FBS, with the exception of MCF-10A, which was maintained in DMEM with 10% FBS. Luria-Bertani medium was used to grow P. aeruginosa before transferring to experimental medium of 0.5% minimal glucose medium (MGM) without antibiotics.

[0083] P. aeruginosa quantification: This was performed using turbidimetric method after establishing a standard growth curve of P. aeruginosa with 0.5% MGM. An optical density (OD) of 0.3 correlated with the mid-logarithmic phase of bacterial growth and was chosen as the standard OD at 600 nm in all experiments.

[0084] Expression and purification of azurin: The cloning, expression of the azu gene in E. coli, and purification of recombinant azurin were previously described1.

[0085] Co-incubation of P. aeruginosa and human cancer cells: P. aeruginosa strain 8822 was grown overnight at 37°C in LB medium and then transferred to 0.5% MGM at 1:100 v:v without antibiotic supplementation. The OD of the medium was measured until the experimental target OD was reached. Human cells were grown in the appropriate medium as reported above. Cells were trypsinized and counted using a Coulter Counter® cell and particle analyzer. The required number of cells were washed twice with PBS and finally suspended in 0.5% MGM. For the direct co-incubation assay, human cells and P. aeruginosa were co-incubated for 30 min with P. aeruginosa at a concentration of 500,000 human cells / ml and an optical density (OD) of 0.3. The required number of cells was transferred based on the details of each experiment. For the indirect co-incubation assay, Corning Transwell® polyester membrane cell culture plates and inserts (sterile 24 mm Transwell with 0.4 μm pores, TC-treated, Sigma Millipore) were used. Human cells were immobilized in the lower compartment (well) and bacteria were incubated in the upper compartment (insert) for 30 min at 37°C, with a 0.4 μm pore semipermeable membrane separating the two compartments. Wild-type (WT) P. aeruginosa PAO1 and its azure ヌル The mutant cells were used for aldolase secretion assay. The mutant P. aeruginosa was maintained on LB agar plates containing spectinomycin. Other experimental conditions were the same as above.

[0086] Protein extraction: Comparative analysis of secreted proteins in the medium was performed as described previously. Equal volumes of medium were collected from the cell suspension (direct co-incubation assay) or from the wells of the lower compartment (indirect co-incubation assay) after incubation and centrifuged at high speed (12,000 x g). The supernatant was then filtered using an Amicon® 0.22 μm pore filter. An equal volume of trichloroacetic acid (TCA, final concentration 20%) was added to the supernatant and incubated on ice for 30 min. After centrifugation at 18,000 x g, the TCA was decanted and two 100% acetone precipitations with 5 min incubations were performed. In the last step, the acetone was decanted and the protein pellet was dried at room temperature for 15 min and resuspended in PBS. Equal volumes of each sample were loaded per lane.

[0087] Western blotting: After NuPAGE, proteins were transferred to PVDF membranes (BioRad Laboratories Inc, Hercules, CA) and blocked with SuperBlock (T20 TBS buffer, Thermo Fisher) for 1 h. For secreted proteins, equal loading was confirmed by Ponceau staining of the membranes. PVDF membranes were incubated overnight at 4°C in polyclonal rabbit anti-azurin antibody 1 (1:5000) or monoclonal mouse anti-aldolase A (1:200, Santa Cruz Biotechnology, TX). For cell lysates, GAPDH was used as a loading control (Novus Biologicals). Secondary antibody was added (1:1000, polyclonal goat anti-rabbit IgG-HRP, Santa Cruz Biotechnology). Signals were detected using enhanced chemiluminescence (ECL, Thermo Fisher Scientific). Quantitative analysis of bands was performed by computer-assisted imaging densitometer (UN-SCAN-IT Gel version 5.1).

[0088] Host cell adhesion assay: The assay was performed essentially as described previously38. Cancer cells were co-incubated with P. aeruginosa 8822 in the presence or absence of various concentrations of aldolase A in 96-well plates for 2 h. Cancer cell monolayers were washed extensively with PBS to remove unbound P. aeruginosa. Monolayers were homogenized with 0.1% saponin (Sigma Millipore) in PBS. P. aeruginosa were enumerated by serial dilution of the homogenized suspension followed by plating on LB agar plates to determine colony forming units (CFU / ml). The control value of CFU / ml is expressed as 100%. siRNA-induced silencing of aldolase and MUC1 genes in MDA-MB-231 and Mel2 cells was performed as previously described. 80, 81 Briefly, SMARTpool human ALDOA, MUC1, and non-targeting siRNA pools (non-targeting siRNA) (Dharmacon, PA) were used as siRNAs targeting aldolase A, MUC1, and negative control, respectively. MDA-MB-231 and Mel2 cells were seeded in 96-well plates, grown to 80-90% confluence, transfected with 120 nM siRNA for 48 h using FuGENE-HD (Promega) according to the manufacturer's protocol, and used for host cell adhesion assays. Knockdown of ALDOA and MUC1 by siRNA transfection was examined by Western blot analysis using monoclonal mouse anti-aldolase A and MUC1 antibodies (1:200, Santa Cruz Biotechnology).

[0089] Detection of azurin gene in human tumors: Breast and melanoma tumor samples were collected at the University of Illinois at Chicago. All patients included in the analysis were diagnosed with either breast cancer or melanoma. Supplementary Figure 10 includes relevant patient information with similar age range and median age. Detection of azu gene in human tumors was performed by PCR with azu-specific primers 5'-CAGTTCACCGTCAACCTGTCC-3' (SEQ ID NO: 5) and 5'-TGGTGTGGGCGATGACACG-3' (SEQ ID NO: 6). Human GAPDH was also amplified as a loading control; 5'-AACGGGAAGCTTGTCATCAA-3' (SEQ ID NO: 7) and 5'-TGGACTCCACGACGTACTCA-3' (SEQ ID NO: 8). We used ultraclean equipment, kits, and reagents to minimize and control contamination. Template DNA was prepared using the DNeasy Blood & Tissue kit (Qiagen). The amplified PCR products were identified as single bands on 2% agarose gels. The products were purified using a QIAquick PCR Purification Kit (Qiagen) and sequenced by an ABI Prism 3700 DNA analyzer. Fisher's exact test was performed to determine whether the positive rates were significantly different between subjects with primary and metastatic tumors.

[0090] Visualization of P. aeruginosa and azurin by TEM: Frozen tumors were processed for immunogold transmission electron microscopy according to published methods with minor modifications. Tumor samples, along with their survival data, were obtained from patients who signed informed consent (IRB#H-96-772). Frozen samples were chopped into 1 × 1 mm cubes on dry ice and then fixed by incubation in PBS containing 4% paraformaldehyde and 0.75% glutaraldehyde at 4°C for 72 hours. Samples were washed extensively with PBS and then incubated with PBS containing 0.1% saponin on a rotary mixer (Ted Pella, Inc, Redding, CA) for 1 hour at room temperature. After the samples were thoroughly washed with PBS, they were incubated with PBS containing 5% bovine serum albumin (BSA), 0.1% cold water fish skin gelatin (CWFS), and 5% normal goat serum (NGS) (goat gold conjugate blocking solution, Electron Microscopy Sciences, Hatfield, PA) for 1 h at 4°C. Samples were washed extensively with PBS containing 0.1% acetylated bovine serum albumin (BSA-c) (Aurion BSA-c, Electron Microscopy Sciences) and then incubated with either mouse anti-Pseudomonas aeruginosa antibody (clone B11, 1:50 dilution, Thermo Fisher Scientific, Waltham, MA) or rabbit anti-azurin antibody (1:100 dilution) for 48 h at 4°C. After extensive washing, samples were incubated with either goat anti-mouse or goat anti-rabbit secondary antibodies in 0.1% BSA-c / PBS for 24 h at 4°C.To visualize each specific primary antibody, these antibodies were labeled with 10 nm gold particles (1:50 dilution, Electron Microscopy Sciences). Samples were washed with PBS-0.1% BSA-c and deionized water, then incubated with 2.5% glutaraldehyde (Electron Microscopy Sciences) in deionized water for 1 h at room temperature. After extensive washing with deionized water, sections were fixed with 0.5% osmium tetroxide and 1.5% potassium ferricyanide (Electron Microscopy Sciences) in deionized water for 15 min in the dark. Samples were then dehydrated by incubation in an ascending series of ethanol (25, 50, 75, 95, 100%, Electron Microscopy Sciences). They were then incubated in a 1:1 ratio of 100% ethanol to epoxy resin (EMbed 812, consisting of a mixture of methylnadic anhydride, dodecenylsuccinic anhydride, and 2,4,6-tris(dimethylaminomethyl)phenol, Electron Microscopy Sciences) for 12 hours at room temperature on a rotary mixer (Ted Pella, Inc). Samples were incubated with 100% epoxy resin on a rotary mixer (Ted Pella, Inc) for 12 hours at room temperature. The epoxy resin was replaced and samples were incubated on the rotary mixer for 2 hours at room temperature. The samples were placed into a flat embedding mold filled with epoxy resin and then polymerized at 70° C. for 24 hours.Ultrathin sections (70 nm) were cut with an ultramicrotome (EM UC7, Leica Microsystems, Buffalo Grove, IL), mounted on Formvar carbon-coated 200 mesh copper grids (Electron Microscopy Sciences), and stained with filtered 1% uranyl acetate prior to imaging. Samples were imaged with a Philips CM 120 transmission electron microscope (TSS Microscopy, Hillsboro, OR) equipped with a BioSprint 16 megapixel digital camera (Advanced Microscopy Techniques, Woburn, MA).

[0091] Patient survival: The survival of each patient with primary melanoma and breast cancer was measured from the date of diagnosis to death from any cause or last follow-up. The study was based on a randomized trial, and the investigators were blinded to the allocation in the study and outcome assessment. Two independent sample studies were performed to compare the survival times of the azu gene positive and negative groups. The Shapiro-Wilk test showed that there were significant violations of normality in the data of melanoma patients. The Wilcoxon rank sum test of the median of melanoma survival data was chosen because of its robustness against deviations from normality. In breast cancer patients, the Shapiro-Wilk test showed that the survival times of both the azu gene positive and negative groups could be well modeled by a normal distribution. Since the equality of variance between the two groups was appropriate and confirmed by F-test, a pooled t-test of the means was performed to compare the survival times between the azu gene positive and negative groups. Data were analyzed using R (v.4.0.2) and RStudio (v.1.3.1093).

[0092] Effects of azurin on transgenic mice: Hemizygous MMTV-PyMT (mouse mammary tumor virus-polyoma middle tumor antigen) female mice were obtained from Jackson Laboratory. This mouse model has been widely used and well characterized because MMTV-PyMT mice spontaneously develop mammary tumors that closely resemble the progression and morphology of human breast cancer. 84 Four-week-old mice were randomized into control (N=7) and azurin-treated (N=5) groups. Control animals received PBS, and azurin-treated animals received 2.5 mg / kg azurin in sterile PBS intraperitoneally three times a week for nearly two months (80 days of age). Based on azurin levels in the serum of CF patients, the highest level of azurin was 32 μg / ml. To reach this level of azurin in mice (25 g body weight, 2 ml total blood volume), the azurin dose was determined to be 2.5 mg / kg. Tumor volumes and body weights were measured three times weekly. All tumors were excised and weighed at the end of the study. All experiments were approved by the University of Illinois at Chicago (UIC) Institutional Animal Care and Use Committee (IACUC) and adhered to the US Animal Welfare Act and guidelines established by the National Institutes of Health.

[0093] Statistics and reproducibility: Student's paired t-test, one-way ANOVA were used for comparisons. P-values ​​<0.05 were considered significant. The number of replicates (N) can be found in the figure legends. Data were analyzed using Graphpad Prism software (version 8), R (version 4.0.2), and RStudio (version 1.3.1093).

[0094] RNA extraction and real-time PCR of azurin expression: RT-PCR was performed with 1 μg of total RNA from control and treated samples. SuperScript First-Strand Synthesis System for Real-Time Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) (Invitrogen, Carlsbad, CA) and SYBR® Green PCR Master Mix (Applied Biosystems, Foster City, CA) were used. Primers were designed using software from Integrated DNA Technologies (Coralville, IA). Reactions were performed as a two-step RT-PCR method using the RT-PCR Reagent Kit from Invitrogen and the ABI 7500FAST Sequence Detection System (Applied Biosystems, Foster City, CA). After the last amplification cycle, the PCR products were denatured and dissociation curves were generated. Fluorescent signals were detected and analyzed by ABI Prism® 7500 sequence detection software (Applied Biosystems, Foster City, CA). Threshold cycles (Ct) were calculated by the software and used to determine the relative expression of genes.

[0095] Protein Identification by Mass Spectrometry: Mel-2 and P. aeruginosa 8822 were co-incubated using a Transwell system and the secretions were concentrated using Amicon Centriprep YM-3 centrifugal filters (Thermo Fisher Scientific). The concentrated samples were run on SDS-PAGE. Gels were stained with Coomassie Brilliant Blue R-250 (CBB) (0.1% CBB R-250, 20% methanol, 0.5% acetic acid) and destained for 1 h (BioRad). Mel-2 and P. aeruginosa samples showed additional bands compared to the Mel-2 only sample. The additional protein bands were excised from the gel. In-gel tryptic digestion and matrix-assisted laser desorption / ionization time-of-flight (MALDI-TOF) mass spectrometry of tryptic peptides were performed as follows. The gel plugs were washed with 50% acetonitrile, sulfide bonds were reduced with 60 mM DTT, and free sulfhydryl groups were alkylated with iodoacetamide, 50 mM ammonium bicarbonate (pH 8.0), and 5 mM EDTA, followed by overnight incubation in trypsin (2 μg / 100 μl in 50 mM ammonium bicarbonate (pH 8.0)). For MALDI-TOF, the remaining peptides were extracted, spotted on a MALDI-TOF target, and analyzed in the positive ion reflector mode with delayed extraction in the m / z range of 700-4000. A Voyager DE-PRO mass spectrometer equipped with a nitrogen laser (Applied Biosystems, Foster City, CA) was used. Spectra were externally and internally calibrated. Peptide mass results were used to identify proteins. The MASCOT peptide fingerprint link was used.

[0096] Cell counting and MTT assay: Growth of Mel-2 cells after co-incubation with P. aeruginosa 8822 was assessed by counting the number of cells at the desired time points and comparing the cell number with the initial cell count. Cells were plated in 24-well cell culture plates at a density of 4 x 105 cells / well in MEME supplemented with FBS, L-glutamine, and non-essential amino acids. They were incubated at 37°C in a 5% CO humidified atmosphere and allowed to adhere overnight. P. aeruginosa (OD = 0.3) was co-cultured with Mel-2 for 30 min. After incubation, P. aeruginosa were aspirated and Mel-2 were washed with PBS, trypsinized, made into single cell suspension, diluted with Isoton II diluent, and counted in triplicate using a Coulter Counter cell and particle analyzer. Total cell counts were obtained considering the dilution factor and total volume of cell suspension from each well. Potential antiproliferative effects of P. aeruginosa 8822 on Mel-2 viability were also assessed using the 3-[4,5-dimethylthiaolyl]-2,5-diphenyl-tetrazolium bromide (MTT) assay (TACS-MTT Cell Proliferation Assay Kit, Trevigen, Gaithersburg, MD). Cells were seeded at a density of 5,000 cells / well in 96-well plates. Cell viability was analyzed after co-incubation with P. aeruginosa for 30 min. After co-incubation with P. aeruginosa, Mel-2 was incubated with MTT tetrazolium reagent for 2 h at 37°C, and then the absorbance of formazan was measured at 570 nm. Each treatment was performed in triplicate, and the percentage of inhibited cell growth was calculated by comparison of the absorbance readings of the control (Mel-2 only) to Mel-2 co-incubated with P. aeruginosa.

[0097] Gene expression and intracellular levels of aldolase A. MDA-MB-231 and Mel-2 were treated with azurin for 30 min. Total RNA was extracted from cancer cells, and cDNA was synthesized using a High Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific). The conditions for reverse transcription were as follows: 25°C for 10 min, 37°C for 120 min, and 85°C for 5 min. The expression levels of aldolase A were measured by RTPCR using the SYBR Green assay according to the manufacturer's instructions [PowerUp SYBR Green Master Mix (Life Technologies, USA)]. The relative expression of aldolase A was calculated using the comparative Ct method. The data (N=3) were normalized with the β-actin gene as the housekeeping gene. ΔCt=CtAldolase A-Ctβ-actin. The change of treatment in aldose A signal compared to the total amount of cDNA was expressed as ΔΔCt=Cttreatment-Ctcontrol. The relative change of treatment was then calculated as 2-ΔΔCt. The following primer sequences were used: aldolase A: forward 5'-CGG GAA GGA GAA CCT G-3' (SEQ ID NO: 9) and reverse 5'-GAC CGC TCG GAG TGT ACT TT-3' (SEQ ID NO: 10), and β-actin: forward 5'-ACT GGA ACG GTG AAG GTG AC-3' (SEQ ID NO: 11) and reverse 5'-AGA GAA GTG GGG TGG CTT TT-3' (SEQ ID NO: 12). Real-time PCR was performed under the following conditions: 50°C for 2 min, 95°C for 2 min; 40 cycles of 95°C for 15 s and 60°C for 1 min; and 95°C for 15 s, 60°C for 1 min, and 95°C for 15 s.

[0098] NIR imaging of transgenic mice: Near infrared red fluorescent dye (IR800, Licor, NE) was conjugated with azurin according to the manufacturer's instructions. Transgenic mice with spontaneously developed mammary tumors were given NIR dye-conjugated azurin intraperitoneally once at 5 mg / kg. After 24 hours, the specific fluorescent signal at 800 nm was recorded by a PDE-neo® NIR camera system (Hamamatsu Photonics, Mitaka-USA).

[0099] While preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will occur to those skilled in the art without departing from the invention. It is to be understood that various alternatives to the embodiments of the invention described herein may be employed in the practice of the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

1. An isolated auracyanin peptide that regulates the apoptotic pathway mediated by mitochondria in eukaryotic cells.

2. The auracyanin peptide of claim 1, wherein the auracyanin is a C. aurantiacus protein.

3. The peptide is amino acids 62 to 89 of auracyanin A protein, or 2. The auracyanin peptide of claim 1, wherein the peptide is amino acids aa 61 to 81 of auracyanin B protein.

4. The peptide localizes to the mitochondrial membrane of the eukaryotic cell and binds to the gamma subunit of human mitochondrial ATP synthase protein; or 2. The auracyanin peptide of claim 1, wherein the peptide targets the apoptotic pathway mediated by mitochondria in cancer cells but not in healthy cells.

5. The auracyanin peptide described in claim 1, wherein the peptide has a sequence of sequence number 1 or 2.

6. The auracyanin peptide of claim 1, wherein the peptide has a pI of approximately 3.42 and a molecular weight of approximately 2400 to 2700 Da.

7. Administering a therapeutically effective amount of auracyanin peptide, which is amino acids aa61-81 of auracyanin B protein, to eukaryotic cells. Including, wherein the auracyanin peptide induces a decrease in mitochondrial membrane potential, resulting in the induction of apoptotic pathways mediated by caspases; The mediated cell death induced by the auracyanin peptide is independent of the tumor suppressor protein p53 pathway. A method for modulating the mitochondrial-mediated apoptotic pathway in eukaryotic cells.

8. A method of treating cancer by administering to a patient a therapeutically effective amount of an auracyanin peptide in a pharmaceutically acceptable form to enhance the mitochondrial-mediated apoptotic pathway in cancer cells, administering said effective amount of auracyanin peptide to said subject. Including, wherein the auracyanin peptide induces the caspase-mediated apoptotic pathway in cancer cells; A method of administering a therapeutically effective amount of an auracyanin peptide to a subject in need of treatment for cancer.

9. The method described in claim 8, wherein the auracyanin peptide is amino acids aa61 to 81 of auracyanin B protein.

10. A method of treating cancer in a subject, comprising bringing P. aeruginosa into proximity with the cancer, such that bidirectional communication between the P. aeruginosa and the cancer cells results in azurin secreted by a P. aeruginosa inhibiting cancer growth.

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