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[Cancer Biology & Therapy 3:8, 708-714, August 2004]; ©2004 Landes Bioscience

708 Cancer Biology & Therapy 2004; Vol. 3 Issue 8

Vasu Punj1

Djenann Saint-Dic2

Sharon Daghfal2

J.R. Kanwar3

1Department of Microbiology and Immunology; 2Department of Surgical Oncology;

College of Medicine; University of Illinois at Chicago; Chicago, Illinois USA

3Department of Molecular Medicine and Pathology; Faculty of Medicine and Health

Science; The University of Auckland; Auckland, New Zealand

*Correspondence to: Vasu Punj; Department of Microbiology and Immunology;

835 S. Wolcott Ave.; College of Medicine; University of Illinois at Chicago; Chicago,

Illinois 60612 USA; Tel.: 312.996.2011; Fax: 312.996.6415; Email: [email protected]

Received 04/08/04; Accepted 05/10/04

Previously published online as a Cancer Biology & Therapy E-publication:

http://www.landesbioscience.com/journals/cbt/abstract.php?id=964

KEY WORDS

microorganisms, p53, apoptosis, tumor inhibi-tion, protein-protein interaction

ACKNOWLEDGEMENTS

This investigation was supported by a researchgrant from the (American Cancer Society, IRG)UIC, Cancer Center to VP. The help of Dr. C.Vasu, Department of Microbiology and

Immunology, UIC Chicago in the completion of some experiments is acknowledged.

We also thank Drs. A.M. Chakrabarty and T.K.Das Gupta for their help and support.

Review

Microbial-Based Therapy of Cancer

A New Twist to Age Old Practice

ABSTRACTThe use of bacteria in the regression of tumors has long been known. Various approaches

for using bacteria in cancer therapy include the use of bacteria as sensitizing agents forchemotherapy, as delivery agents for cancer drugs and as agents for gene therapy. Thetumor regression stimulated by infecting microorganisms has been attributed to activationof the immune system of the host. However, recent studies indicate that when tumorharboring mice with defective immune systems are infected with certain microorganismsthe regression of the tumor is still observed, suggesting that there are other host factorscontributing to the microbial associated regression of tumors. Since the use of live orattenuated bacteria for tumor regression has associated toxic effects, studies are inprogress to identify a pure microbial metabolite or any component of the microbial celthat might have anti-cancer activity. It has now been demonstrated that a redox proteinfrom Pseudomonas aeruginosa, a cupredoxin, can enter into human cancer cells and triggerthe apoptotic cell death. In vivo, this cupredoxin can lead to the regression of tumorgrowth in immunodeficient mice harboring xenografted melanomas and breast cancertumors without inducing significant toxic effects, suggesting that it has potential anti-canceractivity. This bacterial protein interacts with p53 and modulates mammalian cellular activity.Hence, it could potentially be used as an anti-cancer agent for solid tumors and has translational value in tumor-targeted or in combinational-biochemotherapy strategies for cancertreatments. Here, we focus on diverse approaches to cancer biotherapy, includingbacteriolytic and bacterially-derived anti-cancer agents with an emphasis on their mechanism of action and therapeutic potential.

INTRODUCTION

In general the growth of tumors or tumor kinetics depends on several closely related

factors:1. Cell cycle time, or the average time for a mitotically dividing cell. This determines the

maximum growth rate of a tumor.

2. Growth fraction, or the fraction of cells undergoing cell division.

3. The total number of cancer cells in the population, which is an indicator of total canceburden. As the number of cancer cells increases, so does the number of resistant cells,

which leads to reducing the effectiveness of an anti-tumor agent.

Variations in these factors are responsible for the variable rates of tumor growthobserved in different tumors. Tumors grow most rapidly at small volumes. As they becomelarge, growth slows down based on a complex process dependent on cell loss and tumorblood and oxygen supply. In order to have the best chances for a cure, anti-tumor agentsmust be given in a manner in which they can achieve a fractional cell killing in a logarithmicfashion.

Current strategies for cancer therapy are based on radiation treatment and the use ofdrugs. However, most solid tumors contain poorly vascularized areas characterized byhypoxia or anoxia, and these areas are relatively less sensitive to radiation in the absence ofoxygen. Moreover, since drugs are transported by blood, the delivery of chemotherapeuticagents is more difficult in poorly vascularized tumors. Despite major scientific and tech-nological progress in combinatorial chemistry, drugs derived from natural products stillmake an enormous contribution to drug development today.

Microorganisms have an inexhaustible metabolic potential and can adapt to variousenvironmental conditions. Tumors with a metabolically compromised microenvironmentprovide a haven for a number of anaerobic bacteria. In the past several years, many strainsof facultative and obligate anaerobic bacteria have been shown to localize and cause lysisin transplanted tumors in animals.1-3 There are several reports suggesting a correlation

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between bacterial infection and tumor regression. To our knowledgethe first scientific report of tumor shrinkage due to therapeuticinfection was published in Berlin, Germany, in 1868.4 Some yearslater in the United States, William Coley 5 discovered that many cancer patients could be cured of advanced metastatic cancer by repeated infection or injection of bacterial extracts consisting of gram-positive Streptococcus pyogenes alone or in combination withgram-negative Serratia marcescens . Since then, a number of studies

have suggested a direct correlation between various live/attenuatedbacteria or their components and the regression of tumors.3 An idealanti-cancer bacterium should be nontoxic to the host, only able toreplicate in the tumor, able to disperse evenly throughout the tumor,be nonimunogenic and have the ability to cause lysis of the tumor.

A significant progress has been made on each of these points.Bacteria were tested as nonspecific immuno-stimulators to boostimmunity against cancer. A variety of test models were used in suchstudies, which include sarcoma, rhabdomyo-sarcoma, carcinoma,melanoma and breast cancer. The bacteria that were tested for invivo regression of solid tumors include: Clostridium (obligatory anaerobes such as C. histolyticum, C. tetani, C. butyricum, C. aceto-butylicum, C. pectinovorum, C. tyrobutyricum, and C. novyi );

Bifidobacterium (obligatory anaerobes including B. bifidum, B. infantis and B. longum); Salmonella typhimurium (a facultative anaerobe) andClostridium parvum (an obligate anaerobe). B. bifidum is an exceptionin that it is a useful part of the microflora of the human intestine andis used to prepare bacterial medicine and fermented milk products.In these experimental studies, some test animals with malignanttumors completely recovered, while regression of tumor growthoccurred in other animals. It has been demonstrated previously, thata protozoan, Toxoplasma gondii , when injected into melanoma-bearing mice, caused tumor regression by blocking angiogenesis. It wasassumed that the parasitic infection induced the release of anti-angiogenic soluble factors that created hypoxic conditions inthe tumor and led to necrosis.6 Modern molecular biology techniquescan be used to manipulate bacteria for efficient and safe use in tumor

therapy. One such example comes from studies by Dang et al.7 whoused heat shock to eliminate lethal toxin genes from Clostridiumnovyi and then used such an attenuated bacterium in combination

with the chemotherapeutic agent mitomycin C and the anti-vascularagent dolastatin-10 to treat colorectal cancer. The rationale behindthis combined therapy (called combination bacteriolytic therapy, orCOBALT) was that the anaerobic bacteria grew in the anaerobiczone within tumor cores; the anti-vascular agent would create moreextensive hypoxic areas for bacterial growth, thereby depriving thetumors of oxygen and essential nutrients while the chemotherapeuticagent attacked and destroyed the remaining well-perfused tumorcells.8 The results of this study were highly significant: in the absenceof bacteria, but in the presence of mitomycin C and dolastatin -10,

the tumors persisted for a much longer time and showed limitedregression, while inclusion of bacteria led to extensive disappearanceof the tumors within a short period of time and, in some cases, com-plete tumor dissolution that left the animal tumor-free. The majorlimitation of this study was its associated toxicity: 15–45% of themice died within few days of beginning treatment, presumably dueto the release of highly toxic metabolic products from the disinte-grating tumors. Others and we have demonstrated the up-regulationof survivin and angiogenic factors induced by Helicobacter pylori ingastric cancer as well as in solid tumors and their surrounding areas,suggesting the importance of survivin for the inhibition of apoptosisin the pathogenesis of gastric cancer.9,10 The intra-tumor plasmid

gene therapy with survivin antagonists could block the expression ofsurvivin and angiogenic factors could enhance the anti-tumor activ-ity.11 The expression of survivin in gastric cancer was associated withreduced apoptosis and COX-2 expression.

This review will describe how an in-depth knowledge of proteomicsand biochemistry, coupled with an understanding of the p53 path-

way, is allowing the development of novel strategies to manipulatethe therapeutic potential of bacterial products. The use of pure

bacterial products might help to lower the risk factors associated with the use of live/attenuated bacteria. Work in this field representsan outstanding illustration of how basic scientific research can betranslated into clinical applications for cancer therapy and the manychallenges involved.

ADJUVANT CANCER THERAPY WITH BACTERIA

Despite setbacks and inconsistent data, the work with bacterialextracts led to the approval of Bacillus Calmette-Guerin (BCG),

which is widely used for the local treatment of bladder cancer.12

Several studies have shown a clear relationship between the use ofBCG immunoprophylaxis after surgical removal of the tumor and adecreased recurrence rate or delayed period during which recurrencecould occur.13,14 The mechanism of anti-cancer activity of BCG hasbeen attributed to its effect on the immune system, with CD4 andCD8 T lymphocytes playing a major role.15 In addition to its use asan immunoprophylatic agent, BCG has been widely used as an adju-vant for modified cancer cell vaccines for several different types oftumors including central nervous system (CNS) malignancies. BCGis also included as an immunoadjuvant for CancerVax TM (CancervaxCorp., CA, USA), a polyvalent tumor cell vaccine that has beenshown to improve overall survival in stage II melanoma patients andin patients treated after curative resection of distant melanoma.16 Onthe other hand, BCG –induced production of interferon (IFN)-γ hasbeen suggested to be the effector mechanism for the anti-tumor effectof BCG on melanoma.17 In addition, an in vitro human system was

used to analyze the role of Natural Killer (NK) cells in BCG-inducedcellular cytotoxicity: mononuclear cells were treated with BCG forseven days and BCG-activated NK cells significantly destroyed bladdertumor cells. Similarly, using C57BL/6 wild type mice, NK-deficientmice and mice treated with anti-NK-1.1 monoclonal antibody,Brandau et al.18 demonstrated that the use of BCG significantlyprolonged survival in wild type mice. BCG therapy was completelyineffective in NK deficient mice or mice treated with Nk1.1 antibody.These studies suggest a key role for NK cells in BCG immunotherapyBCG stimulation of the human innate immune system—as judgedby altered levels of interleukin-12 (IL-12), IL-8, IL-10 and IFN-γ inthe blood of patients with lung cancer—also demonstrated thatBCG has a role in the modulation of the immune system.19

However BCG is effective predominantly in superficial cancers suchas bladder cancer, and similar effects have not been observed inlung 20 cancer or melanoma.21 Such differential effects may be attrib-uted to cytokine network modulation or functional TLR receptor(Toll like receptors) repertoire expressed by surrounding tissues,recruited cells, or the nature of malignant cells themselves. Membersof the toll like receptor gene family convey the signals induced byvarious bacterial factors such as lipopolysaccharides, lipoproteinstoxins, peptidoglycans and CpG nucleic acids, activates signaltransduction pathways that result in transcriptional regulation andstimulate immune function. Because of their ability to modulateadaptive immunity, Toll like receptors represent strategic targets for

MICROBIAL THERAPY OF CANCER

www.landesbioscience.com Cancer Biology & Therapy 709

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cancer and other diseases that involve inappropriate adaptiveimmune responses such as allergy.22

LIVE ATTENUATED BACTERIA AS A VECTORFOR CANCER TREATMENT

With the advances in molecular biology, the scope of use of bacteria in cancer therapy has widened and various possibilities now

include the use of bacteria as sensitizing agents for chemotherapy, asdelivery agents for cancer drugs and as vectors for gene therapy. Themost promising approaches are the use of genetically modifiedbacteria for selective destruction of tumors and bacterial gene-directedenzyme therapy.23 Live attenuated bacteria such as Listeria andSalmonella have shown great potential as a multivalent vaccine forthe delivery of heterologous antigens.24,25 It has also been recently described that bacteria can act as delivery systems for DNA vaccines.Plasmids that encode antigens under the control of a viral or eukary-otic promoter can be incorporated into Salmonella or Listeria .Genetically improved attenuated strains of Salmonella , which cantarget tumor cells rather than normal cells at a ratio of 1000:1, havebeen used as vectors to introduce specific enzymes such as cytosine

deaminase into tumors. Cytosine deaminase converts the nontoxicprodrug 5-fluorocytosine into the highly toxic anti-cancer drug 5-fluorouracil. Thus, injection of such attenuated Salmonella intomice harboring lung tumors, that were subsequently given injectionsof 5-fluorocytosine, has been reported to result in a significantreduction in tumor size. TAPET (Tumor amplified protein expressiontherapy) uses a genetically altered strain of Salmonella as a bacterialvector or vehicle to preferentially deliver an anti-cancer drug to solidtumor.23 More recently, bacterial strains, that specifically targettumors, such as Bifidobacterium, Clostridium and Salmonella, havebeen shown to preferentially replicate within solid tumors and havebeen used to transport and amplify genes encoding factors such aspro drug-converting enzymes, toxins, angiogenesis inhibitors andcytokines.26

BACTERIA-DERIVED ANTI-CANCER AGENTS

The normal mammalian cell cycle is composed of four phases.Cells that are committed to divide enter the G1 phase. Preliminary cellular processes are accomplished to prepare the cell to enter DNA synthesis—the S phase. Specific protein signals regulate the cell cycleand allow replication of the genome where the DNA contentbecomes tetraploid (4N). After completion of the S phase, the cellenters a second resting phase, G2, prior to undergoing mitosis. Thenthe cell progresses to the mitotic (M) phase, in which the chromosomescondense and separate leading to cell division, producing twodaughter cells. Anti-tumor agents may be cell cycle specific or non-

specific. Cell cycle nonspecific anti-cancer agents, like alkylating agents have a linear dose-response curve; the number of cells killedincreases linearly with the dose. However most of the bacteria-derived agents are cell cycle specific.

The use of live/attenuated bacteria in cancer therapy has a limitedapplication because of associated toxic effects. Here, we will point to

ways to achieve better effects with less toxicity by using a commonbacterial component. Various microbial products have been used asanti-tumor agents; among these are members of the anthracyclin,bleomycin, actinomycin, mitomycin and aureolic acid families.Clinically useful agents from these families are daunomycin-relatedagents, the peptolides (e.g., dactinomycin), the mitosanes (e.g.,

mitomycin C) and the glycosylated anthracenone mithramycin.These microbial derived anti-tumor agents have been described in areview by Rocha et al.27 Rhizoxin, a microbial metabolite containingmore than one epoxide group, has been demonstrated to haveanti-angiogenic activity and has anti-tubulin activity.28 The use oflive microorganisms in cancer therapy has often been associated withunpredictable efficacy, a strong immune response, and associatedside effects and toxicity. The human immune system is able to recog-

nize bacterial DNA based on recognition of unmethylated cytidine-guanosine dinucleotides within a specific pattern of flanking bases(CpG motif ).29,30 Such CpG motifs are potent inductors of deacti-vation and maturation of B cell and T cell responses.31 Hence, studiesare in progress to identify pure metabolites of microbial origin or anycomponent of the microbial cell, excluding DNA, that might haveanti-tumor activity.

Some reports described the varying anti-tumor efficacy of certaindetoxified bacterial LPS preparations with or without additionalcomponents,32 including some preparations of Pseudomonas aerugi-nosa .33 Bacterial lipid A induces various transcription factors via anintracellular signaling cascade. Its anti-tumor effect has been demon-strated in animals as well as in clinical trials.34 Lipotechoic acid(LTA) from Streptococcus pyogenes and Entercoccous faecalis has had atumor protective effect both, alone and in combination with pepti-doglycan of Moraxella catarrhalis and LPS from E. coli . This abilityof LTA to enhance and/or amplify the anti-tumor mechanisms inducedby primary stimuli may be related to its capability to trigger in vitrolong-lasting anti-tumor activity in bone marrow macrophages.35,36

Some species of infectious microorganisms produce powerfulimmunostimulatory and disease- causing toxins, which are called-super-antigens due to their ability to polyclonally activate large frac-tions of T-cell populations at very minute concentrations. The bacte-rial super-antigen Staphylococcal enterotoxin-A (SEA), produced bysome strains of Staphylococcus aureus , induces proliferation of cyto-toxic T lymphocytes and leads to cytokine production in vivo. Suchsuper-antigens have been shown to be highly efficient for antibody-

targeted therapy in various tumor models.37 The anti-tumor propertyof staphylococcal exotoxin-A is well documented in various tumors

A combination of protein A and super-antigen have an additiveeffect in prolonging the immune response in vivo and promoting amaximal anti-tumor effect. Another classical example is the identifi-cation of the secondary metabolites, epothilones (microtubule de-polymerization inhibitors) of the myxobacterium Sorangium cellulosum

which have shown both in vitro and in vivo cytotoxicity in variouscancer cells.38,39 Epothilones are anti-microtubule agents, and theirmechanism of action is more or less similar to taxol. However,epothilones have several advantages over taxol. They are more water-soluble than taxol and most importantly, the epothilones are betterable to inhibit the growth of tumor cells overexpressing the P-glyco-

protein, a factor responsible for drug resistance in many tumors.Hence, epothilone-like compounds may be more useful than taxol inthe treatment of multi-drug resistant tumors something which isproblematic with taxol. Many analogues of epothilones have beensynthesized such as Desoxyepothilone B, which are more promisingbecause of their efficacy against a range of tumors and relative lackof toxicity.40,41 Some studies have also demonstrated the role of theanthrax toxin as an anti-tumor agent. The anthrax toxin has a role inMEK signaling (MAP/ ERK kinase) and offers a novel strategy to usein cancer treatment.42

The cell death induced by microorganism-derived substancesraises an important question—what could be the mechanism of



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mammalian cell death with wholemicroorganisms or their constituents,and can this cell death be differentially regulated in normal cells? The physio-logical mechanism involved is based onapoptosis. The intracellular location of the pathogen is not a prerequisite forthe induction of apoptosis. The

pathogen can inject toxic proteins intoa target cell upon contact with it. Thistype of delivery of cytotoxic proteinsrequires cell-to-cell contact and isreferred to as a type III secretion mech-anism.43 It is used by Pseudomonas aeruginosa for the induction of apoptosisin macrophages and epithelial cells.44,45

It has been recently reported that a bacterial redox protein, azurin, of Pseudomonas aeruginosa acts as ananti-cancer agent both in vitro and invivo in a nude mouse model.46 Azurin

is a copper-containing oxido-reductasethat is normally involved in the denitri-fication process in P. aeruginosa . Azurinis a member of a group of proteinscollectively called cupredoxins, whichare small, soluble redox proteins whoseactive site contains a type 1 copperprotein. Azurin itself is involved inelectron transfer during denitrification.

We have reported that purified azurin induces cytotoxicity in J774macrophages, a sarcoma derived cell line, by somehow forming a complex with the tumor suppressor protein p53, thereby stabilizing it and enhancing its function as an inducer of pro-apoptotic activity.47

The redox activity of azurin is not important for its cytotoxic activity.48

Azurin has been reported to induce apoptosis not only in macrophagesbut also in human melanoma cells (Mel 2), which harbor a func-tional tumor suppressor p53. The extent of apoptosis was muchlower in the p53 null cell line Mel 6.48 Azurin was shown to beinternalized in Mel 2 cells and was localized predominantly in thecytosol and nuclear fraction. However in Mel 6 cells, azurin waslocalized primarily in the cytosolic fraction suggesting that intracel-lular trafficking of azurin to the nucleus may be p53-dependent.

The p53-tumor suppressor protein is a potent inhibitor of cellgrowth that induces cell cycle arrest and apoptosis49 and mainly actsvia transcriptional activity. p53 function is not required for normalcell growth and development of the cell; however, it plays an impor-tant role in preventing tumor development. It is activated by several

types of stress signals and induces expression of several proteins thatare involved in inhibition of cell proliferation or in promoting celldeath by apoptosis. The importance of p53-induced apoptosis intumor suppression is further evidenced by some tumor-derived p53mutants that have a selective loss of function only for apoptosis andnot for G1 arrest.50 The choice of activation of particular targetgenes is controlled by covalent p53 modification and protein-proteininteractions. Such interactions could make the difference betweenthe life or apoptotic death of a cell.51

In the cell, p53 activity is tightly regulated and controlled by a negative feed back loop involving Mdm2 (murine double minute 2).Structurally and functionally, p53 consists of three well-defined

regions, each having a specified function: The N-terminal region-consists of a transactivation domain (amino acids 1–70) and a pro-line-rich stretch (amino acids 60-97). This region also contains anMdm2 protein-binding site. The central region consists of ahydrophobic DNA binding domain (amino acids 94–312), a con-

served region of p53 in various species. The C-terminal regionconsists of an oligomerization domain (amino acids 320–360) and abasic C-terminal domain (amino acids 360–393). In normal andnonstressed cells, the half-life of p53 is very short due to an auto-regulatory feedback loop mechanism in which the Mdm2 proteinplays a key role. This p53-Mdm2 auto-regulatory loop has provideda central target for therapeutic drug developments. Agents, that canalter the p53-Mdm2 pathway and protect p53 from Mdm2 degra-dation, are potential candidates for anticancer therapy. A recentstudy by Vassillev et al.52 is a classical example demonstrating thatthe use of such protein-protein interactions could be valuable inp53-based therapy. The study reported the selection of a syntheticmolecule that can interact with Mdm2, and rescue p53; this moleculecould be used for therapeutic purposes. Recent studies have suggested

that the p53 response is differentially regulated in normal andcancer cells,53,54 and have reinforced the idea that p53 stabilizingmolecules can be specifically used in controlling cancer cells. Anincreasing understanding of the biochemistry of the p53 pathway isallowing us to develop novel therapeutic strategies by manipulatingthis pathway. This illustrates how basic research is being applied toclinical applications in cancer.55 Indeed, the first commercialp53-based production of gene therapy drug named Gendicine hasbeen approved recently in China. The Food and Drug

Administration of China has licensed Shenzhen SiBono GenTechto produce and market recombinant Ad-p53 gene therapy for headand neck squamous cell carcinoma.56



Figure 1. A representative flow diagram of apoptotic events and the role of the bacterial redox protein azurinin mammalian cell death.

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Azurin from Pseudomonas aeruginosa can physically interact withp53.57 It preferentially binds to the N-terminal and middle regionof the p53 but only weakly to the C-terminal.58 To the best of our

knowledge azurin is the first bacterial protein reported to be inter-acting with p53, and hence it has the potential of being used inp53-based anti-cancer therapies. It modulates p53 activity and inducesthe apoptotic cell death (Fig. 1). Interestingly intraperitoneal injec-tions of 0.5 mg of azurin to a group of immunodeficient nude miceharboring xenografted Mel 2 tumors allowed tumor regression to theextent of 59% in 22 days. Multivariate analysis based on a randomcoefficient model showed that the difference in tumor volumebetween treated and control animals was statistically significant.46

Moreover, when two hydrophobic amino acids at the 44 and 64positions in azurin were replaced with more polar amino acids togenerate a M44KM64E double mutant, it was found to havereduced cytotoxicity towards Mel 2 cells as compared to wild typeazurin. This mutant triggered the inhibition of cell cycle progression

in mammalian cells but little apoptosis.46,48,59 The oligomerizationdomain of p53 is located in the central to the C-terminal region of p53; it might therefore be possible that the M44KM64E mutantazurin binds to p53 in this region and somehow directs the speci-ficity of p53 for p21. p53 is a very well characterized molecule. Theuse of synthetic nonoverlapping p53 peptides of known functionmay help in characterization of their interaction with azurin/ ormutant azurin. Alternatively, gene array studies can be used as a toolto identify mammalian cell cycle genes, which are differentially reg-ulated in cancer cells treated with azurin.

More recently, the entry of azurin into the human breast cancercell line MCF-7 followed by induction of apoptosis has beenreported.58 MCF-7 cells containing wild type p53 were highly sus-

ceptible to azurin treatment compared to the p53 null cell lines. Theintracellular p53 level increased after azurin treatment in MCF 7cells; however a fraction of p53 as well as a small amount of azurin

was also translocated to the mitochondria. Mitochondrial p53 is asso-ciated with nontranscriptional activity to modulate cell functions.60

Hence further studies are needed to study the role if any of mitochon-drial p53 in cell death associated with azurin treatment and analyzethe relative involvement of transcriptional and nontranscriptionalp53 activity. Current studies are directed at analyzing the relativeimportance of transcriptional and nontranscriptional p53-dependentcell death in response to azurin treatment. A clinically importantfinding of Punj et al.58 was that azurin allowed significant regression

of breast cancer in vivo in athymic mice injected with 1 mg of azurindaily as compared to untreated mice. A mean tumor volume of 15%

was observed 29 days after the start of the experiment in treated mice

as compared to controls, demonstrating 85% regression of tumorgrowth. There was no apparent loss of body weight of mice or anyother visual side effects of azurin treatment. The immuno-histologicalexaminations of tumors showed apoptotic cell death in tumors, witha marked increase of disintegrated parenchyma and reduction inproliferating cells in azurin-treated mice over untreated control.

One of the major concerns in using foreign proteins as therapeuticagents is their associated immunogenic and inflammatory response.Immune responses to therapeutic proteins can affect therapy byinducing inflammation and unwanted immune responses such asautoimmunity. The mitogenic effect of some bacterial proteins canalso induce the polyclonal activation of T and B cells with possibleclinical complications. In view of this, we tested if azurin can inducean immune response in C57BL/6 mice treated with 1 mg/ml of

azurin, intra-peritoneally daily for 28 days, as reported in previousstudies.46,58 The mice were monitored for 40 days, bled every 10days, and tested for azurin specific antibody response. After 40 days,mice treated with azurin demonstrated a very low level of azurin-specific antibody (titer of 1:800), predominantly of the IgG class,

which is insignificant considering that these mice had receivedrepeated injections of azurin. Spleen cells from these mice showed nosignificant proliferative response as determined by 3H-thymidineincorporation assay. There was no change in the number of T cellsub-populations (CD4+ and CD8+), B cells (CD19+) and T cellactivation markers (CD62L) as determined by FACS analysis,suggesting that azurin has no significant antigenic or mitogenicactivity.61

It is interesting to note that Punj et al.58 found that azurin has lessapoptotic effect in nontransformed cells such as MCF10F, HBL 100or foreskin cells as compared to MCF-7 cells, suggesting that perhapscancer cells are preferential targets for azurin treatment. It is still notknown how azurin enters into cancer cells and if any receptors areinvolved in azurin trafficking. Studies on the mechanism of entry ofazurin into mammalian cells might explain the affinity of azurin fortransformed cells. Certain antibacterial peptides have been found tohave impressive in vitro62,63 and in vivo64 activity against cancer cells

without damaging the normal eukaryotic cells. Expression of anti-microbial peptides such as cecropin B1 (CB1) and melittin wasfound to produce anti-tumor properties.65 CB1 is a derivative of a



Figure 2. (A) Schematic representation of azurin fragments fused with His-tag; +, binding with full-length p53; -, no interaction. (B) His pull down assay wascarried out to study the interaction with His-tagged azurin fragments with full length p53 using Ni-NTA agarose beads following standard protocol. 71 Theproteins were separated on 8–16% SDS-PAGE, transferred to a PVDF membrane and immunoblotted with p53 antibody (IB:p53). The full length azurin andits fragments containing amino acids 30–94 and 71–150 showed the interaction with p53. His immunoblot (IB:His) was used as internal loading controlfor azurin fragment.

A B

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natural antibacterial peptide Cecropin B, and has a higher specificactivity against cancer cells than its natural form Cecropin A (CA).This specific activity has been attributed to unique structure of CB1consisting of two-ampipathic helical segments.66 However themechanism by which peptides kill both prokaryotic and transformedeukaryotic cells is not well understood. It is believed that the extentof secondary structure as they approach the cell membrane, followedby the contents of cationic residues, may play an important role in

the lysis of cell membranes.67,68

However it is not clear whether it isthe protein structure or the sequence which plays a predominantrole in the lysis of the cell membrane.

An ideal anti-cancer agent will be as small a molecule as possible with low levels of side effects. Azurin induces a low level of cell deathin normal cells. It might be possible that only a portion of the azurinmolecule may be required for interaction with p53 leading toinduction of cell death. The His-tag fused truncated azurin proteins

were purified using Ni-NTA spin columns, and used for His-pull-down assays to study the interaction with p53. Azurin fragmentsencompassing amino acids 30–94 and 71–150 (corresponding tothe predicted amino acid sequence) showed binding with full-lengthp53 (Fig. 2). It is interesting to note that azurin fragment 30–94 is

an amphipathic peptide with both hydrophobic and hydrophilicdomains. Such peptide could be targeted with a tumor specificantigen and used in combinational-bio-chemotherapy in order tooptimize the dose. One of the important example of biochemotherapy is the use of Dacarbazine (DTIC) and Interferon-α (IFN-α), whichis an FDA approved and is widely used therapy in metastaticmelanoma.69 The clinical significance of this azurin fragment shouldbe investigated in order to develop a peptide-based anti-cancer agent.

The question which arises,- is why bacteria would secrete such a redox protein to kill mammalian cells? It has recently been proposedthat it is the physical presence of redox enzymes rather than theirenzymatic activities, in the cytosol of the mammalian cells, whichsends a signal to the cell of an impending energy catastrophe.Generally, redox proteins such as AIF (apoptosis inducing factor)and cytochrome c are present in the mitochondrial inter-membraneand not in the cytosol. Mitochondria, which are believed to haveprokaryotic ancestry from protobacteria, are the energy storehouseof the mammalian cells. In response to death stimuli, mitochondrialredox proteins are released into the cytosol and trigger cell death. Itis interesting to note that ancestors of protobacteria, the mitochondria,and the present day bacteria such as P. aeruginosa retain the evolu-tionary conserved function of releasing redox proteins to triggerapoptosis in mammalian cells.70

CONCLUSION AND PERSPECTIVES

Bacterial products such as epothilones, lipotechoic acid, peptido-

glycans, toxins and especially, the redox proteins of prokaryotes suchas Pseudomonas aeruginosa in particular can modulate the mammaliancell functions. These products lead to cell death, by entering the hostcells either in p53-dependent or independent mechanisms. Sinceazurin showed in vivo regression of tumor growth without inducing any significant toxic side effects, this promising anti-cancer agentshould be further studied in order to understand its translationalsignificance. Further understanding, the mechanism of action of azurin and its p53 interaction will provide us with the basis to furtherimprove the potency of this agent and optimize its dose. It should bepossible to preferentially develop a short azurin-based syntheticpeptide, which could be used in the fight against cancer. Further in

vivo studies are in progress to determine if azurin can be used as analternative means of gene therapy in solid tumors.

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