Ritonavir (RIT) is a widely used antiviral drug that acts as an HIV protease inhibitor with emerging potential in anticancer therapies. RIT causes inhibition of P-glycoprotein, which plays an important role in multidrug resistance (MDR) in cancer cells when overexpressed. Moreover, RIT causes mitochondrial dysfunction, leading to decreased ATP production and reduction of caveolin I expression, which can affect cell migration and tumor progression. To increase its direct antitumor activity, decrease severe side effects induced by the use of free RIT and improve its pharmacokinetics, ritonavir 5-methyl-4-oxohexanoate (RTV) was synthesized and conjugated to a tumor-targeted polymer carrier based on a N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer. Here we demonstrated that polymer-bound RTV enhanced the internalization of polymer-RTV conjugates, differing in RTV content from 4 to 15 wt%, in HeLa cancer cells compared with polymer without RTV. The most efficient influx and internalization properties were determined for the polymer conjugate bearing 11 wt% of RTV. This conjugate was internalized by cells using both caveolin- and clathrin-dependent endocytic pathways in contrast to the RTV-free polymer, which was preferentially internalized only by clathrin-mediated endocytosis. Moreover, we found the co-localization of the RTV-conjugate with mitochondria and a significant decrease of ATP production in treated cells. Thus, the impact on mitochondrial mechanism can influence the function of ATP-dependent P-glycoprotein and also the cell viability of MDR cancer cells. Overall, this study demonstrated that the polymer-RTV conjugate is a promising polymer-based nanotherapeutic, suitable for antitumor combination therapy with other anticancer drugs and a potential mitochondrial drug delivery system.
- MeSH
- adenosintrifosfát biosyntéza MeSH
- antitumorózní látky aplikace a dávkování chemie MeSH
- chemorezistence účinky léků MeSH
- endocytóza účinky léků MeSH
- HeLa buňky MeSH
- kaveolin 1 biosyntéza genetika MeSH
- klathrin farmakologie MeSH
- koncentrace vodíkových iontů MeSH
- lidé MeSH
- methakryláty chemie MeSH
- nanostruktury chemie MeSH
- P-glykoprotein účinky léků metabolismus MeSH
- polymery MeSH
- ritonavir aplikace a dávkování analogy a deriváty chemie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Mouse polyomavirus (PyV) virions enter cells by internalization into smooth monopinocytic vesicles, which fuse under the cell membrane with larger endosomes. Caveolin-1 was detected on monopinocytic vesicles carrying PyV particles in mouse fibroblasts and epithelial cells (33). Here, we show that PyV can be efficiently internalized by Jurkat cells, which do not express caveolin-1 and lack caveolae, and that overexpression of a caveolin-1 dominant-negative mutant in mouse epithelial cells does not prevent their productive infection. Strong colocalization of VP1 with early endosome antigen 1 (EEA1) and of EEA1 with caveolin-1 in mouse fibroblasts and epithelial cells suggests that the monopinocytic vesicles carrying the virus (and vesicles containing caveolin-1) fuse with EEA1-positive early endosomes. In contrast to SV40, PyV infection is dependent on the acidic pH of endosomes. Bafilomycin A1 abolished PyV infection, and an increase in endosomal pH by NH4Cl markedly reduced its efficiency when drugs were applied during virion transport towards the cell nucleus. The block of acidification resulted in the retention of a fraction of virions in early endosomes. To monitor further trafficking of PyV, we used fluorescent resonance energy transfer (FRET) to determine mutual localization of PyV VP1 with transferrin and Rab11 GTPase at a 2- to 10-nm resolution. Positive FRET between PyV VP1 and transferrin cargo and between PyV VP1 and Rab11 suggests that during later times postinfection (1.5 to 3 h), the virus meets up with transferrin in the Rab11-positive recycling endosome. These results point to a convergence of the virus and the cargo internalized by different pathways in common transitional compartments.
- MeSH
- buněčné linie MeSH
- časové faktory MeSH
- elektronová mikroskopie MeSH
- endozomy metabolismus virologie MeSH
- financování organizované MeSH
- fúze membrán MeSH
- kaveolin 1 genetika metabolismus MeSH
- koncentrace vodíkových iontů MeSH
- myši MeSH
- Polyomavirus fyziologie MeSH
- rab proteiny vázající GTP metabolismus MeSH
- rezonanční přenos fluorescenční energie MeSH
- transferin metabolismus MeSH
- transport proteinů MeSH
- vazba proteinů MeSH
- virion metabolismus MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
