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Dextrin-Phospholipase A2: Synthesis and Evaluation as a Bioresponsive Anticancer Conjugate Elaine L. Ferguson*,† and Ruth Duncan Centre for Polymer Therapeutics, Welsh School of Pharmacy, Cardiff University, King Edward VII Avenue, Cardiff, CF10 3XF, United Kingdom Received November 12, 2008; Revised Manuscript Received March 18, 2009
There is still an urgent need for improved treatments for metastatic cancer. Although the phospholipase A2 (PLA2) crotoxin, an antitumor protein that appears to act by interaction with epidermal growth factor receptors (EGFR), has recently shown activity in breast cancer in phase I clinical trials, it also displayed nonspecific neurotoxicity. Therefore, the aim of this study was to apply a novel concept called polymer-masked-unmasked-protein therapy (PUMPT) to give a bioresponsive dextrin-PLA2 conjugate that would reduce PLA2 systemic toxicity but retain antitumor activity following R-amylase triggered degradation of dextrin in the tumor interstitium. Dextrin (Mw ∼ 60000 g/mol; ∼22 mol % succinoylation) and PLA2 (from honey bee venom) were chosen as models for these initial studies, and the conjugates synthesized contained 6.1 wt % PLA2, with MCF-7 (IC50 ) 62.9 µg/mL) > B16F10 (IC50 ) 133.3 µg/mL)). Both free and dextrin-conjugated PLA2 were most toxic in HT29 cells and they have the highest level of EGFR expression. Hemolytic Activity. Free PLA2 was found to be very hemolytic at all concentrations >100 µg/mL PLA2. However, no hemolysis was seen for the dextrin-PLA2 conjugate up to
Figure 4. Measurement of PLA2 and dextrin-PLA2 conjugate enzyme activity. (a,b) PLA2 activity using an egg yolk emulsion assay. (a) PLA2 (0.5 mg/mL) activity with increasing substrate concentrations. (b) PLA2 (0.5 mg/mL) and dextrin-PLA2 conjugate (0.5 mg/mL PLA2 equiv; (R-amylase). Data show activity as mean ( SD, n ) 3; (*) indicates significance p < 0.05 compared to free PLA2 control.
concentrations of 500 µg/mL PLA2 equiv (Figure 6a). Control experiments with dextrin showed B16F10, and it was significantly more toxic than PLA2 in MCF-7 cells. This may be surprising as native PLA2 would be expected to hydrolyze phospholipids immediately (turnover rate (Kcat) ) 130 s-1),26 whereas the dextrin-PLA2 conjugate is likely to release active
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PLA2 more slowly over the 72 h incubation. Although the delayed release of PLA2 by “unmasking” of the conjugate might be expected to reduce PLA2’s cytotoxicity compared to the native enzyme, this is probably more than counter-balanced by the enhanced stability of PLA2 following dextrin conjugation. Enhanced stability against proteolytic attack and autolysis has previously been demonstrated using dextrin-trypsin and dextrin-rhEGF conjugates.24,25 None of the cells produce R-amylase constitutively and FCS added to the cell culture media contained 7.2 IU/L R-amylase according to the Phadebas assay. This is considerably lower than the R-amylase concentration found in normal human serum (40-125 IU/L), so unmasking would be expected to occur more rapidly in vivo. Given that MCF-7 cell culture medium was supplemented with a 50% lower concentration of FCS, and therefore, R-amylase, than HT29 and B16F10 cells (0.36 IU/L compared to 0.72 IU/L, respectively), it was interesting to see that the dextrin-PLA2 conjugate was more cytotoxic than the free enzyme only in these cells. It is, therefore, also impossible that the enhanced cytotoxicity of dextrin-PLA2 in MCF-7 cells compared to B16F10 cells be attributable to the difference in serum R-amylase. Moreover, while B16F10 and HT29 cells were supplemented with equal quantities of FCS, an 8-fold difference in cytotoxicity was observed. At present, the main source of polymer degradation in these in vitro experiments remains unclear; lysosomal glycosidases are also able to degrade dextrin and it has not been determined whether these are excreted from the cells. PLA2 enzymes demonstrate preference toward certain phospholipids (phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin) that are found at higher levels in cancer cells.27,28 An explanation for differential cytotoxicity seen in the three cell lines here could be differences in their membrane composition. Alternatively, PLA2 does act via the EGFR3, and both PLA2 and dextrin-PLA2 conjugate cytotoxicity did show partial correlation with the EGFR expression here, that is, HT29 > MCF-7 cells. Donato et al.3 also noticed that crotoxin caused the most effective growth suppression in cells expressing high intrinsic levels of EGFR. Here, DiFi rectal carcinoma cells, having ∼100-fold greater EGFR expression than HT29 cells, demonstrated ∼20-fold greater sensitivity to crotoxin than HT29 cells (IC50 ) 2.2 µg/mL (DiFi), 42 µg/mL (HT29)). Further experiments to explore the precise mechanism of action of dextrin-PLA2 conjugates are planned to understand better the involvement of the EGFR and help realize the full potential of dextrin-PLA2 as an anticancer therapy. Hemolytic Activity. Having confirmed that dextrin-PLA2 conjugates could retain biological activity in a physiological setting it was important to consider their possible toxicological profile prior to future in vivo and clinical testing. Hemocompatibility of dextrin-PLA2 conjugates would be essential for safe systemic administration by IV injection that would be needed to benefit from the EPR effect. While a 72 h incubation period was chosen for measuring cytotoxicity, 1 h was used for the hemolysis studies since it was expected that a dextrin-PLA2 conjugate would accumulate within tumors by the EPR effect so the exposure to RBCs would be limited. Preliminary experiments conducted here showed that concentration-dependent hemolysis displayed by native PLA2 was abolished by dextrin conjugation (Figure 6), indicative of an improved safety profile for IV administration. As a PLA2 conformational change appears to be required for interfacial activation,29,30 dextrin conjugation would interfere with this mechanism as well as providing a more general steric hindrance
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to access. Even after R-amylase-unmasking of the conjugate, hemolytic activity was not restored. Because R-amylase will cleave dextrin randomly, it is probable that there will be residual saccharide/oligosaccharide units left attached to the enzyme’s surface. These could prevent the conformational changes required for hemolytic activity. The addition of Ca2+ (8 mM), a cofactor for PLA2 activity, enhanced hemolysis by native PLA2 toward RBCs, however, it did not influence the toxicity of dextrin-PLA2 conjugates (Figure 6b). It is proposed that dextrin, and indeed maltose, following R-amylase degradation of dextrin, could block calcium coordination with the calcium-binding loop of PLA2 by steric hindrance. Final Considerations. Bee venom PLA2 was chosen as a model for these proof of concept studies since its anticancer potential has been demonstrated22 and its monomeric structure facilitates characterization of the conjugate. While the greatest evidence for PLA2’s anticancer activity relates to crotoxin, this enzyme is composed of two subunits that must dissociate prior to activation,2 requiring more complex characterization of conjugates and specific release kinetics. Synthesis of a dextrin-crotoxin conjugate for comparison to the bee venom PLA2 conjugate described here would yield interesting results that could be applicable to the published clinical studies with crotoxin, while polymer conjugation may be capable of reducing the nonspecific neurotoxicity seen in phase I clinical trials.2
Conclusions This study reports the first bioresponsive, PLA2-containing polymer therapeutic designed as an anticancer nanomedicine. The dextrin-PLA2 conjugate demonstrated significant toxicity toward human breast and colon and murine melanoma cancer cells and seemed to correlate, in part, with EGFR status. However, while the enzyme’s hydrolytic activity was enhanced by R-amylase exposure the dextrin-PLA2 conjugate was less hemolytic than native enzyme, even after exposure to R-amylase. Dextrin-PLA2 conjugates have potential for further in vivo evaluation as novel anticancer agents. Acknowledgment. E.L.F. would like to thank the Welsh School of Pharmacy and the Centre for Polymer Therapeutics for funding her Ph.D. studies, and we would like to acknowledge support from EPSRC Platform Grant No. EP/C 013220/1.
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