Download BRADIKYNIN AFFECTS THE INVASIVENESS OF MURINE

BRADIKYNIN AFFECTS THE INVASIVENESS OF MURINE MELANOMA AND MAMMARY
ADENOCARCINOMA CELLS. 1Fernandez, C.F, 1Paschoalin, T, 1Rodrigues, E.G. 1Experimental
Oncology Unit (UNONEX), Department of Micro, Immuno and Parasitology, Federal University of
São Paulo (UNIFESP), São Paulo/SP.
Aim: Generation and cleavage of factors by proteolytic enzymes, that may modulate tumor
development, is observed at all stages of tumor progression. The nonapeptide Bradykinin (BK) is
modulated by proteases present in the tumor microenvironment and may be involved in tumor
progression. BK participates as a primary mediator of tumor angiogenesis, and the involvement of
this molecule in the induction of innate and adaptive immune responses is now being
unveiled. The presence of kinin receptors (B1R and B2R) has been demonstrated in several tumor
cell lineages, and interaction with their ligands induces cell signaling cascades, that may interfere
with the cell growth in vitro or induce the production of factors involved in the metastatic potential
of these cells, as metalloproteases. Our goal was to verify the presence of B1R or B2R
receptors in murine melanoma B16F10 and breast adenocarcinoma 4T1 cells, and determine
the functionality of these receptors in these cells by verifying primarily the growth and metastatic
potential in vitro after incubation with the peptide.
Methods and Results: A real-time PCR with murine specific primers showed the expression of
B1R and B2R in B16F10-Nex2 and 4T1 murine cell lines. To determine the effect of BK on tumor
cell proliferation, 5x103 B16F10-Nex2 and 4T1 cells were seeded in 96-well plates and incubated
with several concentrations of BK for 24, 48 and 72h. The Cell Proliferation Kit I [Roche, based on
MTT (3 - (4,5-Dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide) method] and counting viable
cells in the presence of Trypan blue in a Neubauer chamber were used to evaluate B16F10-Nex2
and 4T1 cell viability, respectively. The results showed that BK has no effect on the proliferation of
both tested cell lines. To verify the effect of BK on the metastatic potential of these cells, in vitro
migration and invasion assays were performed. Cells (2x105 ) were seeded in transwell chambers
precoated with 50 g of extracellular matrix (Matrigel, BD Biosciences), 10μM or 100μM of BK was
added to the bottom well, and incubated for 24h. The number of invading cells was evaluated
using the Leica Qwin software. BK inhibited B16F10-Nex2 invasion and had a contrary effect on
4T1 cells, increasing the number of invading cells. The wound healing assay was performed to
test the effect of BK on cell migration. B16F10-Nex2 (2x105) cells were seeded on 12-well plates,
when 90% confluence was reached a wound was inflicted and cells were incubated in the
presence of 10μM or 100μM of BK for 24h. Images obtained from 0 to 24h showed that BK
reduced significantly the migration of these cells compared with untreated control.
Conclusion: B16F10-Nex2 cells showed the expression of B1R and B2R, and suggested that the
interaction of one or both receptors with BK reduced the invasive potential, but had no effect on in
vitro proliferation, of this tumor cell. 4T1 cells also showed the expression of both receptors, and
interaction with BK increased the invasiveness, but had no effect on proliferation, of this murine
cell line.
Sources of research support: FAPESP, CNPq