Glioblastoma Multiforme: Novel Therapeutic Approaches

The current therapy for glioblastoma multiforme involves total surgical resection followed by combination of radiation therapy and temozolomide. Unfortunately, the efficacy for such current therapy is limited, and newer approaches are sorely needed to treat this deadly disease. We have recently described the isolation of bacterial proteins and peptides with anticancer activity. In phase I human clinical trials, one such peptide, p28, derived from a bacterial protein azurin, showed partial and complete regression of tumors in several patients among 15 advanced-stage cancer patients with refractory metastatic tumors where the tumors were no longer responsive to current conventional drugs. An azurin-like protein called Laz derived from Neisseria meningitides demonstrates efficient entry and high cytotoxicity towards glioblastoma cells. Laz differs from azurin in having an additional 39-amino-acid peptide called an H.8 epitope, which allows entry and high cytotoxicity towards glioblastoma cells. Since p28 has been shown to have very little toxicity and high anti-tumor activity in advanced-stage cancer patients, it will be worthwhile to explore the use of H.8-p28, H.8-azurin, and Laz in toxicity studies and glioblastoma therapy in preclinical and human clinical trials

Perturbing the Dynamics and Organization of Cell Membrane Components: A New Paradigm for Cancer-Targeted Therapies

Cancer is a multi-process disease where different mechanisms exist in parallel to ensure cell survival and constant adaptation to the extracellular environment. To adapt rapidly, cancer cells re-arrange their plasma membranes to sustain proliferation, avoid apoptosis and resist anticancer drugs. In this review, we discuss novel approaches based on the modifications and manipulations that new classes of molecules can exert in the plasma membrane lateral organization and order of cancer cells, affecting growth factor signaling, invasiveness, and drug resistance. Furthermore, we present azurin, an anticancer protein from bacterial origin, as a new approach in the development of therapeutic strategies that target the cell membrane to improve the existing standard therapies

Exploring the anticancer potential of the bacterial protein azurin

Bacterial proteins and their derivative peptides have emerged as promising anticancer agents. Nowadays they represent a valuable set of candidate drugs with different origins and modes of action. Among these, monomeric cupredoxins, which are metalloproteins involved in the electron transport chain of prokaryotes, have been shown to possess potent anticancer activities. In particular, much attention has been focused on azurin produced by the pathogenic bacterium Pseudomonas aeruginosa. More recently, several in vitro and in vivo studies have reported the multi-targeting anticancer properties of azurin. Moreover, p28, a peptide derived from azurin, has completed two phase I clinical trials in cancer patients with promising results. In this updated review, we examine the current knowledge regarding azurin’s modes of action as an anticancer therapeutic protein. We also review the clinical trial results of p28 emphasizing findings that make it suited (alone or in combination) as a therapeutic agent for cancer treatment. Finally we discuss and address the challenges of using the human microbiome to discover novel and unique therapeutic cupredoxin-like proteins

A BCAM0223 Mutant of <em>Burkholderia cenocepacia</em> Is Deficient in Hemagglutination, Serum Resistance, Adhesion to Epithelial Cells and Virulence

<div><p><em>Burkholderia cepacia</em> complex (Bcc) bacteria are a problematic group of microorganisms causing severe infections in patients with Cystic Fibrosis. In early stages of infection, Bcc bacteria must be able to adhere to and colonize the respiratory epithelium. Although this is not fully understood, this primary stage of infection is believed to be in part mediated by a specific type of adhesins, named trimeric autotransporter adhesins (TAAs). These homotrimeric proteins exist on the surface of many Gram negative pathogens and often mediate a number of critical functions, including biofilm formation, serum resistance and adherence to an invasion of host cells. We have previously identified in the genome of the epidemic clinical isolate <em>B. cenocepacia</em> J2315, a novel cluster of genes putatively encoding three TAAs (BCAM0219, BCAM0223 and BCAM0224). In this study, the genomic organization of the TAA cluster has been determined. To further address the direct role of the putative TAA BCAM0223 in <em>B. cenocepacia</em> pathogenicity, an isogenic mutant was constructed via insertional inactivation. The <em>BCAM0223::Tp</em> mutant is deficient in hemagglutination, affected in adherence to vitronectin and in biofilm formation and showed attenuated virulence in the <em>Galleria mellonella</em> model of infection. Moreover, the <em>BCAM0223::Tp</em> mutant also showed a significant reduction in its resistance to human serum as well as in adherence, but not in invasion of, cultured human bronchial epithelial cells. Altogether these results demonstrate that the BCAM0223 protein is a multifunctional virulence factor that may contribute to the pathogenicity of <em>B. cenocepacia.</em></p> </div

Gene organization of the TAA gene cluster in <i>B. cenocepacia</i> K56-2. A

<p>) Genomic organization and operon mapping of the TAA gene cluster. The regions amplified by RT-PCR are indicated by filled arrows and the respective primers location is shown in the genes. The regions co-transcribed are identified with dashed arrows. Hyp – hypothetical protein, HK- histidine kinase, TAA – trimeric autotransporter adhesin, OMP – outer membrane protein, RR – response regulator. <b>B</b>) Results from the RT-PCR analysis, showing that no amplification was obtained for regions c), d) and f). Regions a), b) and e) showed amplification indicating that genes are co-trasncribed. Genomic DNA was used as positive control (+). A negative control was performed with RNA without reverse transcriptase, and the result was always negative. M, molecular mass marker.</p

Effect of the <i>BCAM0223</i> mutation on serum resistance.

<p>The serum bactericidal assay performed using 30% normal human serum (NHS) incubated with <i>B. cenocepacia</i> K56-2 (black bars) and <i>BCAM0223</i> mutant (open bars) showed significant sensitivity of the mutant to serum killing (*<i>P</i><0.001). Heat inactivated NHS (hNHS) was used as control of the experiment showing no killing activity. Determination of the pathway involved in serum-killing of <i>BCAM0223::Tp</i> mutant, was performed using three distinct sera: NHS containing 10 mM MgCl₂ plus 10 mM EGTA that block both, the lectin and classical pathways (LC-NHS), a C1q-depleted serum (C-NHS) (blocks classical pathway) (C<b>-</b>NHS) and factor B-depleted human serum (B<b>-</b>NHS) which contains solely the alternative complement pathway blocked. The wild-type <i>B. cenocepacia</i> K56-2 (filled bars) showed resistance to all three sera, whereas <i>BCAM0223::Tp</i> mutant (open bars) was resistant to LC-NHS and C<b>-</b>NHS but sensitive to B<b>-</b>NHS (*<i>P</i><0.001), suggesting that the killing of the serum-sensitive <i>BCAM0223::Tp</i> mutant is mediated by the classical complement pathway. All the results are from three independent experiments.</p

Hemagglutinin motifs in the BCAM0223 protein and effect of the <i>BCAM0223</i> mutation on hemagglutination ability.

<p><b>A</b>) Schematic illustration of the domain organization and coiled-coil prediction of BCAM0223 protein from <i>B. cenocepacia</i> J2315 using data program (<a href="http://toolkit.tuebingen.mpg.de/dataa" target="_blank">http://toolkit.tuebingen.mpg.de/dataa</a>). The protein comprises the typical membrane-anchor domain of TAAs at the C-terminal, a signal peptide (SP) at the N-terminal, two YadA-like heads domains and several connector domains (Neck and DALL). Using Pfam database (<a href="http://pfam.sanger.ac.uk/" target="_blank">http://pfam.sanger.ac.uk/</a>) numerous Hep-Hag repeats and hemagglutinin domains (Him) were found in BCAM0223 protein. Pfam also predicted the YadA-like C-terminal corresponding to the membrane-anchor and an extended SP. <b>B</b>) Hemagglutination ability of the wild type <i>B. cenocepacia</i> K56-2 and <i>BCAM0223::Tp</i> mutant, using 3% sheep red blood cells. PBS was used as a negative control. The highest dilution giving hemagglutination was taken as the titre.</p

Effect of the <i>BCAM0223</i> mutation on adherence to extracellular matrix proteins and biofilm formation. A

<p>) Adherence of wild-type <i>B. cenocepacia</i> K56-2 (filled bars) and <i>BCAM0223::Tp</i> mutant (open bars) to ECM proteins, laminin, fibronectin, collagen type I and IV and vitronectin. Only the binding capacity to vitronectin was significantly decreased in the mutant (*<i>P</i><0.001). <b>B</b>) Static biofilm formation in polystyrene microtiter plates by wild-type <i>B. cenocepacia</i> K56-2 (filled bars) and <i>BCAM0223::Tp</i> mutant (open bars) at 24 and 48 h. Biofilm growth was quantified by the solubilization of crystal violet-stained cells with ethanol. Either after 24 h or 48 h incubation, the <i>BCAM0223</i> mutant strain exhibited a statistically significant reduction in biofilm formation (*<i>P</i><0.001 and **<i>P</i><0.05). All the results are from three independent experiments; bars indicate SD.</p