Produção recombinante e estudos funcionais de três novas cistatinas da cana-de-açúcar e sua utilização em estudos de inibição da adesão, proliferação, migração e invasão celular
Resumo
ABSTRACT
Tumor metastasis is usually a major cause of death among cancer patients.
Degradation and invasion of the extracellular matrix and basement membrane, key
steps in the metastatic process, rely on the activity of proteolytic enzymes. The levels
and activities of cathepsins B and L are considerably increased in cancer cells, and
have been related to the invasive potential of these cells. Moreover, the levels of their
endogenous inhibitors, the cystatins, are decreased, which results in an imbalance
that contributes to the development of the metastatic phenotype. Thus, the potential
use of cystatins as therapeutic agents in novel anti cancer strategies has been
suggested. The sugarcane genome project (SUCEST FAPESP) allowed the
identification of about 20 putative cystatins in this plant. In the present study, we
describe the heterologous expression, purification and characterization of three
cystatins from sugarcane, dubbed as CaneCPI-2, CaneCPI-3 and CaneCPI-4, which
showed different inhibitory activities against human cathepsins B and L. While the
three recombinant cystatins inhibited cathepsin L activity with Ki values of 0.17, 0.6
and 0.021 nM, respectively, only CaneCPI-4 was capable of efficiently inhibiting
cathepsin B activity (Ki = 0.83 nM). Considering the involvement of these cathepsins
in tumor cell invasion, the effect of the cystatins on the invasive ability of human
breast cancer MDA-MB-231 cells was assessed after the addition of the recombinant
cystatins to the cells, and in cell clones expressing the sugarcane cystatins. Overall,
the Ki values correlated with the ability of the cystatins to inhibiting the cysteine
cathepsin activity of the tumor cells as well as the cell invasion through a Matrigel
matrix. A slight reduction in the invasive ability was observed in the MDA-MB-231
cells expressing CaneCPI-4. In addition, a substantial reduction of ~ 60% in the cell
invasion was obtained in the presence of 2 µM recombinant CaneCPI-2 or CaneCPI-
3, or 0.2 µM of CaneCPI-4. Finally, the cystatins showed negligible effects on the
adhesion and proliferation of the cells. Our results open the possibility of considering
these as well as other phytocystatins as therapeutics agents in anti-cancer
strategies.