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| Author(s): |
Maria Raquel Unterkircher Galheigo
Total Authors: 1
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| Document type: | Doctoral Thesis |
| Press: | São Paulo. |
| Institution: | Universidade de São Paulo (USP). Instituto de Ciências Biomédicas (ICB/SDI) |
| Defense date: | 2021-04-29 |
| Examining board members: |
Ruy Gastaldoni Jaeger;
Hernandes Faustino de Carvalho;
Maria Aparecida da Silva Pinhal;
Marinilce Fagundes dos Santos
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| Advisor: | Ruy Gastaldoni Jaeger |
| Abstract | |
Breast cancer is among the most frequent worldwide mortality. Tumor microenvironment undertakes important role during cancer progression. Different cells enmeshed in the extracellular matrix (ECM) compose such microenvironment. Laminin 111 is a major ECM glycoprotein and exhibits different bioactive peptides influencing tumor biology. We have demonstrated that the laminin-derived peptide C16 (KAFDITYVRLKF, short arm of gamma 1 chain) regulates migration, invasion, and invadopodia formation in different cancer cells. Our findings indicate that regulatory mechanisms underlying C16 effects are related to β 1 integrin. This prompted us to investigate the interaction between C16 peptide and β 1 integrin in breast cancer cells as well as its morpho functional effects. These cells attach to C16 peptide and this attachment is inhibited by β 1 integrin depletion by siRNA. Moreover, rhodamine-C16 colocalized with activated β 1 integrins in breast cancer cells. Flow cytometry assay showed that C16 increased β 1 activation after 20 minutes in these cells. Cellular localization of C16 was carried out by transmission electron microscopy (TEM) and time-lapse fluorescence microscopy. TEM showed nanogold-conjugated C16 decorating cell membrane and inside cellular vesicles. The presence of C16 inside vesicles would imply peptide endocytosis. Time-lapse confocal microscopy displayed peptide C16 internalized by breast cells over time and decreased internalization of this peptide by dynasore in MCF-7 cells. Moreover, fluorescence microscopy also showed that a caveolin-1 depletion by siRNA decreases C16 internalization. According to these results, we hypothesized whether the endocytosed peptide would be directed to the endosome-lysosome pathway for degradation. Time-lapse and immunofluorescence illustrated part of the internalized peptide colocalized with early endosomes and lysosomes in the studied cells. Taken together, our results suggested that after membrane interaction and β 1 integrin activation, breast cells would internalize peptide C16, which, at least in part, will be degraded in lysosomes. (AU) | |
| FAPESP's process: | 16/20228-4 - Interaction between the laminin-derived peptide C16 and beta 1 integrin in breast cancer cells. Mechanisms involved and functional consequences. |
| Grantee: | Maria Raquel Unterkircher Galheigo |
| Support Opportunities: | Scholarships in Brazil - Doctorate |