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Sci. Pharm.Scientia Pharmaceutica
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29 September 2026

17 Pages

Lysosomal Exocytosis and Drug Resistance in K562 Cells

and
Department of Anatomy, Faculty of Medicine and Dentistry, Palacky University Olomouc, Hnevotinska 3, 77515 Olomouc, Czech Republic
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Sci. Pharm.2026, 94(4), 88;https://doi.org/10.3390/scipharm94040088
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Abstract

Lysosomal exocytosis induced by the sequestration of hydrophobic weak-base drugs has been proposed as a mechanism of drug resistance (DR), although direct evidence for its quantitative relevance is lacking. Here, we compared the kinetics of lysosomal exocytosis with those of ABCB1-mediated active drug efflux and passive drug efflux by diffusion in K562 cells. Drug panels were selected according to the process investigated, with passive efflux assessed using compounds representing both the lysosomal exocytosis and ABCB1 substrate groups. Lysosomal exocytosis was quantified using FITC-dextran-labelled lysosomes and flow cytometry. K562 cells with high or moderate ABCB1 expression were used to determine active efflux, whereas parental K562 cells lacking detectable ABCB1 expression were used to determine passive efflux. Drug-induced lysosomal exocytosis was slow, with the intracellular FITC-dextran content decreasing to approximately 50% of its initial value after 5–8 h at 30 μM and 8–12 h at 10 μM. In contrast, ABCB1-mediated and passive efflux exhibited half-times of approximately 12–20 and 23–36 min, respectively. Moreover, measurable lysosomal exocytosis occurred only at high micromolar drug concentrations associated with off-target cytotoxicity. Thus, lysosomal exocytosis is unlikely to represent a quantitatively relevant mechanism of drug resistance in K562 cells.

1. Background

Our understanding of lysosomal structure and function has expanded considerably since the discovery of lysosomes by Christian de Duve in 1955 [1]. Initially regarded primarily as static organelles involved in cellular catabolism, lysosomes are now recognized as dynamic organelles involved in numerous physiological processes, including the regulation of cell proliferation, gene expression, metabolism, cellular stress responses, autophagy, cell death, immune responses, membrane repair, and synaptic plasticity. They also play important roles in pathological conditions, including cancer, lysosomal storage disorders, neurodegenerative diseases, obesity, and infections [1,2,3,4,5,6,7,8].
Lysosomal exocytosis is one of the mechanisms by which lysosomes contribute to cellular homeostasis [9,10,11]. Initially, this process was thought to occur predominantly in specialized cells, such as neutrophils, mast cells, and cytotoxic T lymphocytes, which contain secretory lysosomes [9]. It was subsequently demonstrated in many other cell types, where it contributes, among other functions, to plasma membrane repair and secretion. During this Ca2+-dependent process, lysosomes translocate towards the cell periphery, fuse with the plasma membrane, and release their contents into the extracellular space [10,11].
Lysosomal exocytosis has also been implicated in tumor progression, particularly in processes associated with tumor cell invasion and metastasis [8,12]. In addition, several studies have proposed that lysosomal exocytosis may contribute to drug resistance (DR). Klohs and Steinkampf were among the first to suggest that acidic vesicle-mediated exocytosis might contribute to DR in tumor cells exhibiting a multidrug-resistant (MDR) phenotype [13]. Subsequently, increased lysosomal exocytosis was observed in MDR lymphoblastic leukemia cells overexpressing P-glycoprotein (ABCB1) compared with drug-sensitive cells [14]. This was accompanied by a marked reduction in the cellular content of lysosomal hydrolases, leading the authors to propose that lysosomal exocytosis might contribute to the elimination of the hydrophobic weak-base anticancer drug vinblastine from ABCB1-expressing MDR leukemia cells [14].
Later, Zhitomirsky and Assaraf demonstrated that lysosomal accumulation of hydrophobic weak-base anticancer drugs at high concentrations can trigger lysosomal exocytosis in drug-sensitive HeLa cells [15]. Although the quantitative contribution of this process to drug elimination was not determined, the authors proposed lysosomal exocytosis as a component of lysosome-mediated MDR [15]. Several subsequent studies have implicated lysosomal exocytosis as a potential mechanism of resistance to hydrophobic weak-base drugs as well as to cisplatin [16,17,18,19,20,21,22]. However, these studies do not directly demonstrate that lysosomal exocytosis removes sufficient amounts of drug to substantially reduce its availability at intracellular target sites and thereby confer DR.
In the present study, we therefore examined the quantitative characteristics of drug-induced lysosomal exocytosis in K562 leukemia cells and compared its kinetics with those of two other routes of drug elimination: active efflux mediated by the ABCB1 transporter and passive efflux by diffusion. The drugs were selected according to the process investigated. For the analysis of lysosomal exocytosis, hydrophobic weak-base drugs known to accumulate extensively in lysosomes were selected [15,23]; for three of the five compounds examined—chloroquine, gefitinib, and vinblastine—induction of lysosomal exocytosis had also been reported previously [23,24]. The compounds were additionally selected for their suitability for the experimental system, including the absence of interference with FITC-dextran fluorescence. For the analysis of ABCB1-mediated efflux, established ABCB1 substrates that also undergo lysosomal accumulation were used [23,25]. Passive efflux was examined using compounds representing both groups, allowing its kinetics to be compared with those of lysosomal exocytosis and ABCB1-mediated active efflux. Thus, the aim was not to compare identical sets of drugs across the experimental systems, but to characterize each process using compounds suitable for analysis of the respective pathway. Our results show that lysosomal exocytosis is a very slow process and is induced only at relatively high concentrations of hydrophobic weak-base drugs, arguing against its role as a quantitatively relevant mechanism of DR under these experimental conditions.

2. Materials and Methods

2.1. Chemicals

Clomipramine hydrochloride (CLO, purity ≥ 99.5%), doxorubicin hydrochloride (DOX, purity ≥ 99%), imatinib mesylate (IM, purity ≥ 99.8%), and nigericin sodium salt (purity ≥ 98.4%) were purchased from Selleckchem (Houston, TX, USA). Chloroquine diphosphate salt (CHQ, purity ≥ 98.5%) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Gefitinib (GF, purity ≥ 99.9%), vinblastine sulfate (VIN, purity 99.6%), topotecan hydrochloride (TPT, purity 99.9%), nocodazole (NC, purity 99.6%), and aloxistatin (E64d, ALX; purity 99.9%), a cell-permeable irreversible broad-spectrum cysteine protease inhibitor, were obtained from MedChemExpress (Monmouth Junction, NJ, USA). Cyclosporin A (CsA, purity ≥ 98%) was obtained from Enzo Life Sciences AG (Lausen, Switzerland). All drugs were dissolved in DMSO. The final concentration of DMSO in the culture medium was approximately 0.1%. Fluorescein isothiocyanate-dextran (FITC-dextran; MW 40,000) was obtained from Thermo Fisher Scientific (Waltham, MA, USA).

2.2. Cell Culture

K562 cells, originally isolated from the bone marrow of a 53-year-old female patient with chronic myelogenous leukemia in blast crisis, were obtained from the European Collection of Authenticated Cell Cultures (ECACC, Salisbury, UK). K562 cells grow in suspension and are characterized by the presence of the Bcr-Abl fusion gene. Cells were cultured in RPMI-1640 medium supplemented with 10% fetal calf serum, 100 U/mL penicillin, and 100 μg/mL streptomycin at 37 °C in a humidified atmosphere containing 5% CO2.
K562/Dox cells overexpressing ABCB1 (P-glycoprotein, P-gp) were kindly provided by Prof. J.P. Marie (University of Paris 6, Paris, France) and were cultured under the same conditions. A more detailed characterization of the K562/Dox cell line has been reported previously [26].
K562/DoxDR2 cells with moderate ABCB1 expression were established by stable transfection of K562/Dox cells with a plasmid vector expressing shRNA targeting ABCB1, as described previously [27,28].
Cell density was determined using a Vi-CELL automated cell analyzer (Beckman Coulter, Indianapolis, IN, USA). Cell viability was routinely assessed by the trypan blue exclusion method using the same analyzer [29].

2.3. Determination of Mitochondrial Transmembrane Potential

Mitochondrial transmembrane potential (Δψm) was used to distinguish viable from dead cells. Cells with high Δψm were classified as viable, whereas cells with low/dissipated Δψm were classified as dead, as established and validated in our previous studies [30,31]. This approach enables detection of dead cells before loss of plasma membrane integrity and subsequent cell disintegration. Cells at densities up to 2 × 105 cells/mL were incubated with 100 nM tetramethylrhodamine ethyl ester (TMRE) in standard growth medium for 20 min in a standard cell-culture incubator (37 °C, 5% CO2). The cells were subsequently analyzed by flow cytometry without washing, with TMRE remaining present in the medium during the analysis. Fluorescence was measured using a Cytomics FC 500 flow cytometer (Beckman Coulter) with excitation at 488 nm and detection in the FL2 channel, as described previously [31,32]. At least 10,000 cells were analyzed per sample.

2.4. Analysis of ABCB1 Expression

ABCB1 expression was determined using a Human P-glycoprotein (P-gp) ELISA kit (Reddot Biotech, Houston, TX, USA) according to the manufacturer’s instructions.

2.5. Lysosomal Exocytosis

Lysosomal exocytosis was quantified by measuring the amount of FITC-dextran remaining in cells after drug treatment. Cells were loaded with FITC-dextran using a protocol adapted from Eriksson et al. [33] and previously employed in our study of lysosomal pH [34]. Briefly, cells were incubated in standard growth medium containing FITC-dextran (MW 40,000; 0.1 mg/mL) for 24 h under standard culture conditions. The medium was then replaced with fresh FITC-dextran-free growth medium, and the cells were incubated for an additional 2 h. After this 2-h chase period, an aliquot was taken as the common time-zero sample for all experimental conditions. The remaining cells were then divided into treatment groups, and the indicated hydrophobic weak-base drugs or vehicle alone were added for the specified periods. At the indicated time points, cells were washed with ice-cold PBS and resuspended in Britton–Robinson buffer (pH 6.0) supplemented with 50 mM 2-deoxyglucose, 5 mM sodium azide, and 10 μM nigericin. After 10 min of equilibration on ice, samples were analyzed by flow cytometry. By equilibrating intracellular pH to pH 6.0 in all samples, the potential influence of drug-induced changes in lysosomal pH on FITC fluorescence was eliminated, allowing the fluorescence signal to reflect solely the amount of FITC-dextran remaining in the cells [33]. FITC-dextran fluorescence was analyzed only within the gated population of viable cells; dead cells were excluded from the analysis based on their distinct FSC/SSC characteristics.

2.6. Efflux Kinetics of Drugs in K562 Cells Expressing ABCB1

Cells were incubated with the indicated drug in growth medium containing 5 μM cyclosporin A (CsA) for 4 h under standard culture conditions. CsA was included during drug loading to competitively inhibit ABCB1-mediated drug efflux and thereby allow intracellular drug accumulation. An aliquot of the cell suspension was then collected to determine the initial intracellular drug concentration. The remaining cells were collected by centrifugation (1600 rpm, 5 min, 25 °C) and transferred to fresh drug- and CsA-free medium. Intracellular drug levels were subsequently determined at the indicated time points to assess the kinetics of ABCB1-mediated drug efflux.

2.7. Efflux Kinetics of Drugs in Parental K562 Cells

Cells were incubated with the indicated drug for 4 h under standard culture conditions. An aliquot of the cell suspension was then collected to determine the initial intracellular drug concentration. The remaining cells were collected by centrifugation (1600 rpm, 5 min, 25 °C) and transferred to fresh drug-free medium. Intracellular drug levels were subsequently determined at the indicated time points. As these cells lack detectable ABCB1 expression, the decrease in intracellular drug levels after transfer to drug-free medium was used as an estimate of passive drug efflux.

2.8. Assay for Determination of Intracellular Drug Levels

Intracellular drug levels were determined using methods described previously [35,36]. Briefly, cells were separated from the growth medium by centrifugation through a layer of silicone oil. Cell pellets were extracted with ice-cold extraction solution: 4% (w/v) formic acid (FA) in water for DOX and IM, and 1% (w/v) FA in 50% (v/v) methanol in water for CHQ, CLO, GF, TPT, and VIN. Cell extracts were clarified by centrifugation (40,000× g, 10 min, 4 °C), diluted with the corresponding extraction solution, and analyzed by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS).

2.9. Statistical Analysis

Data are presented as means ± SE from three independent experiments. For Figure 1 and Figure 2, statistical analyses were performed separately for each drug using one-way ANOVA followed by multiple comparisons. Dunnett’s test was used to compare each drug concentration with the vehicle control, whereas pre-specified comparisons between each drug concentration and the corresponding drug + inhibitor treatment were performed using the Holm–Sidak method. Differences in ABCB1 expression between K562/Dox and K562/DoxDR2 cells (Figure 3) were evaluated using an unpaired two-tailed Student’s t-test. A p value < 0.05 was considered statistically significant. Statistical analyses were performed using SigmaPlot 11.2 (Systat Software Inc., San Jose, CA, USA).
Figure 1. Concentration-dependent effect of weak-base drugs on lysosomal exocytosis. FITC-dextran-loaded cells were treated with various concentrations of weak-base drugs in the presence or absence of nocodazole (NC). After 4 h of incubation, intracellular FITC-dextran content was determined by flow cytometry. Cells treated with DMSO alone served as the vehicle control (Co). (a) Chloroquine (CHQ); (b) clomipramine (CLO); (c) gefitinib (GF); (d) imatinib (IM); (e) topotecan (TPT). Columns represent means ± SE from three independent experiments. */** indicate p < 0.05 and p < 0.01, respectively, compared with the vehicle control; #/## indicate p < 0.05 and p < 0.01, respectively, compared with the corresponding drug-only group.
Figure 2. Effect of weak-base drugs on cell death induction. Cells were treated with various concentrations of weak-base drugs alone or in combination with aloxistatin (ALX), and cell death was determined after 12 h of incubation by flow-cytometric analysis of mitochondrial transmembrane potential (Δψm), with cells exhibiting low/dissipated Δψm classified as dead. Cells treated with DMSO alone served as the vehicle control (Co). (a) Chloroquine (CHQ); (b) clomipramine (CLO); (c) gefitinib (GF); (d) imatinib (IM); (e) topotecan (TPT). Columns represent means ± SE from three independent experiments. */** indicate p < 0.05 and p < 0.01, respectively, compared with the vehicle control; # indicates p < 0.05 compared with the corresponding drug-only group.
Figure 3. Analysis of ABCB1 expression in K562/Dox and K562/DoxDR2 cells. Columns represent means ± SE from three independent experiments. Statistical significance between the two cell lines was evaluated using an unpaired two-tailed Student’s t-test. ** p < 0.01.

3. Results

To assess the potential quantitative contribution of lysosomal exocytosis to drug elimination, we first examined its concentration dependence and kinetics in K562 cells and subsequently compared these characteristics with those of ABCB1-mediated and passive drug efflux.
Drug-induced lysosomal exocytosis increased with increasing concentrations of the hydrophobic weak-base drugs examined (Figure 1a–e). Measurable exocytosis was observed only at concentrations of 10 μM or higher. Nocodazole (NC), which disrupts microtubule-dependent lysosomal trafficking towards the plasma membrane and thereby inhibits lysosomal exocytosis [15], markedly inhibited the drug-induced decrease in intracellular FITC-dextran (Figure 1a–e). This inhibitory effect further supports the interpretation that the drug-induced loss of intracellular FITC-dextran reflects lysosomal exocytosis.
The kinetics of lysosomal exocytosis were slow (Figure 4). At drug concentrations of 30 μM, the time required for the intracellular FITC-dextran content to decrease to approximately 50% of its initial value was 5–8 h, whereas at 10 μM it was approximately 8–12 h.
Figure 4. Time course of lysosomal exocytosis induced by weak-base drugs. FITC-dextran-loaded cells were treated with weak-base drugs at the indicated concentrations, and intracellular FITC-dextran content was determined by flow cytometry at the indicated time points. Cells treated with DMSO alone served as the vehicle control (crosses). Chloroquine, triangles; clomipramine, circles; gefitinib, squares; imatinib, diamonds; topotecan, hexagons. Filled symbols represent a drug concentration of 10 μM and open symbols a concentration of 30 μM. Data represent means from three independent experiments; SE values ranged from approximately 4% to 14% of the corresponding measured values and did not exceed 14%. Error bars are omitted for clarity.
The high drug concentrations required to induce measurable lysosomal exocytosis were also associated with rapid cell death (Figure 3). Cell death induced by these concentrations was partially inhibited by aloxistatin (ALX), a cell-permeable cysteine protease inhibitor. These findings are consistent with the involvement of lysosomal cysteine proteases in the rapid cytotoxic response.
We next examined drug efflux in K562/Dox and K562/DoxDR2 cells, characterized by high and moderate ABCB1 expression, respectively. The difference in ABCB1 expression between the two cell lines was confirmed by ELISA (Figure 4). ABCB1-mediated drug efflux was rapid, with estimated half-times of approximately 12–15 min in K562/Dox cells and 15–20 min in K562/DoxDR2 cells (Figure 5).
Figure 5. Drug efflux mediated by ABCB1. Cells expressing high or intermediate levels of ABCB1 were incubated with the indicated weak-base drugs (10 μM) in the presence of CsA for 4 h and then transferred to drug- and CsA-free medium. Intracellular drug levels were determined by LC-MS/MS at the indicated time points. Gefitinib, squares; imatinib, diamonds; doxorubicin, crosses; vinblastine, stars. Filled symbols represent K562/Dox cells and open symbols K562/DoxDR2 cells. Data represent means from three independent experiments; SE values did not exceed 12% of the corresponding measured values. Error bars are omitted for clarity.
Finally, drug efflux was examined in K562 cells lacking detectable ABCB1 expression (Figure 6), providing an estimate of predominantly passive drug efflux. For most drugs examined, this process was slower than ABCB1-mediated efflux but remained relatively rapid, with estimated half-times of approximately 23–36 min (Figure 6). DOX represented a notable exception, showing substantially slower elimination from the cells.
Figure 6. Passive drug efflux in K562 cells. K562 cells were incubated with the indicated weak-base drugs (10 μM) for 4 h and then transferred to drug-free medium. Intracellular drug levels were determined by LC-MS/MS at the indicated time points. Chloroquine, triangles; clomipramine, circles; gefitinib, squares; imatinib, diamonds; doxorubicin, crosses; vinblastine, stars. Data represent means from three independent experiments; SE values did not exceed 10% of the corresponding measured values. Error bars are omitted for clarity.

4. Discussion

Lysosomes have been implicated in DR through several direct and indirect mechanisms. Their indirect contribution through cytoprotective autophagy is well established, as autophagy can promote tumor cell survival under therapeutic stress [37,38,39,40,41]. In contrast, lysosomal sequestration and lysosomal exocytosis have been proposed as direct mechanisms of DR, but their quantitative contribution remains uncertain. Although extensive lysosomal accumulation of hydrophobic weak-base drugs has been repeatedly demonstrated, evidence that sequestration itself reduces drug availability at intracellular target sites sufficiently to confer resistance remains limited [23,34,42,43,44,45,46,47]. Similarly, drug-induced lysosomal exocytosis has been demonstrated experimentally [15], and recent studies continue to implicate lysosomal exocytosis in the modulation of anticancer drug sensitivity [48]. However, such observations do not by themselves establish that lysosomal exocytosis removes sufficient amounts of drug to substantially reduce its availability at intracellular target sites and thereby confer resistance. Thus, the critical question is not whether hydrophobic weak-base drugs accumulate in lysosomes or can induce lysosomal exocytosis, but whether these processes are quantitatively capable of reducing drug availability at intracellular target sites to an extent sufficient to confer resistance. In the present study, we addressed this question specifically for lysosomal exocytosis by examining its concentration dependence and kinetics and comparing them with those of ABCB1-mediated and passive drug efflux.
Our results revealed several quantitative limitations of drug-induced lysosomal exocytosis. First, measurable exocytosis was observed only at relatively high drug concentrations, with 10 μM representing approximately the lowest effective concentration among the compounds examined (Figure 1), consistent with the observations of Zhitomirsky and Assaraf [15]. Moreover, the concentrations required to induce measurable lysosomal exocytosis are relatively high compared with clinically achievable systemic concentrations reported for some of the compounds examined, further questioning the potential clinical relevance of this process as a mechanism of DR [49,50]. Second, and more importantly, lysosomal exocytosis was a slow process. At 10 μM, the time required for the intracellular FITC-dextran content to decrease to approximately 50% of its initial value was 8–12 h, whereas at 30 μM it was approximately 5–8 h (Figure 2). In comparison, ABCB1-mediated drug efflux occurred with half-times of approximately 12–20 min (Figure 5), and predominantly passive drug efflux with half-times of approximately 23–36 min for most compounds examined (Figure 6). Thus, these routes of drug elimination operate on a markedly shorter timescale than drug-induced lysosomal exocytosis.
A further limitation is that the drug concentrations required to induce measurable lysosomal exocytosis were themselves markedly cytotoxic. At these concentrations, all compounds examined induced rapid cell death, which was partially inhibited by ALX, consistent with the involvement of lysosomal cysteine proteases in the early cytotoxic response (Figure 3). One possible explanation is that extensive accumulation of hydrophobic weak-base compounds within lysosomes at high concentrations contributes to lysosomal swelling and membrane destabilization [51], ultimately leading to lysosomal membrane permeabilization (LMP) and subsequent release of lysosomal proteases into the cytosol. LMP and the release of cathepsins and other lysosomal hydrolases are well-established mechanisms of lysosome-dependent cell death and can be induced by lysosomotropic compounds [52,53]. However, LMP was not directly examined in the present study, and this interpretation therefore remains speculative. Importantly, prolonged exposure resulted in a second, slower wave of cell death, affecting 20–60% of cells treated with CHQ or CLO and becoming essentially lethal with GF, IM, and TPT (unpublished data). Thus, even if lysosomal exocytosis removes a fraction of the lysosomally accumulated drug, the concentrations required to activate this process are associated with substantial acute and delayed cytotoxicity, arguing against an effective protective role under these conditions.
The potential contribution of lysosomal exocytosis may be further constrained by its limited capacity. Lysosomes represent a heterogeneous population with respect to their intracellular localization and mobility, with peripheral lysosomes being more mobile and more readily available for exocytosis [8]. Interestingly, the kinetic curves in Figure 2 tended to reach a plateau when approximately 50% of the initial intracellular FITC-dextran remained in the cells. One possible interpretation is that only a subpopulation of FITC-dextran-containing lysosomes is readily available for exocytosis. Although this interpretation requires direct experimental verification, the observed plateau is consistent with the possibility that the capacity of lysosomal exocytosis to remove lysosomal contents is intrinsically limited.
The rapid ABCB1-mediated drug efflux observed in the present study (Figure 5) is consistent with previous kinetic studies showing fast P-glycoprotein-mediated transport of anticancer drugs in living cells, including K562-derived models [54,55]. The relatively small difference between ABCB1-mediated and predominantly passive drug efflux is also noteworthy. These findings are consistent with the proposed hydrophobic vacuum cleaner/flippase model, according to which hydrophobic substrates partition into the plasma membrane and are recognized by ABCB1 within the inner leaflet and translocated towards the outer leaflet or extracellular side [56,57,58]. Subsequent partitioning into the extracellular medium can occur spontaneously. Thus, passive diffusion and ABCB1-mediated transport need not be regarded as completely independent processes; rather, ABCB1 accelerates the elimination of suitable substrates by promoting their translocation across the plasma membrane. DOX was a notable exception, showing markedly slower passive efflux, most likely because its strong intracellular binding, particularly to DNA [59,60], reduces the freely diffusible drug fraction.
More fundamentally, for lysosomal exocytosis to confer DR, it must remove sufficient amounts of drug to meaningfully decrease its concentration at intracellular target sites. Although lysosomal sequestration can generate high intralysosomal drug concentrations, the relevant parameter for resistance is not the amount accumulated in lysosomes per se, but the extent to which its subsequent exocytosis reduces drug availability in the target-containing compartment. The conventional ion-trapping model assumes that the uncharged forms of hydrophobic weak-base drugs can readily cross biological membranes by passive diffusion, allowing redistribution between the extracellular medium, cytosol, and intracellular compartments [23,42]. Within such a system, removal of a fraction of lysosomally accumulated drug by exocytosis would be followed by redistribution of the remaining drug and would not necessarily produce a corresponding decrease in drug concentration at its molecular target. Moreover, cellular uptake of many drugs may involve membrane transporters rather than, or in addition to, simple passive diffusion [61]. Such transporter-mediated uptake would make the relationship between lysosomal drug removal and drug concentration at intracellular target sites even more complex and further argues against assuming that lysosomal exocytosis necessarily results in an equivalent reduction in intracellular drug exposure. Considering that substantial lysosomal exocytosis requires several hours, whereas the drug-efflux processes examined here operate within tens of minutes, our results provide no evidence that lysosomal exocytosis contributes sufficiently to net drug elimination to meet this requirement in K562 cells.
Taken together, these limitations—including the high drug concentrations required, slow kinetics, associated cytotoxicity, potentially limited exocytotic capacity, and uncertain impact on drug concentrations at intracellular target sites—make it difficult to envisage lysosomal exocytosis as a quantitatively important mechanism of resistance to small hydrophobic weak-base drugs in K562 cells.
These conclusions should not be generalized to all materials entering the lysosomal compartment. Lysosomal exocytosis may be considerably more important for nanoparticles and drug carriers internalized by endocytosis, which, unlike small hydrophobic molecules, cannot readily redistribute across biological membranes by passive diffusion. Indeed, lysosomal exocytosis has been implicated in their cellular elimination, and its inhibition can increase intracellular retention and biological effects [62,63,64,65]. Thus, lysosomal exocytosis may be an important determinant of intracellular exposure to endocytosed drug-delivery systems even if its contribution to the elimination of small hydrophobic weak-base drugs is limited.

5. Conclusions

Drug-induced lysosomal exocytosis in K562 cells requires relatively high drug concentrations and proceeds over several hours, whereas ABCB1-mediated and predominantly passive drug efflux occur within tens of minutes. Moreover, the concentrations required to induce measurable lysosomal exocytosis are themselves associated with substantial cytotoxicity. Taken together, these findings indicate that lysosomal exocytosis is unlikely to represent a quantitatively relevant mechanism of drug resistance in K562 cells under the conditions examined.

Author Contributions

Conceptualization, P.M., methodology, P.M., investigation, P.M. writing—original draft preparation, P.M., writing—review, proofreading and editing, P.M., supervision, P.M., funding acquisition, P.M., and validation, P.D., formal analysis, P.D., investigation, P.D., data curation P.D. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by Palacky University Olomouc, grant number IGA_LF_2025_023 and grant number IGA_LF_2026_033.

Data Availability Statement

The original contributions presented in this study are included in the article. Further inquiries can be directed to the corresponding author.

Conflicts of Interest

The authors declare no competing interests.

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