Vicrostatin – An Anti-Invasive Multi-Integrin Targeting Chimeric Disintegrin with Tumor Anti-Angiogenic and Pro-Apoptotic Activities

Similar to other integrin-targeting strategies, disintegrins have previously shown good efficacy in animal cancer models with favorable pharmacological attributes and translational potential. Nonetheless, these polypeptides are notoriously difficult to produce recombinantly due to their particular structure requiring the correct pairing of multiple disulfide bonds for biological activity. Here, we show that a sequence-engineered disintegrin (called vicrostatin or VCN) can be reliably produced in large scale amounts directly in the oxidative cytoplasm of Origami B E. coli. Through multiple integrin ligation (i.e., αvβ3, αvβ5, and α5β1), VCN targets both endothelial and cancer cells significantly inhibiting their motility through a reconstituted basement membrane. Interestingly, in a manner distinct from other integrin ligands but reminiscent of some ECM-derived endogenous anti-angiogenic fragments previously described in the literature, VCN profoundly disrupts the actin cytoskeleton of endothelial cells (EC) inducing a rapid disassembly of stress fibers and actin reorganization, ultimately interfering with EC's ability to invade and form tubes (tubulogenesis). Moreover, here we show for the first time that the addition of a disintegrin to tubulogenic EC sandwiched in vitro between two Matrigel layers negatively impacts their survival despite the presence of abundant haptotactic cues. A liposomal formulation of VCN (LVCN) was further evaluated in vivo in two animal cancer models with different growth characteristics. Our data demonstrate that LVCN is well tolerated while exerting a significant delay in tumor growth and an increase in the survival of treated animals. These results can be partially explained by potent tumor anti-angiogenic and pro-apoptotic effects induced by LVCN

The expression, purification and initial characterization of VCN as an active disintegrin.

<p><b>Panel A –</b> The production of Trx-VCN was assessed in different <i>E. coli</i> strains that were transformed, grown, induced, and processed under identical conditions. The same amount (5 µl) of cell lysates from each induced strain was loaded on a precast gel and Coomassie stained. Unlike the BL21 (DE3) strain, the lysates from both AD494 (DE3) and Origami B (DE3) strains generate a unique and consistent Trx-VCN band (shown by the arrow). By employing a modified media recipe, the Origami B (DE3) Trx-VCN transformants achieve higher cell densities at the end of the induction time, generating up to 200 mg of soluble VCN per L of bacterial culture after purification. <b>Panel B –</b> Coomassie stained gel showing the migration of Trx-VCN before and after TEV proteolysis (lanes 2 and 3, respectively) versus C18 reverse phase-HPLC purified VCN (lane 4). <b>Panel C </b><b>–</b> VCN and native CN exhibit an almost identical dose-dependent inhibitory effect against ADP-induced platelet aggregation when incubated with human platelet-rich plasma (with a calculated IC₅₀ of ∼60 nM). In contrast, the rCN construct, which is also expressed as a soluble polypeptide in Origami B (DE3), shows no inhibitory activity. <b>Panel D </b><b>–</b> The agonistic activity of VCN (FAK activation) was assessed in serum-starved non-migratory MDA-MB-435 cells kept in suspension and exposed to increasing concentrations of disintegrins for 30 min. Similar to dimeric CN, VCN is also shown to engage integrins agonistically (outside-in signaling).</p

LVCN treatment shows enhanced tumor apoptosis in the breast MDA-MB-231 xenograft model.

<p>For this experiment, MDA-MB-231 xenografts were allowed to grow to a significantly larger volume (4 weeks after inoculation) before treatments were initiated. The animals received either liposomal VCN (at the dose-equivalent of 100 µg of VCN per injection) or Avastin (400 µg per injection) administered intravenously every other day and compared to a control group that received saline only. All animals were sacrificed after receiving 6 consecutive doses of each treatment. (<b>i</b>) To assess the impact of VCN on cell death, tumor cryostat sections from each group were stained with FITC-TUNEL, and counterstained with Hoechst 33342. Representative confocal images from multiple experiments taken at ×250 magnification are shown above (scale bar, 100 µm; panels A₁-C₁ - TUNEL-Hoechst, panels A₂-C₂ - TUNEL only). (<b>ii</b>) The amount of cell death was quantitated as ‘number of TUNEL⁺ nuclei/total number of nuclei x 100’ by counting all nuclei in ‘hotspot’ areas from multiple fields using a computer-assisted approach (the ‘SimplePCI’ imaging software). The liposomal VCN group shows a significantly increased amount of cell death compared to either Avastin or control. (<b>iii</b>) The impact of VCN treatment on tumor proliferation was assessed by Ki-67 immunoperoxidase staining. Representative Ki-67 images are shown above (scale bar, 200 µm). (<b>iv</b>) Cell proliferation was quantitated using the same approach as for TUNEL staining. The differences in cell proliferation between the treatment groups were much smaller than those observed for cell death. The data was analyzed with ANOVA followed by <i>post-hoc</i> tests (* signifies a P<0.01).</p

Disintegrin-integrin biding kinetics by fluorescence polarization.

<p>The binding kinetics were calculated from the fluorescence anisotropy data generated by the steady state binding of FITC-labeled disintegrins to either purified (αvβ3 and αvβ5) or recombinant (α5β1) functional human integrins. The dissociation constants for interactions of either CN or VCN with soluble integrins were determined by Scatchard analysis using a non-linear curve fit.</p

VCN inhibits HUVEC tube formation (tubulogenesis).

<p>(<b>i</b>) HUVEC were plated on ‘Endothelial Cell Tube Formation’ plates (BD Biosciences) in the presence of various concentrations of either CN or VCN (1–1000 nM) and allowed to form tubes after incubation for 12–16 hr at 37°C in the presence of 5%CO₂. Suramin, a known tube formation inhibitor, was used as a positive control at two different concentrations (50 and 100 µM). At the end of the incubation period, cells were stained with Calcein AM and imaged by confocal microscopy (magnification, ×25). Representative figures from three independent experiments are shown above (scale bar, 200 µm). (<b>ii</b>) The degree of tubulogenesis was assessed by capturing multiple photomicrographs for all conditions on which the total length of the tubes was measured and computed in multiple fields using the Zeiss LSM Image Browser (Carl Zeiss MicroImaging GmbH) and then averaged to form each data point. The data presented above was assembled from three independent experiments.</p

VCN does not affect the viability of cells plated on top of Matrigel.

<p>HUVEC, MDA-MB-231 or MDA-MB-435 cells were seeded in serum-free media in multiwell chamber slides on complete Matrigel and allowed to adhere for 1 hr. Once adherent, the cells were incubated for up to 48 hr with either CN or VCN up to a maximum concentration of 1 µM. Untreated cells or cells exposed to the apoptosis inducer Staurosporine (STSP) at a concentration of 1 µM were used as controls. The cells were fixed, TUNEL-stained and counterstained with Hoechst 33342. The amount of cell death was plotted for each condition by digitally counting the apoptosis events in random fields from images taken from multiple experiments for each condition.</p

VCN induces massive actin cytoskeleton reorganization in HUVEC seeded on Matrigel.

<p>HUVEC were seeded in serum-free media in multiwell chamber slides on complete Matrigel, allowed to adhere, and then treated for 3 hr with various concentrations of cRGDfV peptide (1 and 10 µM) or FITC-VCN (10 and 100 nM) or the monoclonal antibodies 7E3 (100 nM) or LM609 (100 nM). When cells were incubated with integrin-binding antibodies (7E3 or LM609), a FITC-conjugated secondary antibody was used to track these treatments. The actin modifier Cytochalasin D was used as a positive control (50 nM). At the end of the incubation period, the cells from all conditions were fixed in 4% formaldehyde, permeabilized in 0.1% Triton X-100, stained with Rhodamine-Phalloidin and Hoechst 33342, and imaged by confocal microscopy. The cells exposed to FITC-labeled treatments (VCN or integrin-binding antibodies) are triple stained. The images shown above are Rhodamine-Hoechst only (panels A–B and E–F), FITC-Hoechst (panels C₂-D₂ and G₂-H₂) or overlayed three fluorophores (panels C₁-D₁ and G₁-H₁). Representative confocal images from multiple experiments taken at the same magnification (×630) are shown above (scale bar, 20 µm).</p

Binding analysis of FITC-labeled disintegrins by flow cytometry.

<p><b>Panel A –</b> Both CN and VCN are detected at cell surface when probed with CN polyclonal antiserum. MDA-MB-435 cells were incubated with either VCN (red) or CN (green) followed by CN rabbit polyclonal antiserum and an anti-rabbit FITC-labeled secondary antibody. The controls included cells incubated with either anti-rabbit FITC-labeled secondary only (grey) or CN antiserum followed by the FITC-labeled secondary (blue). <b>Panels B, C </b><b>–</b> FITC-CN (panel B) or FITC-VCN (panel C) fail to bind to cells prewashed in EDTA media, but once bound in regular media the subsequent addition of EDTA does not displace them from integrins. MDA-MB-435 cells were either incubated with FITC-labeled disintegrins only (grey) or with labeled disintegrins and then washed and resuspended in 3 mM EDTA media (green) or preincubated in 3 mM EDTA and then washed and exposed to labeled disintegrins (red) or just probed with an FITC-labeled irrelevant antibody control (blue). <b>Panels D, E and F –</b> Labeled disintegrins bind in a similar manner to cells with different integrin profiles. MDA-MB-435, MDA-MB-231 or HUVEC were either incubated with FITC-CN (green) or FITC-VCN (red) or probed with an FITC-labeled irrelevant antibody control (blue). <b>Panels G, H, I and J </b><b>–</b> FITC-labeled disintegrins fail to bind to cells pretreated with either unlabeled disintegrins or an antibody competitor. MDA-MB-435 or MDA-MB-231 cells (panels G and H) were either incubated with FITC-CN only (grey) or an FITC-labeled irrelevant antibody control only (blue) or preincubated with unlabeled VCN (green) or 7E3 (red) and then probed with FITC-CN. Similarly, MDA-MB-435 or MDA-MB-231 cells (panels I and J) were either incubated with FITC-VCN only (grey) or an FITC-labeled irrelevant antibody control only (blue) or preincubated with unlabeled CN (green) or 7E3 (red) and then probed with FITC-VCN. The data are representative of four independent experiments.</p

Comparison between contortrostatin (CN), recombinant contortrostatin (rCN), vicrostatin (VCN), and echistatin sequences.

<p>Mass spectrometry and crystallographic data have confirmed that CN is a dimer with two identical chains oriented in an antiparallel fashion and held together by two interchain disulfide bonds. Unlike native CN, mass spectrometry showed that both rCN and VCN are monomers. In the above sequences, the Arg-Gly-Asp tripeptide motif is depicted in bold whereas the non-native amino acids in rCN and VCN are both italicized and underlined.</p