|M.Sc Student||Turgeman Yamit|
|Subject||Overcoming Multidrug Resistance via Imidazoacridinone-|
Dependent Lysosomal Photodestruction
|Department||Department of Biology||Supervisor||Professor Yehuda Assaraf|
|Full Thesis text|
A major primary hindrance to curative cancer chemotherapy is the development of multidrug resistance (MDR) in tumor cells, which display cross-resistant to a broad range of structurally and functionally unrelated agents, making it difficult to treat these tumors. Thus, introduction of novel strategies to overcome MDR is of supreme clinical significance. A recent microscopy observation in our lab revealed that members of the imidazoacridinone (IA) family, bearing an intrinsic fluorescence, dramatically accumulate within acidic lysosomal compartments, and that upon illumination, a rapid lysosomal photodestruction occurs, via generation of ROS, thereby resulting in lysosome rupture and rapid cell death. Hence, the aim of the current research was to establish the phenomenon of IAs-based lysosomal photodestruction. To this end, we here describe a new strategy to overcome MDR, based upon lysosomal sequestration of cytotoxic agents of the IAs family and consequent lysosomal photodestruction following illumination with visible light. We specifically find that IAs that are hydrophobic fluorochrome weak bases, highly concentrate within lysosomes. Lysosomal alkalinization with the H-ATPase inhibitor bafilomycin A1 or the lysosomotropic agent ammonium chloride, abrogated intralysosomal accumulation of IAs and supported the conclusion that the pH gradient between the lysosome and the cytoplasm is the driving force for lysosomal sequestration of IAs. We found that other established anticancer drugs including hydrophobic weak base drugs bearing intrinsic fluorescence, such as doxorubicin and sunitinib, also accumulate in lysosomes and upon illumination, induce lysosomal photodestruction. Quantification of lysosome number per A549/K1.5 MDR tumor cell with ABCG2 overexpression using established viable lysotracker red and IAs staining revealed a 6-fold increase, relative to A549 parental cells. We propose that the basis for 2.5-fold intrinsic resistance of MDR cells to the IA C-1330, compared to parental cells, is their ability to sequester this drug in lysosomes and hence prevent it from reaching its targets, topoisomerase II and DNA. Chloroquine, a lysosomotropic alkalinization anti-malarial agent, induced a marked increase in both the number and volume of lysosomes in A549 parental cells, hence resulting in 23-49-fold enhancement in C-1330 cytotoxicity upon illumination. Morphological characteristics of cell death were already apparent three hours after illumination. In summary, these novel findings identify intralysosomal sequestration of photosensitizer hydrophobic weak base chemotherapeutics as an organelle "Achilles Hill" of MDR cells, that can be readily exploited to efficiently eliminate malignant cells via lysosomal photodestruction.