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?-Actin-binding complementarity-determining region 2 of variable heavy chain from monoclonal antibody C7 induces apoptosis in several human tumor cells and is protective against metastatic melanoma.

ABSTRACT: Complementarity-determining regions (CDRs) from monoclonal antibodies tested as synthetic peptides display anti-infective and antitumor activities, independent of the specificity of the native antibody. Previously, we have shown that the synthetic peptide C7H2, based on the heavy chain CDR 2 from monoclonal antibody C7, a mAb directed to a mannoprotein of Candida albicans, significantly reduced B16F10 melanoma growth and lung colony formation by triggering tumor apoptosis. The mechanism, however, by which C7H2 induced apoptosis in tumor cells remained unknown. Here, we demonstrate that C7H2 interacts with components of the tumor cells cytoskeleton, being rapidly internalized after binding to the tumor cell surface. Mass spectrometry analysis and in vitro validation revealed that ?-actin is the receptor of C7H2 in the tumor cells. C7H2 induces ?-actin polymerization and F-actin stabilization, linked with abundant generation of superoxide anions and apoptosis. Major phenotypes following peptide binding were chromatin condensation, DNA fragmentation, annexin V binding, lamin disruption, caspase 8 and 3 activation, and organelle alterations. Finally, we evaluated the cytotoxic efficacy of C7H2 in a panel of human tumor cell lines. All tumor cell lines studied were equally susceptible to C7H2 in vitro. The C7H2 amide without further derivatization significantly reduced lung metastasis of mice endovenously challenged with B16F10-Nex2 melanoma cells. No significant cytotoxicity was observed toward nontumorigenic cell lines on short incubation in vitro or in naïve mice injected with a high dose of the peptide. We believe that C7H2 is a promising peptide to be developed as an anticancer drug.

SUBMITTER: Arruda DC

PROVIDER: S-EPMC3340220 | biostudies-literature | 2012 Apr

REPOSITORIES: biostudies-literature

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β-Actin-binding complementarity-determining region 2 of variable heavy chain from monoclonal antibody C7 induces apoptosis in several human tumor cells and is protective against metastatic melanoma.

Arruda Denise C DC Santos Luana C P LC Melo Filipe M FM Pereira Felipe V FV Figueiredo Carlos R CR Matsuo Alisson L AL Mortara Renato A RA Juliano Maria A MA Rodrigues Elaine G EG Dobroff Andrey S AS Polonelli Luciano L Travassos Luiz R LR

The Journal of biological chemistry 20120213 18

Complementarity-determining regions (CDRs) from monoclonal antibodies tested as synthetic peptides display anti-infective and antitumor activities, independent of the specificity of the native antibody. Previously, we have shown that the synthetic peptide C7H2, based on the heavy chain CDR 2 from monoclonal antibody C7, a mAb directed to a mannoprotein of Candida albicans, significantly reduced B16F10 melanoma growth and lung colony formation by triggering tumor apoptosis. The mechanism, however ...[more]

PMID: 22334655

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Project description:Extensive diversity has been identified in the human heavy chain immunoglobulin locus, including allelic variation, gene duplication, and insertion/deletion events. Several genes have been suggested to be deleted in many haplotypes. Such findings have commonly been based on inference of the germline repertoire from data sets covering antibody heavy chain encoding transcripts. The inference process operates under conditions that may limit identification of genes transcribed at low levels. The presence of rare transcripts that would indicate the existence of poorly expressed alleles in haplotypes that otherwise appear to have deleted these genes has been assessed in the present study. Alleles IGHV1-2*05, IGHV1-3*02, IGHV4-4*01, and IGHV7-4-1*01 were all identified as being expressed from multiple haplotypes, but only at low levels, haplotypes that by inference often appeared not to express these genes at all. These genes are thus not as commonly deleted as previously thought. An assessment of the 5' untranslated region (up to and including the TATA-box), the signal peptide-encoding part of the gene, and the 3'-heptamer suggests that the alleles have no or minimal sequence difference in these regions in comparison to highly expressed alleles. This suggest that they may be able to participate in immunoglobulin gene rearrangement, transcription and translation. However, all four poorly expressed alleles harbor unusual sequence variants within their coding region that may compromise the functionality of the encoded products, thereby limiting their incorporation into the immunoglobulin repertoire. Transcripts based on IGHV7-4-1*01 that had undergone somatic hypermutation and class switch had mutated the codon that encoded the unusual residue in framework region 3 (cysteine 92; located far from the antigen binding site). This finding further supports the poor compatibility of this unusual residue in a fully functional protein product. Indications of a linkage disequilibrium were identified as IGHV1-2*05 and IGHV4-4*01 co-localized to the same haplotypes. Furthermore, transcripts of two of the poorly expressed alleles (IGHV1-3*02 and IGHV4-4*01) mostly do not encode in-frame, functional products, suggesting that these alleles might be essentially non-functional. It is proposed that the functionality status of immunoglobulin genes should also include assessment of their ability to encode functional protein products.

Project description:We have developed a monoclonal antibody (mAb) C7 that reacts with Als3p and enolase present in Candida albicans cell wall and exerts three anti-Candida activities: candidacidal activity and inhibition of both adhesion and filamentation. To investigate the mode of action of mAb C7 on fungal viability, we examined changes in the genome-wide gene expression profile of C. albicans grown in presence of a subinhibitory concentration of mAb C7 (12.5 µg/ml) by using microarrays. A total of 49 genes were found to be differentially expressed upon treatment with mAb C7. Of these, 28 were found to be up-regulated and 21 down-regulated. The categories of up-regulated genes with the largest number of variations were those involved in iron uptake or related to iron homeostasis (42.86%), while the energy-related group accounted for 38.10% of the down-regulated genes (8/21). Results were validated by real Time PCR. Since these effects resembled those found under iron-limited conditions, the activity of mAb C7 on C. albicans mutants with deletions in key genes implicated in the three iron acquisition systems described in this yeast was also assessed. Only mutants lacking TPK1 and TPK2 genes were less sensitive to the candidacidal effect of mAb C7. FeCl3 or hemin at concentrations ≥ 7.8µM reversed the candidacidal effect of mAb C7 on C. albicans, on a concentration dependent manner. The results presented in this study provide evidence that the candidacidal effect of mAb C7 is related to the blockage of the reductive iron uptake pathway of C. albicans. A saturated culture of C. albicans grown overnight was diluted to an optical density at 600 nm of approximately 0.1 and divided in two aliquots. One of them was used untreated as control and the second one was treated with a subinhibitory concentration (12.5 µg/ml) of monoclonal antibody C7 . Both cultures were incubated for 18 h at 37ºC before harvesting cells. Antibody added and control samples were obtained each time. The experiment was repeated once. Dye-swap technique was used for hybridization and four arrays were analyzed to compare the expresion of over six thousands genes in response to antibody C7.

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?-Actin-binding complementarity-determining region 2 of variable heavy chain from monoclonal antibody C7 induces apoptosis in several human tumor cells and is protective against metastatic melanoma.

Publications

β-Actin-binding complementarity-determining region 2 of variable heavy chain from monoclonal antibody C7 induces apoptosis in several human tumor cells and is protective against metastatic melanoma.

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