Discovering and exploiting bacterial proteins as anticancer agents

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UNIVERSIDADE DE LISBOA

FACULDADE DE CIÊNCIAS

DEPARTAMENTO DE BIOLOGIA ANIMAL

Discovering and exploiting bacterial

proteins as anticancer agents

Gonçalo Emanuel Fialho Mourata da Silva

DISSERTAÇÃO

Mestrado em Biologia Humana e Ambiente

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UNIVERSIDADE DE LISBOA

FACULDADE DE CIÊNCIAS

DEPARTAMENTO DE BIOLOGIA ANIMAL

Discovering and exploiting bacterial

proteins as anticancer agents

Gonçalo Emanuel Fialho Mourata da Silva

DISSERTAÇÃO

Mestrado em Biologia Humana e Ambiente

Dissertação orientada por Professor Doutor Arsénio Fialho e Professora Doutora Ana Crespo

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"To myself I am only a child playing on the beach, while vast oceans of truth lie undiscovered before me." Isaac Newton

"I am not apt to follow blindly the lead of other men."

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ACKNOWLEDGMENTS

I would like to express my sincere gratitude to my supervisor Professor Arsénio Fialho (Instituto Superior Técnico), for having granted me this opportunity to work with him and for all the help and support that he gave me, every time I needed, throughout my thesis.

I am also grateful to Professor Ana Crespo, at the Departamento de Biologia Animal (Faculdade de Ciências, Universidade de Lisboa), for having gently accepted to be my supervisor and for being always available whenever I needed.

I would also like to thank Nuno Bernardes, for all the knowledge transmitted and for teaching me everything I have learned inside a laboratory this year.

In addition, I am thankful to the people in the Biological Sciences Research Group (Instituto Superior Técnico), especially to Dalila Mil-Homens and Sofia Abreu which helped me in many occasions.

I sincerely want to address a special thanks to my closest friends Alexsandro Costa, João Guerreiro, Duarte Silva, João Serafim, Ana Catarina, Andreia Ferreira, for always been there, for giving me a kind word, for listening, for making me laugh, and specially for believing in me and my capabilities. I would like to extend this acknowledge also with my friends from my master degree course, especially to Sofia Alves.

I especially want to thank Tânia, for being my fountain of joy and happiness, and for having the amazing ability of making me smile even in the toughest times. I would also like to thank my family, especially my parents and my brother, for all their support, patience and strength they gave me through this entire year. I could not have finished my master degree without them.

I dedicate the achievement of my master degree to my grandparents José and Mariana, for being the reason why I came to science and whose memories remain strictly in my heart and still give me the strength to overcome my fears and difficulties.

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ABSTRACT

Azurin is a low molecular weight protein, produced by Pseudomonas

aeruginosa and possesses several antitumor properties, like causing P-cadherin

levels to decrease in invasive breast cancer cells. In this work, we studied the effect of lysosome and proteasome inhibitors on P-cadherin level, using a breast cancer cell line, expressing high P-cadherin level (MCF-7/AZ.Pcad), previously treated with azurin. Additionally, we evaluated how a cholesterol-depleting agent (MβCD) affects P-cadherin level. The effects of both inhibitors on P-cadherin were observed by western blot and confirmed that azurin mediates P-cadherin degradation through lysosome and proteasome proteolytic pathways. We also described, for the first time, that MβCD causes P-cadherin level to decrease. Together, these findings have increased our understanding of how the bacterial protein azurin is acting as anti-cancer agent.

In this work wehave also studiedthe in vitro cytotoxicity of two other bacterial proteins (MPT 63 and Ndk) against human breast and lung cancer cells. MPT 63 is an antigen secreted by Mycobacterium tuberculosis that induces immunogenic responses in animal models and its cytotoxicity against several tumor cell lines was recently described in a patent. Nucleoside diphosphate kinase (Ndk) is a ubiquitous enzyme which maintains the nucleotide pools within the cells, and can be secreted by P. aeruginosa. A human Ndk, termed Nm23-H1, also showed an anti-metastatic role in different cancer models. In order to test possible antitumor properties of these proteins, MTT cell viability assays were performed in breast and lung cancer models (MCF-7/AZ.Mock and A549) using increasing azurin, MPT 63 and Ndk concentrations, and different exposure times. In addition, matrigel invasion assay was performed in A549 invasive cells treated with Ndk. Both azurin, MPT 63 and Ndk evidenced cytotoxicity against both cancer models in a time and dose dependent manner. Ndk revealed cytotoxic activity and selectivity against tumor cells similar to azurin. We observed a small decrease in cell invasion using this protein. In summary, we promoted a screening of new bacterial proteins that demonstrated antitumor potential, especially Ndk.

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RESUMO

Novas terapias anti-tumorais emergentes baseiam-se em abordagens pouco convencionais, como a utilização de microorganismos, nomeadamente bactérias vivas ou produtos purificados a partir das mesmas, como proteínas. A azurina é uma proteína de baixo peso molecular, produzida por Pseudomonas aeruginosa e possui diversas propriedades anti-tumorais, entre as quais a indução de apoptose em células tumorais pela estabilização da proteína supressora de tumores p53. Mais recentemente, um novo tipo de acção anti-tumoral foi descoberta, tendo sida descrita a sua capacidade de diminuir os níveis de P-caderina em células tumorais invasivas de cancro da mama, sem afectar, no entanto, os níveis de E-caderina. O mecanismo, pelo qual a azurina causa o decréscimo de P-caderina nas células tumorais não é ainda totalmente conhecido, mas esta parece actuar a nível pós-transcripcional dado que não se verificam diferenças na expressão de P-caderina em células tratadas com azurina. Dados relativos a ensaios com análise a microarrays revelaram que a transcrição de genes associados ao lisossoma e processos de transporte mediado por vesículas se encontrava mais activa. No presente trabalho pretendeu-se esclarecer se a diminuição dos níveis de P-caderina, mediada pela acção da azurina, se deve à sua degradação pelos sistemas proteolíticos a nível celular, como o lisossoma e proteossoma. Nesse sentido utilizaram-se células de uma linha celular de cancro da mama, que expressa níveis elevados de P-caderina (MCF-7/AZ.Pcad), e que foram previamente tratadas com azurina antes de serem administrados inibidores de lisossoma (cloreto de amónio) e de proteossoma (MG-132). Do mesmo modo foram também avaliados os efeitos de um agente sequestrador de colesterol (MβCD) e inibidor de entrada da azurina nas células, ao nível da P-caderina nesta linha celular tumoral. Os efeitos de ambos os inibidores, ao nível da degradação da P-caderina, foram observados por western blot e confirmaram que a azurina medeia a degradação da P-caderina por sistemas proteolíticos como o lisossoma e o proteossoma. Descrevemos igualmente, pela primeira vez, que a MβCD provoca a diminuição dos níveis de P-caderina sem afectar os níveis de E-caderina. Conjuntamente, estes resultados permitiram aumentar o nosso conhecimento acerca do modo como a azurina actua como agente anticancerígeno neste caso específico.

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Neste trabalho pretendemos também estudar a citotoxicidade in vitro de duas outras proteínas bacterianas (MPT 63 e Ndk) em células tumorais humanas de cancro da mama e de pulmão. A MPT 63 é uma proteína antigénica secretada por

Mycobacterium tuberculosis e capaz de induzir respostas imunogénicas em

diversos modelos animais. Esta proteína apresenta uma estrutura semelhante a imunoglobulinas, uma característica que é partilhada com a azurina. Recentemente foi descrita como possuindo elevada actividade citotóxica contra várias linhas celulares tumorais, assim como um péptido derivado desta proteína (MB30), tendo esta propriedade de ambas as molécula sido registada numa patente. A nucleosídeo difosfato cinase (Ndk) é uma enzima ubíqua em diversos organismos e que tem como função manter as reservas de nucleótidos das células. Esta proteína pode igualmente ser secretada por várias bactérias como P.

aeruginosa. As Ndks humanas estão agrupadas numa família de proteínas

denominada de Nm23, tendo sidas até hoje descritas dez tipos. A primeira destas proteínas a ser descrita, denominada Nm23-H1, demonstrou possuir adicionalmente uma importante acção anti-metastática em diferentes modelos de cancro. Tendo em conta o vasto leque de acção anti-tumoral da azurina, procurou-se procurou-seleccionar duas proteínas bacterianas (MPT 63 e Ndk) com propriedades interessantes de serem exploradas, no sentido de testar uma possível actividade citotóxica das mesmas em células cancerígenas. Para esse efeito foram realizados ensaios de viabilidade celular (ou ensaios de MTT) em modelos tumorais de cancro da mama e do pulmão (MCF-7/AZ.Mock e A549), usando concentrações crescentes de azurina, MPT 63 e Ndk, bem como diferentes tempos de exposição, com o intuito de entender como estes parâmetros podem afectar o nível de citotoxicidade destas proteínas. Adicionalmente foi testada a actividade anti-metastática da Ndk, realizando um ensaio de invasão em matrigel, usando uma linha celular altamente invasiva de cancro de pulmão, A549. A azurina, assim como a MPT 63 e a Ndk, evidenciaram citotoxicidade contra ambos os modelos tumorais testados, de um modo dependente do tempo e concentrações administradas. A Ndk revelou níveis de actividade citotóxica e selectividade de acção, relativamente a células tumorais, semelhantes à azurina. Observámos ainda um pequeno decréscimo da invasão celular das células tumorais de pulmão A549,

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quando esta proteína foi administrada. Em suma, promovemos um rastreio de novas proteínas bacterianas que demonstraram potencial anti-tumoral, especialmente a Ndk. O conhecimento acerca destas propriedades necessita de ser expandido e aprofundado para que, no futuro, se possa avaliar a sua utilização como agentes anti-cancerígenos úteis, tal como a azurina.

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LIST OF ABBREVIATIONS

BCG - Bacille Calmette-Guérin BSA - Bovine serum albumin DMBA - Dimethyl-benz-anthracene DMSO - Dimethyl sulfoxide

E1 - Ubiquitin-activating enzyme E2 - Ubiquitin-conjugating enzyme E3 - Ubiquitin-ligase enzyme

ELISA - Enzyme-linked immunosorbent assay Eph - Ephrin receptor

FBS - Fetal bovine serum HBS - Hepes buffered saline

HLA-DR - Human Leucocyte Antigen

HUVEC - Human umbilical vein endothelial cells INF-γ - Gamma interferon

IPTG - Isopropyl-β-D-Thiogalactopyranoside KSR - Kinase suppressor of ras

Laz - Lipid-modified azurin LB - Luria Broth

LPS - Lipopolysaccharide

MEM - Minimum Essential Medium MMP - Matrix metalloproteases

MTT - 3-(4,5 dimethylthiazol-2-yl-2,5 tetrazolium bromide) MβCD - Methyl-β-cyclodextrin

NaCl - Sodium chloride

NCBI - National Center for Biotechnology Information Ndk - Nucleoside diphosphate kinase

NDPs - Nucleoside diphosphates NH4Cl – Ammonium chloride

NSCLC - Non-small cell lung cancer NTPs - Nucleoside triphosphates OD640 nm - Optical density

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PBMNCs - Peripheral blood mononuclear cells PBS - Phosphate buffered saline

PDB - Protein Data Bank

PTD - Protein transduction domain PVP - Polyvinylpyrrolidone

SCLC - Small cell lung cancer

SDS-PAGE - Sodium dodecyl sulfate polyacrylamide gel electrophoresis sP-cad - Soluble P-cadherin fragment

STRAP - Serine-threonine kinase receptor-associated protein SURE - Stop Unwanted Rearrangement Events

Th1 - T helper 1

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INDEX OF FIGURES

Figure 1 – Current diverse strategies to use bacteria and bacterial products in

cancer treatment (Bernardes et al, 2013) [11]. ... 3

Figure 2 – The three-dimensional structure of azurin from P. aeruginosa

(Bonander et al, 1997) [18]. ... 4

Figure 3 – Azurin as a promiscuous protein, which possess both anticancer,

antiparasite and antiviral activities (Fialho et al, 2007) [37]. ... 10

Figure 4 – Schematic representation of the classical cadherin-catenin complex

and the structural components of cadherins (adapted from Albergaria et al, 2011) [40]. ... 11

Figure 5– Structure of M. tuberculosis MPT 63 protein (adapted from Goulding

et al, 2002) [58] ... 17

Figure 6 – Three-dimensional structures of human A. (A) Diagram of

Ndk-A monomer. (B) Diagram of the homo-hexamer (Han et al, 2010) [79]. ... 22

Figure 7 – (A): Western Blot analysis of the effect of lysossome inhibitor,

ammonium chloride (NH4Cl), on E and P-cadherins of MCF-7/AZ.Pcad cells,

previously treated with azurin at 0, 50 and 100 μM. Azurin and the inhibitor were administrated at 0 and 32 hours, respectively, in a 48 hours assay. (B): Charts illustrated represent average percentage values of protein level for E and P-cadherins which signal was normalized with actin levels. ... 41

Figure 8 – (A): Western Blot analysis of the effect of proteasome inhibitor,

MG-132, on E and P-cadherins of MCF-7/AZ.Pcad cells, previously treated with azurin at 0, 50 and 100 μM. Azurin and the inhibitor were administrated at 0 and 32 hours, respectively, in a 48 hours assay. (B): Charts illustrated represent average percentage values of protein level for E and P-cadherins which signal was normalized with actin levels... 42

Figure 9 - Effects of MβCD on azurin’s internalization on MCF-7/AZ.Pcad cells

after 8 hours of exposure to this inhibitor and 24 hours with fresh medium (visualized by western blot). PBS was used as double negative control. DMSO was used as negative control. Azurin was tested at two different concentrations (50 and 100 μM). ... 43

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Figure 10 – Western blot showing the effect of azurin’s internalization inhibitor

MβCD on E- and P-cadherin levels in MCF-7/AZ.Pcad cells after 8 hours of exposure and 24 hours with fresh medium. PBS was used as double negative control. DMSO was used as negative control. A decrease in P-cadherin level, between control samples and samples treated with MβCD and azurin, is shown in the figure. ... 45

Figure 11 - 15% SDS-PAGE of MPT 63 and Ndk purified from E. coli SURE

strain. Proteins appear as a single band at approximately 15 kDa, according to their expected molecular weight. ... 46

Figure 12 – Cytotoxicity (%) caused by azurin on MCF-7/AZ.Mock during 48 h

(one dose) and 72 h (three doses), and A549 48 h (one and two doses). Both cell lines were tested with 5 different azurin concentrations (0, 10, 25, 50, and 100 μM). Significant values, p-value<0.05 with the Student t-test, are shown as asterisks (*). ... 48

Figure 13 – Cytotoxicity (%) caused by MPT 63 on MCF-7/AZ.Mock during 48

h (one dose) and 72 h (three doses), and A549 during 48 h (two doses). Both cell lines were tested with 5 different MPT 63 concentrations (0, 10, 25, 50, and 100 μM). ... 49

Figure 14 - Cytotoxicity (%) caused by Ndk on MCF-7/AZ.Mock during 48 h

(two doses) and 72 h (three doses), A549 48 h (two doses) and 16HBE14o- during 48 h (two doses). Both cell lines were tested with 5 different Ndk concentrations (0, 10, 25, 50, and 100 μM). Significant values, p-value<0.05 with the Student t-test, are shown as asterisks (*). ... 50

Figure 15 – Cell invasion (%) in a Matrigel invasion assay performed on A549

highly invasive tumor cells, which were treated with Ndk at 0, 50 and 100 μM, during 24 hours. ... 51

Figure 16 – ClustalW multiple alignment of human and bacterial Ndks. The

numbers above the sequences indicate the position of amino acid residues. Secondary structure of E. coli Ndk is represented according to PDB code 2HUR. Secondary elements are indicate as: single purple curves (turns), yellow arrows (β-sheets), red lines (bends), blue curved lines (alpha helices), orange curved lines (3/10-helices), and black lines (no secondary structure assigned). NCBI accession

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number for each protein is given below. Human (Homo sapiens) Nm23 proteins: Nm23-H1 (CAG46912), Nm23-H2 (NP_001018147), Nm23-H3 (EAW85629), Nm23-H4 (NP_005000), Nm23-H5 (NP_003542), Nm23-H6 (NP_005784), H7 (Q9Y5B8), H7B (NP_932076), H8 (AAF20909), Nm23-H9 (NP_835231), Nm23-H10 (NP_008846). Bacterial Ndks: P. aeruginosa (EPR01938), M. tuberculosis (EQM19968), M. bovis (AGE68451), E. coli (ERF95659), M. xanthus (P15266), V. cholerae (WP_001162853). ... 58

Figure 17 – Sequence alignment and secondary structure comparison between

human Nm23-H1 and P. aeruginosa Ndk. Symbols in Clustal Consensus sequence indicate standard ClustalW nomenclature: (*) identity, (:) high conservation and (.) conservation. Secondary structure of Nm23-H1 was represented according to PDB code 4ENO. Secondary structure of P. aeruginosa Ndk was predicted using PDB code 3VGU as template. This template was aligned between its 2 to 141 amino acid residues and share a 79% sequence identity with

P. aeruginosa Ndk. Secondary elements are indicate as: single purple curves

(turns), yellow arrows (β-sheets), red lines (bends), blue curved lines (alpha helices), orange curved lines (3/10-helices), and black lines (no secondary structure assigned). NCBI accession number for each protein is given below. Human (Homo sapiens) Nm23-H1 (CAG46912) and P. aeruginosa Ndk (EPR01938). ... 61

Figure 18 – Phylogenetic tree representation of human Nm23 protein family as

well as some bacterial Ndks. Splitstree software was used to construct the tree based on ClustalW multiple alignment generated. All sequences and its NCBI accession numbers used were the same as those displayed in Figure 16. Protein three-dimensional structures represented were retrieved from PDB database: Nm23-H1 (4ENO), Nm23-H2 (3BBF), Nm23-H3 (1ZS6), Nm23-H4 (1EHW), Nm23-H10 (2BX6), and Ndks from M. tuberculosis (1K44), E. coli (2HUR) and

M. xanthus (2NCK). Quaternary structures are represented as homohexamers

(Nm23-H1, Nm23-H2, Nm23-H3 and Nm23-H4 and Ndk from M. tuberculosis) and as homotetramers (Ndk from E. coli and M. xanthus). Tertiary structure is represented as a monomer (Nm23-H10). Ndk from P. aeruginosa is marked with an asterisk (*) since it is a predicted three-dimensional model created using

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model software tools. This representation was modeled using 2 to 141 amino acid residues of the Ndk sequence (79% identity) from Halomonas sp. 593 (3VGU) as template. This template forms a homodimer in its quaternary structure, as well as the model created. ... 62

Figure 19 – Structural alignment between human Nm23-H1 (4ENO) [blue] and P.

aeruginosa Ndk predicted model (green) was performed using PyMOL software.

Three dimensional structures are displayed at different degrees: 0º (A), 90º (B), 180º (C) and 270º (D). ... 63

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INDEX OF TABLES

Table 1 - Phosphate buffer 8x composition ... 29 Table 2 - START and elution buffer compositions ... 29 Table 3 - ÄKTA elution program for protein sample desalting ... 29 Table 4 - PBS composition ... 30 Table 5 - Resolving Gel 8% and 15% composition for one gel ... 34 Table 6 – Percent identity matrix of human Nm23 proteins and different bacterial

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1. INTRODUCTION

1.1 Cancer

Currently cancer represents one of the greatest burdens of our society and will remain a serious and challenging major public health problem in the future years. Each year about 12,7 million people are diagnosed with cancer, and approximately 7,6 million die from it, clearly demonstrating the magnitude of this disease in human population [1].

During the last decades lung cancer has been the most common cancer in the world, as well as the most common cancer in men [1], [2]. This type of cancer alone is accountable for 1,4 million deaths each year (18,2% of overall cancers), being unveiled 1,6 million new cases annually [1]. Among women, lung cancer is the fourth most frequent, as well as the second most common cause of death from cancer [1]. Regarding its histopathologic classification, lung cancer can be divided in 2 major types: Small cell lung cancer (SCLC) and Non-small cell lung cancer (NSCLC), which comprises 3 subtypes (adenocarcinoma, squamous cell carcinoma and large cell carcinoma) and accounts for more than 85% of all lung cancers [3], [4]. NSCLC is typically chemo-resistant and treated primarily by surgery at early stages, while SCLC progresses more rapidly and metastasizes earlier than NSCLC, being usually treated by chemotherapy and radiotherapy [3], [4].

Breast cancer is the second most common cancer worldwide, with 1,4 million cases being diagnosed annually, and the first cause of cancer-related death among women (458,000 deaths/year) [1]. This type of cancer changes the size and/or shape of the breast and can be classified into 2 histopathological categories: ductal and lobular carcinomas. Each one of these carcinomas can be designated as in situ or invasive, according to whether the tumor is confined to the glandular area of the organ or whether it has invaded the stroma [5]. Ductal carcinoma represents 80% of breast cancer cases and it arises from epithelial lining the mammary ducts, whereas lobular carcinoma is a less common form of breast cancer, that is originated in the milk-producing lobules of the breast [5], [6].

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Current cancer treatments rely on surgery, chemo and radiotherapy, or even hormone therapy, in the case of breast cancer [5]. However these therapies can reveal serious and systemic side-effects in patient’s health due to its high toxicity and lack of cancerous tissue specificity [7]. Additionally not every patient responds efficiently to chemotherapy or other treatments, since cancer cells can undergo micro-evolution and rapidly render cancer cells resistant to drug therapy [8], [9]. Another relevant issue is that not always the primary tumor is responsible for the death of cancer patients, but rather the metastases of cancerous cells to secondary sites, as brain, bones or lungs for instance [6], [10]. Therefore today we face new challenges regarding cancer treatment and cancer patients, especially those which do not respond to conventional therapies, demand for new and more efficient and selective drugs or therapies to fight this disease.

1.2 Microorganisms and their products as anti-cancer agents

One of the new paths considered in the search for new anticancer therapies resides on an unconventional approach using microorganisms, namely live bacteria or their purified products (Figure 1). Although it can be considered, at first sight, as a revolutionary method, the use of live bacteria and their products has been investigated and initially proposed a long time ago. In fact, in late nineteenth century, William Coley, a surgeon in the Memorial Hospital in New York, used a mixture of extracts of killed bacteria for the first time against different types of cancer. Surprisingly he observed anti-tumor activity and complete remission of tumor in some cases, although some patients have developed systemic infections and died [11], [12]. More recently this line of work has regained a new insight and several patents, regarding the use of bacteria or its derived products, have been issued [12].

Since Coley’s experiments until now several live, attenuated or engineered bacteria, namely Mycobaterium, Clostridium, Salmonella or Listeria, have shown the ability to act as anticancer agents [13]. The main disadvantage of this method is clearly the risk of originating undesired infections on patients, caused by live bacteria themselves, making it a serious risk if applied on humans. Therefore,

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attenuated or genetic engineered bacteria have been explored in order to minimize that problem.

However not only bacteria may have applications in cancer therapies, since bacterial purified products such as protein, enzymes, immunotoxins, antibiotics or other secondary metabolites have been extensively studied concerning this matter [11]. With these approaches one could overcome the limitation of using living bacteria, eliminating the risk of infection. Moreover, some of these products have proven to cause significant and promising results, such as tumor regression through growth inhibition, cell cycle arrest or even apoptosis induction [12]. The use of some bacterial products (as proteins for instance) which are able to target and lead to the death of tumor cells specifically, would probably overcome the flaws unspecific cancer treatments.

Figure 1– Current diverse strategies to use bacteria and bacterial products in cancer treatment

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1.3 Azurin

Azurin is a low molecular weight (128 aa-14 kDa), water-soluble, type I copper-containing protein that belongs to the cupredoxin family [13], [14], [15]. This periplasmic redox protein is secreted by Pseudomonas aeruginosa and act as electron donor to nitrite reductase during denitrification in this pathogenic bacterium [14], [16].

Azurin possesses a characteristic single domain structure, which consists of a rigid β-sandwich core (an immunoglobulin fold), formed by eight antiparallel strands (Greek key β-barrel structure) stabilized by a disulfide bridge and its tridimensional structure is presented in Figure 2 [13], [17]. In addition, it has a neutral hydrophobic patch surrounding the copper site [17]. Azurin displays structural similarity with variable domains of various immunoglobulins, thereby demonstrating its single antibody-like structure [13].

However a new and interesting role regarding azurin was revealed in 2000, when Zaborina et al. reported azurin cytotoxic and apoptosis-inducing activities towards murine macrophage cell line J774 [19]. Since J774 is a transformed cell line, derived from reticulum cell sarcoma, it was relevant to verify if azurin could

Figure 2 – The three-dimensional structure of azurin from P. aeruginosa (Bonander et al, 1997)

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cause cytotoxic effects in human tumor cell lines. In fact, later it was shown that azurin can also trigger apoptosis and lead to significant cytotoxicity in different human tumor cell lines as breast cancer (MCF-7), melanoma (UISO-Mel-2) and osteosarcoma (U2OS) cells [20], [21], [22]. Moreover, in the referred cell types the elevation of p53 intracellular levels led to enhanced pro-apoptotic Bax formation and a rearrangement in its distribution from the cytosol to the mitochondria, due to azurin treatment, which it is known to occur along with the release of cytochrome c from the mitochondria to the cytosol [21]. In the case of MCF-7 cells it was also shown that not only enhanced Bax formation occurs but also anti-apoptotic Bcl2 levels decrease with the time of azurin treatment [21]. Interestingly azurin exhibits preferentially selectivity against tumor cell lines, showing much less cytotoxic and apoptotic effects towards normal cell lines [21]. A domain with only 28 amino acids, designated Azu 50-77 or p28, was identified as the preferential entry domain of azurin and can act as a potential protein transduction domain (PTD) in cancer cells [23]. Although azurin entry mode remains unclear, recently some studies regarding p28 showed that this peptide requires caveolae-mediated endocytosis in order to enter cells, since microtubule and caveolae-disrupting agents, that inhibit caveosome formation and transport, were able to inhibit considerably the entry of p28 in different cancer cell lines [15], [24]. Caveolae are a 50- to 100-nm subset of lipid raft invaginations of the plasma membrane, which contains caveolin-specific proteins, as caveolin-1, that act as regulators of signal transduction [24]. Another evidence that supports the importance of caveolae in p28 internalization is that p28 co-localizes with caveolin-1 [24], [25]. Moreover cancer cells showed to be more sensitive to the effects of these inhibitors comparing to normal cells, which suggests that a higher number of membrane receptors or structures present in cancer cells, such as caveolae, can help to explain p28 preferential entry in this type of cells [15], [24]. On the other hand several inhibitors of energy-dependent transport mechanisms, as Na+K+ ATPase pump, had no inhibitory effect on p28 penetration, suggesting that non-endocytic pathways may also be involved in the internalization of this peptide [15], [24].

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After entering cancer cells, azurin can be found in the cytosol and nuclear fractions [20]. Azurin has shown the ability to bind and to form a complex with the tumor suppressor protein p53, thereby stabilizing it and favoring its intracellular level to raise [20]. This protein is a central and major player in a complex network responsible for regulating processes like cell growth, genomic stability and cell death [26]. These different processes can be regulated by the same protein as it is a transcription factor that acts as a sequence-specific transcription regulator for many pro-apoptotic genes, namely bax and p21, which encode protein Bax, involved in apoptosis, and protein 21, involved in the inhibition of cell cycle progression as well as growth arrest [26]. Although p53-mediated apoptosis is not yet a fully understood phenomenon, it is clear that p53 has a fundamental role on azurin triggering-apoptosis. As a matter of fact evidence shows that apoptosis rate and Bax levels are almost inexistent in p53 null cell lines or p53 nonfunctional cell lines, thus supporting a mandatory relationship between p53 positive cells and azurin apoptotic induction [21]. Therefore azurin can cause an increase in p53 intracellular level which, ultimately, lead to apoptosis induction in cancer cells, via caspase-mediated mitochondrial pathways [20]. Another interesting fact is that azurin was shown to be localized in cytosol and in mitochondria but not in the nucleus of p53 null cell lines, thereby demonstrating that azurin nuclear transport is p53-dependent [20], [21]. Further studies on this matter revealed that azurin binds preferentially to the N-terminal and central domain of p53, but very weakly to the C-terminal domain of this protein [21]. It was suggested that azurin forms a stable complex by binding the N-terminal domain of p53 in a 4:1 stoichiometry, and that it can also bind the DNA-binding domain of this tumor suppressor protein [15]. MDM2, a repressor oncoprotein which inhibits p53 transcriptional activity and favors its degradation, also binds p53 in the N-terminal region [27]. However it seems that azurin does not overlap the MDM2 binding site, although it may sterically shield p53 from interacting with MDM2 or other ubiquitin ligases [27]. Therefore the increase in p53 in response to azurin results essentially from a reduction in proteasome degradation of the tumor suppressor protein, and not from p53 enhanced transcription [15].

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More recently, azurin in vitro anti-tumor effects were extended to liquid cancers as well. Azurin demonstrated selective entry and cytotoxic effects against HL60, an acute myeloid leukemia cell line, and K562, a chronic myeloid leukemia cell line [28]. It also showed significant effect on arresting K562 cells at G2/M checkpoint, which often leads to induction of apoptosis.

Additionally to its entry specificity in cancer cells, azurin-derived peptide p28 has been shown to be capable of interfering with angiogenesis by inhibiting the formation of capillary tube formation of human umbilical vein endothelial cells (HUVEC), in a dose-related manner [25]. Although p28 could alter p53 intracellular levels in these cells, inhibition of angiogenesis was not suggested to be accomplished by a p53-mediated inhibition of cell cycle. In this study azurin also revealed the same anti-angiogenic activity but not the same efficacy of inhibition as p28.

Azurin, as well as p28, seems additionally capable of interfering in oncogenic transformation, since it was shown to inhibit the development of precancerous lesions in a mouse mammary gland organ culture model, previously exposed to a carcinogen Dimethyl-benz-anthracene (DMBA) [16].

Considering azurin’s remarkable p53-mediated apoptosis and cytotoxic effects against several tumor cell lines as in vitro models, it was of interest to verify whether or not similar results could be observed within in vivo models. On this subject azurin has shown to be effective on tumor growth inhibition in nude (athymic) mice with xenotransplanted UISO-Mel-2 and MCF-7 cells [20], [21]. In both studies tumor regression was justified by an increase of apoptosis in tumor cells. In another study, using a Dalton’s lymphoma bearing ascites mice model, Ramachandran et al. also showed azurin’s ability to induce apoptosis in tumor cells, therefore leading to tumor regression. In this study Bax and caspase-3 levels in tumor cells, treated with azurin, were shown to be increased, whereas Bcl-2 levels were diminished [29]. These results clearly support in vitro evidence, already described, regarding p53-mediated apoptosis by azurin, which alters the balance between pro and anti-apoptotic protein levels in tumor cells in favor of the first. Another important factor, verified in these three in vivo models, was the lack

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of azurin’s toxic effects on the animals [20], [21], [29]. In both cases no body weight changes or any histologic evidence of toxicity were observed.

Since p28 was considered accountable for a significant amount of the overall tumoricidal activity of azurin, this peptide was tested originally in vivo towards MCF-7 xenografs in athymic mice, resulting in tumor growth inhibition [15]. A more recent and broader study evaluated p28 activity against HCT-116 (colon cancer), UISO-Mel-23 (melanoma) and MDA-MB-231 (breast cancer) xenografs in athymic mice [30]. In this study tumor cell proliferation decreased in a dose-related manner in all three xenografs, whereas tumor regression was observed in a dose-related way in UISO-Mel-23 and MDA-MB-231 xenografs [30]. This azurin-derived peptide was shown to be non-immunogenic and non-toxic in mice and non-human primates, which indicates that it can become a promising drug to be used in a near future [30].

Taking into account all these promising findings about p28, it was proposed its entry into a phase I clinical trial in order to access its potential role as an anti-tumor drug in humans. Intravenous p28 was administrated three times a week, for 4 weeks, in 15 adult patients with p53-positive advanced solid tumors (7 melanoma, 4 colon cancer, 1 sarcoma, 1 gastrointestinal stromal cell tumor, 1 prostate cancer and 1 pancreatic cancer) [31]. Five escalating doses of p28 were used although no significant adverse effects, toxicity, nor any immune response were observed in any patient. This phase I clinical trial resulted in one patient with a complete tumor regression, three patients with partial regression, and seven patients with stable disease [31].

Cupredoxins, like azurin, exhibit topological similarity to a eucaryotic family of ligands named ephrins, having a common type of Greek key β-barrel [32]. Ephrins are endogenous ligands that bind with Ephrin receptors (Eph), which constitute the largest family of receptor protein tyrosine kinases [33]. Binding and heterodimerization between an ephrin and its receptor leads to the trans-autophosphorylation of the tyrosine kinase domains of the Eph receptor, leading to several signaling transduction cascades involved in developmental processes that require organized patterning and movement of cells, as in the remodeling of blood vessels [32], [33]. However Eph receptors as well as ephrins have shown

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also to be linked to pathological processes, such as tumor progression, angiogenesis, migration and invasion [32]. Both types of proteins were shown to be up-regulated in several different tumors, like EphB2 in breast carcinoma or lung cancer, for instance [32]. Azurin can bind the EphB2 receptor tyrosine kinase with a higher affinity than its endogenous ligand, ephrinB2, competing for this receptor and diminishing tyrosine phosphorylation, thereby interfering with cell signaling and cancer growth [32].

Despite being involved in the denitrification in P.aeruginosa, an obligatory role of azurin in this process was excluded, thereby the biological role of this protein still requires clarification [23]. It has been suggested, however, that azurin’s physiological function could be involved in bacterial virulence, characteristic of pathogenic bacteria like P.aeruginosa [23]. Experiments involving this bacterium and cancer cells have demonstrated that azurin secretion occurs mainly in the presence of cancer cells in the medium, whereas in the absence of cancer cells very little secretion of this protein was verified [34]. These findings point out the possible existence of a sensing mechanism in bacteria that could lead to azurin secretion in the presence of cancer cells, which they could sense as a threat or competitor to their own growth [35].

Interestingly it was shown that azurin can inhibit not only growth in cancer cells, but also in different pathogens as virus (AIDS virus HIV-1), parasites (Plasmodium falciparum) and protozoans (Toxoplasma gondii) [14], [36]. It appears that azurin’s ability for binding some pathogen surface proteins interferes in the entry and inhibit the growth of different pathogens and cancer cells [14], [36]. This promiscuity in binding different proteins, as seen in Figure 3, may be attributable to its structural similarity with the variable folds of immunoglobulins, which could represent a “progenitor” immune response used by prokaryotes, as suggested by some authors [14].

Azurin’s promiscuity and broader anti-tumor action represent two major aspects of this bacterial protein. So far it has been shown capable of 3 different modes of anti-tumor action: induction of apoptosis through p53 stabilization; inhibition of angiogenesis; and binding ephrin receptor kinases. Acting on different pathways in cancer progression, azurin differentiates itself from any

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other current anti-tumor drug available, which could show to be more effective against cancer cells.

Figure 3 - Azurin as a promiscuous protein, which possess both anticancer, antiparasite and

antiviral activities (Fialho et al, 2007) [37].

1.4 Cadherins

Classical cadherins constitute a family of transmembrane glycoproteins that mediate calcium-dependent cell-cell adhesion and present themselves as the major components of cell-cell adhesive junctions [38], [39]. These particular family includes four different types of cadherins, designated accordingly to their tissue distribution: CDH1/E-cadherin (epithelial), CDH2/N-cadherin (neuronal),

CDH3/cadherin (placental) and CDH4/R-cadherin (retinal) [40]. E- and

P-cadherin can be divided in three major structural domains: 1) an extracellular domain, which is responsible for cadherins adhesion properties since it’s where Ca2+ ions will bind to stabilize cadherins conformation; 2) a single membrane-spanning segment, accountable for protein anchorage to the cellular membrane; 3) a highly conserved cytoplasmic domain that bind directly to α, β and γ-catenins and p120-catenin, forming a complex that acts as a bridge between cadherins and the actin cytoskeleton [38], [39]. This binding is supported by α-catenin and

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provides the molecular basis for stable cell-cell interactions and its represented in Figure 4 [40].

The epithelial-calcium dependent cell-cell adhesion is accomplished by the establishment of hemophilic interactions between proteins, as two cadherin molecules of adjacent cells to form a homodimer [39]. The cadherin/catenin complex stability, as well as the signaling pathways controlled by this structure, is therefore essential for maintaining some cell properties, like cell-cell adhesion and homeostatic tissue architecture [39]. By regulating these properties the cadherin/catenin complex has a major role on processes like cell growth, differentiation, motility and survival [40]. In fact there is wide evidence that alterations in the adhesion properties between adjacent cells provide them with an invasive and migratory phenotype. Several data was reported regarding changes in normal E- and P-cadherin function or expression, which has been associated with all steps involved in tumor progression [39].

E-cadherin is predominantly expressed in all epithelial tissues, playing a major role in the formation of epithelia, and being responsible for the maintenance of cell shape and polarity [40]. E-cadherin gene, CDH1, acts as a tumor suppressor

Figure 4 – Schematic representation of the classical cadherin-catenin complex and the structural

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gene, regulating the invasion and metastasis of tumor cells. In fact loss of expression or abnormal function of E-cadherin, by mutations or loss of heterozygosity, can result in increased ability of tumor cells to invade and to create metastasis in neighbouring tissues, namely in breast cancer [39], [41]. Another relevant factor that inhibits E-cadherin expression is hypermethylation of the promotor region of CDH1, which has been implicated at the same time with the induction of migration in breast cancer cell lines [41]. Reduced expression of E-cadherin was associated with tumor progression in several types of cancer, including breast and stomach carcinomas [38], [39].

P-cadherin is a 118 kDa cadherin, that is expressed in ectodermal tissues, namely in the basal layers of stratified epithelia (as skin, uterine cervix, prostate, lung) and in myoepithelial cells of the breast [38], [40], [42]. This cadherin has been associated in growth and differentiation processes, as those during embryogenesis, for instance, and low levels of this protein were detected in normal tissues [40]. Unlike CDH1, which is undoubtedly recognized as a tumor suppressor gene, CDH3 role in cancer is contradictory and less characterized [40]. Distinct P-cadherin behavior was verified in different types of cancer, since it appears to act as an invasion suppressor in colorectal cancer and melanoma models whereas it resembles an oncogene in other models, as breast cancer, by inducing tumor cell motility and invasiveness when overexpressed [43]. Increased P-cadherin expression was also correlated with cell dedifferentiation and increased cell proliferation [39]. The opposite effects of E- and P-cadherin in breast cancer (as well as other models) are moreover unexpected since these two molecules share more than 67% of homology [40].

Before acquiring an invading behavior, tumor cells often suffer a process termed epithelial-to-mesenchymal transition, on which epithelial cadherins suffers downregulation whereas mesenchymal cadherins are expressed de novo [44]. This cadherin switch results in inhibition of cell-cell contacts and elicits active signals which prompt cell migration and invasion. In breast cancer, many of the highly aggressive tumors do not show, however, this cadherin switch. By contrast these tumors show P-cadherin overexpression while maintaining the normal E-cadherin expression [44]. P-cadherin is preferentially overexpressed in basal like

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carcinomas and it was verified that 20% to 40% of invasive breast carcinomas, as well as 25% of ductal carcinomas in situ showed overexpression of this cadherin [42], [43]. Another relevant observation is that P-cadherin expression has been reported as a marker of poor prognosis and reduced patient survival in high histological grade tumors, with decreased cell polarity [42]. Expression of this cadherin can also have an important role in the prognosis of invasive breast carcinomas that maintains normal E-cadherin expression [39]. An in vitro study supports these findings as it revealed that P-cadherin overexpression, in wild-type E-cadherin breast cancer cell lines, as MCF-7/AZ, can induce increased cell invasion, motility and migration [44]. Moreover it was shown that P-cadherin can only induce invasion in breast cancer cell lines which already express and endogenous cadherin like cadherin, by disrupting the interaction between E-cadherin and two catenins (p120 and β-catenin) [40].

Additionally it was found that the presence of P-cadherin, in these E-cadherin positive cancer cells, can provoke the secretion of pro-invasive factors, as matrix metalloproteases (MMPs), MMP-1 and MMP-2, which in turn lead to P-cadherin ectodomain cleavage [44]. The 80 kDa soluble P-cadherin fragment (sP-cad) formed in that cleavage has pro-invasive activity, being responsible for in vitro invasion of these cancer cells [42], [44]. This protein was found also in body fluids, as milk from the lactating breast, although its biological role in that context is still unknown [42]. Although the mechanism by which P-cadherin overexpression induces the secretion of metalloproteases is unknown, it is clear that these proteases are responsible for extracellular matrix degradation, for instance, which increases cell invasion induction [44].

1.5 Azurin and P-cadherin interactions

Considering azurin’s broader anti-tumor properties and knowing that it exhibits a high affinity level with P-cadherin, it was of interest to evaluate this protein’s activity in an invasive breast cancer cell lines (MCF-7/AZ.Pcad) that express endogenous levels of E-cadherin and higher levels of P-cadherin [45]. After azurin administration with sub-lethal dosages (50-100 µM) to these cells, it was verified, by western blotting, a specific decrease in P-cadherin total protein level

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[45]. P-cadherin distribution across the plasma membrane was analyzed through immunofluorescence, and it was shown that it suffered a decrease, as well, in cells treated with azurin [45]. On the contrary, evidence showed that E-cadherin level and its membrane distribution were not affected by azurin’s treatment. Additionally, it was found that a reduction in P-cadherin’s level (caused by azurin) was correlated with a less invasive behavior of breast cancer cells in a Matrigel system, as well as with a lower activity of metalloproteases MMP-2 and 9 in cell conditioned medium. Another extreme relevant finding was that P-cadherin soluble fragment (sP-cad) level, which has pro-invasive activity, was found to be decreased when the conditioned medium, on which cells treated with azurin where cultured, was analyzed. Complementary assays regarding CDH1 and

CDH3 genes showed that P-cadherin decreased levels were not a consequence of

changes in gene expression [45]. Therefore it was suggested that post-transcriptional regulation processes, mediated by azurin, should be involved in P-cadherin diminished levels. Microarray analysis on mRNA profiles of MCF-7/AZ.Pcad cells treated with azurin showed that genes were associated with membrane organization, vesicle-mediated and endosome transport, and lysosome were up-regulated (Bernardes et al., 2013, submitted for publication).

This work intends to pursue those evidences and to demonstrate that azurin can display an important role on mediating P-cadherin degradation through proteolytic systems.

1.6 Inhibitors

In order to maintain regular homeostasis and meet their nutritional and energetic demands, cells have devolved proteolytic systems which eliminate instable and incorrect folded proteins. These systems are able to protect cells from cytotoxic damage, caused by intracellular accumulation of damaged proteins or organelles, and to recycle amino acids that result from protein degradation, replenishing the intracellular reserve of these macromolecules, which are essential in the absence of nutrients [46]. The two main cellular degradation systems in eukaryotic cells are the autophagy-lysossome system and the

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proteasome system (UPS), although the relative contribution of each one of these pathways may vary greatly between cell types [47], [48].

Lysosomes are single membrane vesicles that contain in their lumen a large diversity of hydrolytic enzymes, such as proteases, lipases, glycosidases and nucleotidases [46]. These enzymes reach their optimal enzymatic activity at the acidic pH verified in the lysosomal lumen, which is maintained by an ATP-dependent proton pump (V-ATPase) present at the lysosomal membrane [46], [47]. The lysosomal degradation pathway is preferentially used in proteolysis of membrane proteins, as receptors or channels, although it can act on cytoplasm proteins as well [47]. In order to reach the lysosomes, tagged proteins must be recognized and delivered to this organelle by three different ways: macroautophagy, microautophagy and chaperone-mediated autophagy [48]. Macroautophagy involves a de novo formed double membrane vesicle or autophagosome, which is responsible for protein sequestering, and that will fuse with late endosomes or lysosomes [46]. In microautophagy, it occurs a lysosomal membrane invagination, resulting in sequestration of regions of the cytosol directly by the lysosomal membrane into its lumen. Finally, chaperone-mediated autophagy is a process that requires the recognition of the target protein by a cytosolic chaperone, and its binding to a lysosomal membrane receptor, which in turn allows target protein translocation into lysosome’s lumen. There are several chemical agents which act as lysosome inhibitors, such as ammonium chloride and chloroquine, two weak bases that accumulate inside the lysosome and dissipate its low acidic pH, by neutralizing H+ ions [49].

The UPS represents the major pathway accountable for the degradation of proteins present in the cytosol, nucleus and endoplasmic reticulum [46]. This proteolytic system is composed by ubiquitin, a small tagging protein that is covalently linked to proteins, through a three step ATP-consuming reaction, that involves E1 (ubiquitin-activating enzyme), E2 (ubiquitin-conjugating enzyme) and E3 (ubiquitin-ligase enzyme), selecting the substrate protein for degradation [48]. These three enzymes are responsible for selecting misfolded proteins to degradation, as well as catalyzing the binding reaction between ubiquitin molecules and target proteins [48]. After ubiquitination target proteins are

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delivered to the proteasome, a multicatalytic enzyme complex composed by the catalytic 20S core and the 19S regulator [47]. The 20S core complex its divided in three catalytic subunits, with distinct activities as chymotrypsin-like, trypsin-like and caspase-like activities [48]. Finally, polyubiquitinated proteins are degraded to peptides by the proteasome and then free ubiquitin is recycled [48]. Currently available proteasome inhibitors interfere with chymotryptic-like of the 20S core complex such as MG132, a reversible but potent peptide aldehyde [48].

Lipid rafts are considered subdomains of cell membrane, and are involved in processes like cell proliferation, differentiation and apoptosis, which often are altered during tumor development [50]. These membrane regions are enriched in cholesterol, or caveolins, for instance. Methyl-β-cyclodextrin (MβCD) is a cyclic oligosaccharide that binds preferentially to cholesterol from the plasma membrane, altering lipid composition and causing cholesterol depletion [51], [52]. By disrupting the assembly of lipid rafts and caveolae, this compound can therefore inhibit caveolae-mediated endocytosis, since caveolin can’t form further vesicle [53], [54].

Since azurin enter cells through caveolae-mediated endocytosis, we sought to use MβCD to inhibit azurin entry into breast cancer cells, in order to evaluate its impact on P-cadherin degradation level.

1.7 MPT 63

MPT 63 is a low molecular weight (16 kDa) protein that comprises 130 amino acid residues preceded by a secretion signal peptide [55], [56]. Firstly described as one of the three most abundant extracellular proteins secreted by Mycobacterium

tuberculosis, MPT 63 was later found to be specific of mycobacteria of the M. tuberculosis complex, as M. africanum, M. bovis and attenuated M. bovis bacille

Calmette-Guérin (BCG) and it is absent in mycobacterial species that do not belong to this complex, as M. avium [56], [57].

The X-ray crystal structure of MPT 63 (represented in Figure 5) was determined by Goulding et al as a sandwich, consisting of two antiparallel sheets, similar to an immunoglobulin-like fold, with and additional antiparallel β-sheet [58]. Interestingly this protein exhibits some structural similarity to cell

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surface-binding proteins such as Homo sapiens β-2 adaptin, bovine arrestin, eukaryotic fibronectin-binding proteins, major histocompability domains or T-cell receptors, for instance [58]. This similarity has suggested a possible role of MPT 63 in cell-host interactions to facilitate endocytosis as well as phagocytosis during bacterial internalization [58]. In fact MPT 63, as well as other proteins secreted by

M. tuberculosis, is considered relevant to the survival of the bacterium within its

host, since it has been shown to be a cell envelope-associated protein that may act as a virulence factor [58]. Moreover, secretion of proteins by intracellular pathogens, as M. bacterium, has a central role in determining pathways of antigen presentation and recognition by effector T-cells involved in protective immunity [58]. Although it shares some structural similarities with proteins of other organisms, this protein doesn’t exhibit a very strong homology with any of them, which leaves its physiological role yet to be unveiled [55].

Figure 5– Structure of M. tuberculosis MPT 63 protein (adapted from Goulding et al, 2002) [58]

As mentioned earlier, MPT 63 is one of the three most abundant extracellular proteins secreted by M. tuberculosis, and the other two most abundant proteins were already described as antigens (antigens 85A and 85B), which can indicate that this protein can also act as an antigen [56]. In fact so far several data have

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been pointing in that direction, as a study that has shown that guinea pigs, previously aerosol infected with virulent M. tuberculosis, were able to induce humoral immune responses, leading to the production of high-level antibodies against MPT 63 [55]. Moreover, guineas pigs injected with purified MPT 63 revealed cutaneous delayed-type hypersensitivity responses, which indicate that this protein can induce immunogenic responses, through different pathways [55]. MPT 63 was also shown to be immunogenic in rabbits, since immunization with the purified protein led to antibodies production against it [59]. Antibodies against this protein were also identified in serum from tuberculosis patients by enzyme-linked immunosorbent assay (ELISA) [60]. In a different study, a DNA vaccine encoding both the MPT 63 and ESAT-6 antigens, has demonstrated to be capable of inducing a robust protective response in mice, namely through generation of gamma interferon (INF-γ)-secreting CD4+ T cells [61]. Since MPT 63 has shown, at some level, structural similarities with major histocompability domains, as well as T-cell receptors, it is important to mention a study where these two factors were tested in humans. In this study it was shown that MPT 63 induced a moderate T helper 1 (Th1) cell reactivity in peripheral blood mononuclear cells (PBMNCs), obtained from M. bovis BCG vaccinated healthy subjects [62]. Th1 cells reactivity were evaluated through proliferation and IFN-γ secretion screening tests. Furthermore the presentation of MPT 63 to Th1 cells were also analyzed, as PBMNCs from MPT 63-responding donors were typed for Human Leucocyte Antigen (HLA-DR) molecules revealing heterogenecity between them [62] . Therefore it seems that MPT 63 exhibits important requirements for a potential use as a vaccine against tuberculosis, since it could be able to induce a positive response throughout heterogeneous groups of donors.

Although little is known about the mechanisms behind this protein’s immunogenic properties it was suggested that these properties could be explained, at least in part, by the first 30 amino acid sequence in its N-terminal region, which has shown a high density of T-cell epitopes recognized in immunized guinea pigs [56].

As mentioned before MPT 63 is not present in mycobacteria which do not belong to the Mycobacterium complex, and this specificity is also supported by

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the fact that MPT 63 lacks epitopes that cross-react serologically with M. avium antigens [55]. Taken altogether, this results regarding the immunogenic properties of MPT 63, led several authors to propose this protein as a target for vaccine design and diagnostic tools development in tuberculosis [57], [60].

MPT 63 and azurin share some important features, as they both are low molecular weight proteins secreted by bacteria. More interestingly, however, is the fact that both their structures are a β-sandwich, which demonstrates evident structural similarities with immunoglobulin-like folds. Another interesting observation is the promiscuity evidenced by these two proteins, which grant them unique properties, allowing them t be capable of binding to different proteins. Additionally to MPT 63’s ability to bind T-cell receptors, it also shares some structural similarity with bovine arrestin, a protein that inhibits receptor activity, by binding to the cytoplasmic surface, occluding the interaction with G-proteins [58]. Taking account that azurin can also bind to surface cell receptors, as Eph receptors in cancer cells, thereby interfering with cancer growth, it is intriguing to verify if these proteins can provoke similar anti-tumor effects.

Recently a 30 amino acid peptide derived from MPT 63 and termed MB30, has shown significant cytotoxic activity against several bladder (HTB-9, UM-UC-3), colon (COLO 205, HCT 116), and cervical (SiHa, CaSki) cancer cell lines [63]. Moreover, MPT 63 and MB30 also showed cytotoxicity in brain (U87), liver (HepG2) and breast (MDA-MB-231) cancer cell lines, although the MB30 peptide has demonstrated an even higher cytotoxicity [63].

Since MPT 63, as well as its derived peptide MB30, has shown cytotoxic effects against different cancer cell lines, our purpose in this work was to unveil a possible role of MPT 63 as an anticancer agent. Therefore we sought to verify if MPT 63 could cause possible cytotoxic effects on a breast (MCF-7/AZ.Mock) and lung (A549) cancer cell lines.

1.8 Nucleoside diphosphate kinase

Nucleoside diphosphate kinase (Ndk) is an enzyme which catalyzes the reversible transfer of the 5’-terminal phosphate from nucleoside triphosphates (NTPs) to nucleoside diphosphates (NDPs), thus playing a key role as a

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housekeeping enzyme, as it maintains the nucleotide pools for the synthesis of nucleic acids [64]. Ndk is a small protein, with approximately 150 amino acids, which form an homohexamer in eukaryotes, archae and gram-positive bacteria, whereas in gram-negative bacteria it forms homotetramers [64], [65]. So far it has been described in humans and others eukaryotes as Droshophila melanogaster, as well as in several bacteria such as P. aeruginosa, Escherichia coli, Myxococcus

xanthus, M. tuberculosis or Vibrio cholerae for instance [66], [67]. Interestingly

there were reported two forms of this protein in P. aeruginosa: a 16 kDa cytoplasmic form (during the early stages of cellular growth), and a 12 kDa membrane-associated form (at the onset of the stationary phase) which is originated from a cleavage in the 16 kDa form, by a periplasmic protease termed elastase [64], [68]. Although the importance of having two forms of this protein is not clear, it is known that the membrane-associated form, constitutes a complex with pyruvate kinase, which predominantly synthesizes GTP [69]. In mammalian cells Ndk can be localized in the cytosol, as membrane-associated or as an ectoenzyme in the cell surface exposed to the outside medium [70].

Despite the fact that Ndk exhibits a different structure in humans and some prokaryotes, it was revealed that Ndk form P. aeruginosa shows 40-45% identity with eukaryotic Ndks, as well as 50-60% identity with other bacterial Ndks, evidencing that it is a highly conserved enzyme among different species [66] .

Extracellular secretion of Ndk was reported in M. bovis BCG, M. tuberculosis,

P. aeruginosa, Trichenella spiralis and V. cholerae [67]. The physiological

relevance for the secretory nature of Ndk in these bacteria is not clear, although it has been mentioned to be an important enzyme in host-pathogen interactions [67]. This suggestion is supported from the fact that Ndk from M. tuberculosis was reported to interfere and block phagosome maturation in murine macrophages [71]. Moreover, Ndks secreted by M. tuberculosis and V. cholerae were shown to display cytotoxic effects on mouse macrophage cell lines, which can indicate that secretable Ndk plays a major role in the modulation of virulence in these pathogenic species [67], [72].

Additionally to its kinase function, Ndk has been associated in further biological functions in eukaryotic cells, namely in humans, where it plays a role in

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normal embryonic development, cell differentiation, tumor progression and cell migration [73], [74]. In humans, 10 genes have been already identified as part of the nm23 (non metastatic) gene family, which represents the human Ndk gene family [75]. Nm23-H1 was the first metastatic gene in humans to be identified in 1988 by Steeg et al. , since the accumulation of correspondent transcripts were shown higher in tumor cells of low metastatic potential in murine melanoma cell lines of varying metastatic potentials [76]. The two more expressed nm23 genes in humans are nm23-H1 and nm23-H2, localized in chromosome 17q21, and encode the Ndk-A and Ndk-B, respectively [10], [75]. The three-dimensional structure of human Ndk-A subunit is shown in Figure 6.

Although it is known mainly by its role in tumor progression, Ndk-B has been also described as a transcription factor for the c-Myc oncogene, and it was proposed to act as a repair protein in humans, on the basis of sequence homology with certain glycosylases [73].

Since Steeg et al. initial observations it has been verified, by several authors, a correlation between reduced Ndk-A expression and high tumor metastatic potential in different carcinomas: liver, melanoma, colon, breast, ovarian, gastric and hepatocellular [75]. Transfection experiments with nm23-H1 and nm23-H2 support these findings, since the overexpression of these genes in breast cancer MDA cell lines resulted in decreased metastatic potential of these cells [77]. The overexpression of this protein in tumor cells was found to reduce tumor cell motility and invasion, promote cellular differentiation and inhibits anchorage-independent growth, as well as adhesion to fibronectin, laminin and vascular endothelial cells [10]. However, although Ndk-A has shown anti-metastatic effects on the majority of carcinomas, there are some exceptions. It was reported that high levels of this protein were detected in aggressive carcinomas derived from thyroid, pancreatic, squamous cell lung carcinomas, neuroblastoma and acute myelogenous leukemia [75]. Moreover high tissue levels of Ndk-A were found in patients with breast carcinoma [78].

Discovering and exploiting bacterial proteins as anticancer agents

UNIVERSIDADE DE LISBOA

FACULDADE DE CIÊNCIAS

DEPARTAMENTO DE BIOLOGIA ANIMAL

Discovering and exploiting bacterial

proteins as anticancer agents

Gonçalo Emanuel Fialho Mourata da Silva

DISSERTAÇÃO

Mestrado em Biologia Humana e Ambiente

UNIVERSIDADE DE LISBOA

FACULDADE DE CIÊNCIAS

DEPARTAMENTO DE BIOLOGIA ANIMAL

Discovering and exploiting bacterial

proteins as anticancer agents

Gonçalo Emanuel Fialho Mourata da Silva

DISSERTAÇÃO

Mestrado em Biologia Humana e Ambiente

ACKNOWLEDGMENTS

ABSTRACT

RESUMO

TABLE OF CONTENTS

LIST OF ABBREVIATIONS

INDEX OF FIGURES

INDEX OF TABLES

1. INTRODUCTION