Targeting the Achilles' heel of cancer cells via integrin-mediated delivery of ROS-generating dihydrolipoamide dehydrogenase

Cancer cells frequently exhibit higher levels of reactive oxygen species (ROS) than normal cells and when ROS levels increase beyond a cellular tolerability threshold, cancer cell death is enhanced. The mitochondrial dihydrolipoamide dehydrogenase (DLDH) is an enzyme which produces ROS in association with its oxidoreductive activity and may be thus utilized as an exogenous anticancer agent. As cancer cells often overexpress integrins that recognize RGD-containing proteins, we have bioengineered the human DLDH with RGD motifs (DLDHRGD) for integrin-mediated drug delivery. The modified protein fully retained its enzyme activity and ROS-production capability. DLDHRGD uptake by cells was shown to depend on the presence of cell-associated integrin alpha v beta 3, as comparatively demonstrated with normal kidney cells (HEK293) transfected with either beta 1 (alpha v beta 1 positive) or beta 3 integrins (alpha v beta 3 positive). The interaction with beta 3 integrins was shown to be competitively inhibited by an RGD peptide. In mice melanoma cells (B16F10), which highly express an endogenous alpha v beta 3 integrin, fast cellular uptake of DLDHRGD which resulted in cell number reduction, apoptosis induction, and a parallel intracellular ROS production was shown. Similar results were obtained with additional human melanoma cell models (A375, WM3314, and WM3682). In contrast, HEK293 beta 3 cells remained intact following DLDHRGD uptake. The high pharmacological safety profile of DLDHRGD has been observed by several modes of administrations in BALB/C or C57B1/6 mouse strains. Treatments with DLDHRGD in a subcutaneous melanoma mice model resulted in significant tumor inhibition. Our study demonstrated, in vitro and in vivo, the development of a unique platform, which targets cancer cells via integrin-mediated drug delivery of an exogenous ROS-generating drug.