The Lysosomal sequestration of weak-base anticancer drugs is one putative mechanism for resistance to chemotherapy but it has never been directly proven. We addressed the question of whether the lysosomal sequestration of tyrosine kinase inhibitors (TKIs) itself contributes to the drug resistance in vitro. Our analysis indicates that lysosomal sequestration of an anticancer drug can significantly reduce the concentration at target sites, only when it simultaneously decreases its extracellular concentration due to equilibrium, since uncharged forms of weak-base drugs freely diffuse across cellular membranes. Even though the studied TKIs, including imatinib, nilotinib, and dasatinib, were extensively accumulated in the lysosomes of cancer cells, their sequestration was insufficient to substantially reduce the extracellular drug concentration. Lysosomal accumulation of TKIs also failed to affect the Bcr-Abl signaling. Cell pre-treatment with sunitinib significantly enhanced the lysosomal accumulation of the TKIs used; however, without apparent lysosomal biogenesis. Importantly, even increased lysosomal sequestration of TKIs neither decreased their extracellular concentrations nor affected the sensitivity of Bcr-Abl to TKIs. In conclusion, our results clearly show that the lysosomal sequestration of TKIs failed to change their concentrations at target sites, and thus, can hardly contribute to drug resistance in vitro.

Department of Anatomy Faculty of Medicine and Dentistry Palacky University Olomouc Hnevotinska 3 Olomouc 77515 Czech Republic

Honorary Professor Department of Chemistry University of Capetown Cape Town 7700 South Africa

Zobrazit více v PubMed

Larsen A.K., Escargueil A.E., Skladanowski A. Resistance mechanisms associated with altered intracellular distribution of anticancer agents. Pharmacol. Ther. 2000;85:217–229. doi: 10.1016/S0163-7258(99)00073-X. PubMed DOI

Wojtkowiak J.W., Verduzco D., Schramm K.J., Gillies R.J. Drug resistance and cellular adaptation to tumor acidic pH microenvironment. Mol. Pharm. 2011;8:2032–2038. doi: 10.1021/mp200292c. PubMed DOI PMC

Raghunand N., Martínez-Zaguilán R., Wright S.H., Gillies R.J. pH and drug resistance. II. Turnover of acidic vesicles and resistance to weakly basic chemotherapeutic drugs. Biochem. Pharmacol. 1999;57:1047–1058. doi: 10.1016/S0006-2952(99)00021-0. PubMed DOI

Zhitomirsky B., Assaraf Y.G. Lysosomal accumulation of anticancer drugs triggers lysosomal exocytosis. Oncotarget. 2017;8:45117–45132. doi: 10.18632/oncotarget.15155. PubMed DOI PMC

Duvvuri M., Krise J.P. Intracellular drug sequestration events associated with the emergence of multidrug resistance: A mechanistic review. Front. Biosci. 2005;10:1499–1509. doi: 10.2741/1634. PubMed DOI

Zhitomirsky B., Assaraf Y.G. Lysosomes as mediators of drug resistance in cancer. Drug Resist. Updat. 2016;24:23–33. doi: 10.1016/j.drup.2015.11.004. PubMed DOI

Shiraishi N., Akiyama S., Kobayashi M., Kuwano M. Lysosomotropic agents reverse multiple drug resistance in human cancer cells. Cancer Lett. 1986;30:251–259. doi: 10.1016/0304-3835(86)90049-2. PubMed DOI

Hindenburg A.A., Gervasoni J.E., Jr., Krishna S., Stewart V.J., Rosado M., Lutzky J., Bhalla K., Baker M.A., Taub R.N. Intracellular distribution and pharmacokinetics of daunorubicin in anthracycline-sensitive and -resistant HL-60 cells. Cancer Res. 1989;49:4607–4614. PubMed

Gervasoni J.E., Jr., Fields S.Z., Krishna S., Baker M.A., Rosado M., Thuraisamy K., Hindenburg A.A., Taub R.N. Subcellular distribution of daunorubicin in P-glycoprotein-positive and -negative drug-resistant cell lines using laser-assisted confocal microscopy. Cancer Res. 1991;51:4955–4963. PubMed

Hurwitz S.J., Terashima M., Mizunuma N., Slapak C.A. Vesicular anthracycline accumulation in doxorubicin-selected U-937 cells: Participation of lysosomes. Blood. 1997;89:3745–3754. doi: 10.1182/blood.V89.10.3745. PubMed DOI

Breuninger L.M., Paul S., Gaughan K., Miki T., Chan A., Aaronson S.A., Kruh G.D. Expression of multidrug resistance-associated protein in NIH/3T3 cells confers multidrug resistance associated with increased drug efflux and altered intracellular drug distribution. Cancer Res. 1995;55:5342–5347. PubMed

Noel G., Peterson C., Trouet A., Tulkens P. Uptake and subcellular localization of daunorubicin and adriamycin in cultured fibroblasts. Eur. J. Cancer. 1978;14:363–368. doi: 10.1016/0014-2964(78)90206-2. PubMed DOI

Loetchutinat C., Priebe W., Garnier-Suillerot A. Drug sequestration in cytoplasmic organelles does not contribute to the diminished sensitivity of anthracyclines in multidrug resistant K562 cells. Eur. J. Biochem. 2001;268:4459–4467. doi: 10.1046/j.1432-1327.2001.02370.x. PubMed DOI

Chapuy B., Panse M., Radunski U., Koch R., Wenzel D., Inagaki N., Haase D., Truemper L., Wulf G.G. ABC transporter A3 facilitates lysosomal sequestration of imatinib and modulates susceptibility of chronic myeloid leukemia cell lines to this drug. Haematologica. 2009;94:1528–1536. doi: 10.3324/haematol.2009.008631. PubMed DOI PMC

Gotink K.J., Broxterman H.J., Labots M., De Haas R.R., Dekker H., Honeywell R.J., Rudek M.A., Beerepoot L.V., Musters R.J., Jansen G., et al. Lysosomal sequestration of sunitinib: A novel mechanism of drug resistance. Clin. Cancer Res. 2011;17:7337–7346. doi: 10.1158/1078-0432.CCR-11-1667. PubMed DOI PMC

Colombo F., Trombetta E., Cetrangolo P., Maggioni M., Razini P., De Santis F., Torrente Y., Prati D., Torresani E., Porretti L. Giant Lysosomes as a Chemotherapy Resistance Mechanism in Hepatocellular Carcinoma Cells. PLoS ONE. 2014;9:e114787. doi: 10.1371/journal.pone.0114787. PubMed DOI PMC

Giuliano S., Cormerais Y., Dufies M., Grépin R., Colosetti P., Belaid A., Parola J., Martin A., Lacas-Gervais S., Mazure N.M., et al. Resistance to sunitinib in renal clear cell carcinoma results from sequestration in lysosomes and inhibition of the autophagic flux. Autophagy. 2015;11:1891–1904. doi: 10.1080/15548627.2015.1085742. PubMed DOI PMC

Gotink K.J., Rovithi M., De Haas R.R., Honeywell R.J., Dekker H., Poel D., Azijli K., Peters G.J., Broxterman H.J., Verheul H.M. Cross-resistance to clinically used tyrosine kinase inhibitors sunitinib, sorafenib and pazopanib. Cell. Oncol. 2015;38:119–129. doi: 10.1007/s13402-015-0218-8. PubMed DOI PMC

Burger H., Den Dekker A.T., Segeletz S., Boersma A.W., De Bruijn P., Debiec-Rychter M., Taguchi T., Sleijfer S., Sparreboom A., Mathijssen R.H., et al. Lysosomal Sequestration Determines Intracellular Imatinib Levels. Mol. Pharmacol. 2015;88:477–487. doi: 10.1124/mol.114.097451. PubMed DOI

Zhitomirsky B., Assaraf Y.G. Lysosomal sequestration of hydrophobic weak base chemotherapeutics triggers lysosomal biogenesis and lysosome-dependent cancer multidrug resistance. Oncotarget. 2015;6:1143–1156. doi: 10.18632/oncotarget.2732. PubMed DOI PMC

Smith D.A., Di L., Kerns E.H. The effect of plasma protein binding on in vivo efficacy: Misconceptions in drug discovery. Nat. Rev. Drug Discov. 2010;9:929–939. doi: 10.1038/nrd3287. PubMed DOI

Smith D.A., Rowland M. Intracellular and Intraorgan Concentrations of Small Molecule Drugs: Theory, Uncertainties in Infectious Diseases and Oncology, and Promise. Drug Metab. Dispos. 2019;47:665–672. doi: 10.1124/dmd.118.085951. PubMed DOI

Mlejnek P., Novak O., Dolezel P. A non-radioactive assay for precise determination of intracellular levels of imatinib and its main metabolite in Bcr-Abl positive cells. Talanta. 2011;83:1466–1471. doi: 10.1016/j.talanta.2010.11.028. PubMed DOI

Kosztyu P., Bukvova R., Dolezel P., Mlejnek P. Resistance to daunorubicin, imatinib, or nilotinib depends on expression levels of ABCB1 and ABCG2 in human leukemia cells. Chem. Biol. Interact. 2014;219:203–210. doi: 10.1016/j.cbi.2014.06.009. PubMed DOI

Kosztyu P., Dolezel P., Mlejnek P. Can P-glycoprotein mediate resistance to nilotinib in human leukaemia cells? Pharmacol. Res. 2013;67:79–83. doi: 10.1016/j.phrs.2012.10.012. PubMed DOI

Mlejnek P., Dolezel P., Ruzickova E. Drug resistance of cancer cells is crucially affected by expression levels of ABC-transporters. BioDiscovery. 2017;20:e11211. doi: 10.3897/biodiscovery.20.e11211. DOI

Mattsson J.P., Väänänen K., Wallmark B., Lorentzon P. Omeprazole and bafilomycin, two proton pump inhibitors: Differentiation of their effects on gastric, kidney and bone H(+)-translocating ATPases. Biochim. Biophys. Acta. 1991;1065:261–268. doi: 10.1016/0005-2736(91)90238-4. PubMed DOI

Frydrych I., Mlejnek P. Serine protease inhibitors N-alpha-tosyl-L-lysinyl-chloromethylketone (TLCK) and N-tosyl-L-phenylalaninyl-chloromethylketone (TPCK) do not inhibit caspase-3 and caspase-7 processing in cells exposed to pro-apoptotic inducing stimuli. J. Cell. Biochem. 2008;105:1501–1506. doi: 10.1002/jcb.21971. PubMed DOI

Bradford M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 1976;72:248–254. doi: 10.1016/0003-2697(76)90527-3. PubMed DOI

Rosti G., Castagnetti F., Gugliotta G., Baccarani M. Tyrosine kinase inhibitors in chronic myeloid leukaemia: Which, when, for whom? Nat. Rev. Clin. Oncol. 2017;14:141–154. doi: 10.1038/nrclinonc.2016.139. PubMed DOI

Picard S., Titier K., Etienne G., Teilhet E., Ducint D., Bernard M.A., Lassalle R., Marit G., Reiffers J., Begaud B., et al. Trough imatinib plasma levels are associated with both cytogenetic and molecular responses to standard-dose imatinib in chronic myeloid leukemia. Blood. 2007;109:3496–3499. doi: 10.1182/blood-2006-07-036012. PubMed DOI

De Duve C., De Barsy T., Poole B., Trouet A., Tulkens P., Van Hoof F. Commentary. Lysosomotropic agents. Biochem. Pharmacol. 1974;23:2495–2531. doi: 10.1016/0006-2952(74)90174-9. PubMed DOI

MacIntyre A.C., Cutler D.J. The potential role of lysosomes in tissue distribution of weak bases. Biopharm. Drug Dispos. 1988;9:513–526. doi: 10.1002/bod.2510090602. PubMed DOI

Sattler M., Salgia R. Role of the adapter protein CRKL in signal transduction of normal hematopoietic and BCR/ABL-transformed cells. Leukemia. 1998;12:637–644. doi: 10.1038/sj.leu.2401010. PubMed DOI

Samanta A., Perazzona B., Chakraborty S., Sun X., Modi H., Bhatia R., Priebe W., Arlinghaus R. Janus kinase 2 regulates Bcr-Abl signaling in chronic myeloid leukemia. Leukemia. 2011;25:463–472. doi: 10.1038/leu.2010.287. PubMed DOI PMC

Lim C.Y., Zoncu R. The lysosome as a command-and-control center for cellular metabolism. J. Cell Biol. 2016;214:653–664. doi: 10.1083/jcb.201607005. PubMed DOI PMC

What Is the Significance of Lysosomal-Mediated Resistance to Imatinib?

Lysosomal Fusion: An Efficient Mechanism Increasing Their Sequestration Capacity for Weak Base Drugs without Apparent Lysosomal Biogenesis