Exosomes have been associated with chemoresistance in various cancers, but such a role in ovarian cancer is not yet clear. Here, using in vitro cell-based and in vivo mouse model experiments, we show that downregulation of O-GlcNAcylation, a key post-translational protein modification, promotes exosome secretion. This increases exosome-mediated efflux of cisplatin from cancer cells resulting in chemoresistance. Mechanistically, our data indicate that downregulation of O-GlcNAclation transferase (OGT) reduces O-GlcNAclation of SNAP-23. Notably, O-GlcNAcylation of SNAP-23 is vital for regulating exosome release in ovarian cancer cells. Reduced O-GlcNAclation of SNAP-23 subsequently promotes the formation of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex consisting of SNAP-23, VAMP8, and Stx4 proteins. This enhances exosome release causing chemoresistance by increasing the efflux of intracellular cisplatin.
Practice of tumor-targeted suicide gene therapy is hampered by unsafe and low efficient delivery of plasmid DNA (pDNA). Using HIV-Tat-derived peptide (Tat) to non-covalently form Tat/pDNA complexes advances the delivery performance. However, this innovative approach is still limited by intracellular delivery efficiency and cell-cycle status. In this study, Tat/pDNA complexes were further condensed into smaller, nontoxic nanoparticles by Ca2+ addition. Formulated Tat/pDNA-Ca2+ nanoparticles mainly use macropinocytosis for intercellular delivery, and their macropinocytic uptake was persisted in mitosis (M-) phase and highly activated in DNA synthesis (S-) phase of cell-cycle. Over-expression or phosphorylation of a mitochondrial chaperone, 75-kDa glucose-regulated protein (GRP75), promoted monopolar spindle kinase 1 (MPS1)-controlled centrosome duplication and cell-cycle progress, but also driven cell-cycle-dependent macropinocytosis of Tat/pDNA-Ca2+ nanoparticles. Further in vivo molecular imaging based on DF (Fluc-eGFP)-TF (RFP-Rluc-HSV-ttk) system showed that Tat/pDNA-Ca2+ nanoparticles exhibited highly suicide gene therapy efficiency in mouse model xenografted with human ovarian cancer. Furthermore, arresting cell-cycle at S-phase markedly enhanced delivery performance of Tat/pDNA-Ca2+ nanoparticles, whereas targeting GRP75 reduced their macropinocytic delivery. More importantly, in vivo targeting GRP75 combined with cell-cycle or macropinocytosis inhibitors exhibited distinct suicide gene therapy efficiency. In summary, our data highlight that mitochondrial chaperone GRP75 moonlights as a biphasic driver underlying cell-cycle-dependent macropinocytosis of Tat/pDNA-Ca2+ nanoparticles in ovarian cancer.
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