Mutant cystathionine gamma-lyase was targeted to phosphatidylserine exposed on tumor vasculature through fusion with annexin A1 or annexin A5. Cystathionine gamma-lyase E58N, R118L, and E338N mutations impart non-native methionine gamma-lyase activity, resulting in tumor-localized generation of highly toxic methylselenol upon systemic administration of non-toxic selenomethionine. The described therapeutic system circumvents systemic toxicity issues using a novel drug delivery/generation approach and avoids the administration of non-native proteins and/or DNA required with other enzyme prodrug systems. The enzyme fusion exhibits strong and stable in vitro binding with dissociation constants in the nanomolar range for both human and mouse breast cancer cells and in a cell model of tumor vascular endothelium. Daily administration of the therapy suppressed growth of highly aggressive triple negative murine 4T1 mammary tumors in immune competent BALB/cJ mice and MDA-MB-231 tumors in SCID mice. Treatment did not result in occurrence of negative side effects or the elicitation of neutralizing antibodies. Based on the vasculature-targeted nature of the therapy, combinations with rapamycin and cyclophosphamide were evaluated. Rapamycin, an MTOR inhibitor, reduces the pro-survival signaling of cells in a hypoxic environment potentially exacerbated by a vasculature-targeted therapy. Immunohistochemistry revealed, unsurprisingly, a significant hypoxic response (increase in hypoxia inducible factor 1 alpha subunit, HIF1A) in the enzyme prodrug treated tumors and a dramatic reduction of HIF1A upon rapamycin treatment. Cyclophosphamide, an immunomodulator at low doses, was combined with the enzyme prodrug therapy and rapamycin; this combination synergistically reduced tumor volumes, inhibited metastatic progression, and enhanced survival. <br />
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