Anti-tumor therapy with macroencapsulated endostatin producer cells

<p>Abstract</p> <p>Background</p> <p>Theracyte is a polytetrafluoroethylene membrane macroencapsulation system designed to induce neovascularization at the tissue interface, protecting the cells from host's immune rejection, thereby circumventing the problem of limited half-life and variation in circulating levels. Endostatin is a potent inhibitor of angiogenesis and tumor growth. Continuous delivery of endostatin improves the efficacy and potency of the antitumoral therapy. The purpose of this study was to determine whether recombinant fibroblasts expressing endostatin encapsulated in Theracyte immunoisolation devices can be used for delivery of this therapeutic protein for treatment of mice bearing B16F10 melanoma and Ehrlich tumors.</p> <p>Results</p> <p>Mice were inoculated subcutaneously with melanoma (B16F10 cells) or Ehrlich tumor cells at the foot pads. Treatment began when tumor thickness had reached 0.5 mm, by subcutaneous implantation of 10⁷recombinant encapsulated or non-encapsulated endostatin producer cells. Similar melanoma growth inhibition was obtained for mice treated with encapsulated or non-encapsulated endostatin-expressing cells. The treatment of mice bearing melanoma tumor with encapsulated endostatin-expressing cells was decreased by 50.0%, whereas a decrease of 56.7% in tumor thickness was obtained for mice treated with non-encapsulated cells. Treatment of Ehrlich tumor-bearing mice with non-encapsulated endostatin-expressing cells reduced tumor thickness by 52.4%, whereas lower tumor growth inhibition was obtained for mice treated with encapsulated endostatin-expressing cells: 24.2%. Encapsulated endostatin-secreting fibroblasts failed to survive until the end of the treatment. However, endostatin release from the devices to the surrounding tissues was confirmed by immunostaining. Decrease in vascular structures, functional vessels and extension of the vascular area were observed in melanoma microenvironments.</p> <p>Conclusions</p> <p>This study indicates that immunoisolation devices containing endostatin-expressing cells are effective for the inhibition of the growth of melanoma and Ehrlich tumors.</p> <p>Macroencapsulation of engineered cells is therefore a reliable platform for the refinement of innovative therapeutic strategies against tumors.</p

VapC from the leptospiral VapBC toxin-antitoxin module displays ribonuclease activity on the initiator tRNA.

The prokaryotic ubiquitous Toxin-Antitoxin (TA) operons encode a stable toxin and an unstable antitoxin. The most accepted hypothesis of the physiological function of the TA system is the reversible cessation of cellular growth under stress conditions. The major TA family, VapBC is present in the spirochaete Leptospira interrogans. VapBC modules are classified based on the presence of a predicted ribonucleasic PIN domain in the VapC toxin. The expression of the leptospiral VapC in E. coli promotes a strong bacterial growth arrestment, making it difficult to express the recombinant protein. Nevertheless, we showed that long term induction of expression in E. coli enabled the recovery of VapC in inclusion bodies. The recombinant protein was successfully refolded by high hydrostatic pressure, providing a new method to obtain the toxin in a soluble and active form. The structural integrity of the recombinant VapB and VapC proteins was assessed by circular dichroism spectroscopy. Physical interaction between the VapC toxin and the VapB antitoxin was demonstrated in vivo and in vitro by pull down and ligand affinity blotting assays, respectively, thereby indicating the ultimate mechanism by which the activity of the toxin is regulated in bacteria. The predicted model of the leptospiral VapC structure closely matches the Shigella's VapC X-ray structure. In agreement, the ribonuclease activity of the leptospiral VapC was similar to the activity described for Shigella's VapC, as demonstrated by the cleavage of tRNAfMet and by the absence of unspecific activity towards E. coli rRNA. This finding suggests that the cleavage of the initiator transfer RNA may represent a common mechanism to a larger group of bacteria and potentially configures a mechanism of post-transcriptional regulation leading to the inhibition of global translation

3D model of leptospiral VapC closely matches the experimental X-ray structure of <i>Shigella</i>'s VapC.

<p>Alignment of the sequences of VapCs from <i>S. flexneri</i> and <i>L. interrogans</i> is shown. On the left panel, the structure model (ribbon) of VapC from <i>L. interrogans</i> (green) was superimposed to the VapC template from <i>S. flexneri</i> (white) (PdB: 3TND-C). The green regions in the target appear almost identical to the template, while the red region does not correspond. The amino acids composing the red region are written in the same color in the sequence alignment. On the right panel, superimposition shows the perfect matching of the conserved threonine and the four acidic residues responsible for coordinating metal ions in the catalytic site, and the cysteine involved in dimerization, positioned in the neighborhood of the catalytic site. These six residues are numbered in the structure according to leptospiral VapC sequence and colored as highlighted in the alignment.</p

VapB and VapC interact <i>in vivo</i> and <i>in vitro</i>.

<p>(<b>A</b>) Pull-down assay. The soluble fraction of <i>E. coli</i> pAE<i>vapBC</i> extract was applied to a Ni⁺²-Sepharose column. Samples were analyzed by SDS-PAGE. Lane 1: initial sample; lane 2: washing; lanes 3–4: elution with 250 mM imidazole. It is important to observe that no VapC was released during the washing step, being co-purified with VapB-His, denoting the <i>in vivo</i> interaction. The arrows indicate the VapB and VapC bands. M - Molecular Weight Marker (kDa). Pull-down assay was perfomed more than 5 times. (<b>B</b>) Ligand affinity blotting. To analyze specific binding between VapB and VapC, the VapC and LipL32 proteins (negative control) were subjected to 15% SDS-PAGE (left panel) and transferred to nitrocellulose membrane (right panel). After blocking, the membrane was incubated with a VapB solution (3 µg ml⁻¹). Following extensive washing, the membrane was incubated with anti-VapB antibodies. M - Prestained Molecular Weight Marker (kDa). VapB in solution bound to both monomeric (15.1 kDa) and dimeric (30.2 kDa) forms of VapC immobilized in the membrane, denoting <i>in vitro</i> interaction. VapB did not bound the negative control, protein LipL32.(<b>C</b>) Western blot control showing that anti-VapB antibodies recognizes specifically VapB, and not VapC.</p

Circular dichroism of VapB and VapC confirmed predicted secondary structure.

<p>(<b>A</b>) The VapB and VapC CD spectra were recorded in the wavelength range of 195–255 nm as average of five scans at 20°C. Measured ellipticities, <i>θ</i> (mdegree), were converted to molar mean residue ellipticities, [<i>θ</i>] (degree.cm².dmol⁻¹). The assays were reproduced with at least 2 samples of each protein. (<b>B</b>) Prediction of secondary structure by the PSIPRED algorithm using the primary sequence of the proteins. The experimental data confirmed the secondary structure predicted by computational analysis.</p

Analysis of purified recombinant proteins by SDS-PAGE revealed VapC dimerization.

<p>(<b>A</b>) VapB purified from the soluble fraction of <i>E. coli</i> extracts (<b>B</b>) VapC expressed as inclusion bodies, before [NP] or after [P] pressurization in buffer containing 0.5 M L-arginine; (<b>C</b>) VapB and VapC purified from the soluble fraction of <i>E. coli</i> co-expressing both proteins. Samples were prepared with or without β-mercaptoethanol ([Reduced] or [NReduced], respectively). M - Molecular Marker (kDa). The arrows indicate purified VapB and monomeric (15.1 kDa) or dimeric (30.2 kDa) forms of VapC. Remarkably, VapC dimers were observed in non-reduced samples of the pressurized protein (B) and in the co-purification with VapB (C). This analysis was made with 3 VapC preparations.</p

Evaluation of VapC models.

<p>VapC structural models were evaluated by QMEAN4 and Z score in order to compare and rank alternative models of the same target. QMAN4 is a reliability score consisting of a combination of four structural descriptors which ranges between 0 and 1 with higher values for better models. Z-score provides an estimate of the “degree of nativeness” of the structural features observed in a model; ‘good-quality’ models reach a mean <i>Z</i>-score of −0.65, ‘medium-quality’ -1.75, and the ‘low-quality’ −3.85. The analysis of the model quality scores showed that <i>Shigella</i> VapC is the only template to render a “good” model for VapC from <i>Leptospira</i> (*), as “good” as the one created for <i>Salmonella</i> VapC (**), which shares 89% identity with the temp<b>l</b>ate. In opposition, <i>Mycobacterium</i> VapC20 model (^#) displayed low scores.</p