Abstract
Palbociclib is a selective inhibitor of cyclin-dependent kinases 4 and 6 (CDK4/6) approved for the treatment of some cancers. The main mechanism of action of palbociclib is to induce cell cycle arrest and senescence on responsive cells. Here, we report that palbociclib concentrates in intracellular acidic vesicles, where it can be readily observed due to its intrinsic fluorescence, and it is released from these vesicles upon dilution or washing out of the extracellular medium. This reversible storage of drugs into acidic vesicles is generally known as lysosomal trapping and, based on this, we uncover novel properties of palbociclib. In particular, a short exposure of cells to palbociclib is sufficient to produce a stable cell-cycle arrest and long-term senescence. Moreover, after washing out the drug, palbociclib-treated cells release the drug to the medium and this conditioned medium is active on susceptible cells. Interestingly, cancer cells resistant to palbociclib also accumulate and release the drug producing paracrine senescence on susceptible cells. Finally, other lysosomotropic drugs, such as chloroquine, interfere with the accumulation of palbociclib into lysosomes, thereby reducing the minimal dose of palbociclib required for cell-cycle arrest and senescence. In summary, lysosomal trapping explains the prolonged temporal activity of palbociclib, the paracrine activity of exposed cells, and the cooperation with lysosomotropic drugs. These are important features that may help to improve the therapeutic dosing and efficacy of palbociclib. Finally, two other clinically approved CDK4/6 inhibitors, ribociclib and abemaciclib, present a similar behavior as palbociclib, suggesting that lysosomal trapping is a property common to all three clinically-approved CDK4/6 inhibitors.
Highlights
The uncontrolled cellular proliferation of tumor cells often depends on the activity of the related kinases CDK4 and CDK6, and these kinases are targets for anti-tumoralThese authors contributed : Diego Megias, Carmen BlancoAparicio
To determine if the palbociclib released to the extracellular milieu could have a biological impact, we examined the senescence-inducing capacity of the conditioned media (CM) obtained from senescent SK-Mel-103 cells
We show that palbociclib is reversibly trapped into lysosomes and, based on this, we demonstrate novel experimental properties of palbociclib that may be relevant to understand its pharmacology (Fig. 6b)
Summary
The uncontrolled cellular proliferation of tumor cells often depends on the activity of the related kinases CDK4 and CDK6, and these kinases are targets for anti-tumoral. Cellular senescence is characterized by prominent phenotypic changes, such as enlarged cellular size and an augmented lysosomal compartment [15] The latter can be visualized by staining cells for lysosomal β-galactosidase activity, which constitutes the basis for the senescence-associated β-galactosidase (SAβgal) activity [16]. The high lysosomal content of senescent cells may contribute to their potent secretory activity, generally known as senescence-associated secretory phenotype (SASP) [18], but not to the cell-cycle arrest. Some drugs are membrane permeable at neutral pH when they are non-protonated, but become membrane impermeable upon protonation at acidic pH These drugs accumulate into lysosomes and other cellular acidic compartments, a process known as lysosomal trapping. The lysosomal entrapment of these drugs is reversible since both forms of the drug, protonated (membrane impermeable) and non-protonated (membrane permeable), establish an equilibrium across cellular membranes. We demonstrate that palbociclib is reversibly sequestered into acidic vesicles and analyze the functional consequences of this feature
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