Selective pharmacological inhibition alters human carcinoma lung cell-derived extracellular vesicle formation

Abstract

Exosomes also termed small extracellular vesicles (sEVs) are important mediators of intercellular communication in many physiological and pathological processes such as protein clearance, immunity, infections, signaling, and cancer. Elevated circulating levels of exosomes have been linked to some viral infections, aggressive cancer, and neurodegenerative diseases. Some pharmacological compounds have been demonstrated to effectively inhibit exosome production pathways. There are very few studies on exosome inhibition and how they influence pathophysiological conditions.

Methods: In the current study, we examined how inhibition of extracellular vesicle release and/or uptake would impact the exosome formation pathway. Using a constellation of improved EV experimental approaches, we evaluated the concentration-based cytotoxicity effects of pharmacological agents (ketoconazole, climbazole, and heparin) on Human Lung Carcinoma (A549) cell viability. We investigated the effect of inhibitor dosages on exosome production and release. Analysis of exosome inhibition includes quantitative analysis and total protein expression of exosome release after pharmacological inhibition; we examined exosome protein level after inhibition.

Results: Selective inhibition of exosomes altered particle sizes, and heparin significantly reduced the total exosomes released. Climbazole and heparin undermined membrane-bound tetraspanin CD63 expression and significantly disrupted ALIX protein (p ≤ 0.0001) and TSG101 (p ≤ 0.001). Azoles and heparin also disrupt transmembrane trafficking by modulating Ras binding protein (p ≤ 0.001).

Conclusion: These findings revealed that pharmacological inhibition of exosomes regulates the endocytic pathway and expression of endosomal sorting complex required for transport mediators, suggesting climbazole and heparin as effective inhibitors of exosome synthesis.

Keywords: ESCRT; Extracellular vesicles; Inhibition; Pathway.

Graphical abstract

Fig. 1

Cytotoxicity evaluation of selected drug concentrations: Analysis of selected inhibitory concentration activities. Bar graphs showing the response of A549 cells (as percent (%) viability) to ketoconazole (1 and 5 μM), climbazole (5 and 10 μM), and heparin (0.176 and 0.88 μM) activities after (A) 6, (B) 12, (C) 24, and (D) 48 h of treatment. Cells were cultured overnight in DMEM and replaced with 2% exosome-free media and then treated with the inhibitors or PBS for 6, 12, 24, and 48 h. MTT dye was added to each well and incubated for 3–4 h. A detailed description of this procedure is described in the “Materials and Methods” section. The graph depicts % viability change compared to untreated controls and represents the mean ± SD of 4 separate experiments.

Fig. 2

Cytotoxicity evaluation of concentrations of pharmacological agents. (A) Bright field and fluorescence images of A549 cell viability after treatment with ketoconazole, climbazole and heparin. Calcein is retained within live cells due to esterase activity emitting green calcein fluorescence. The cell viability graph is depicted by mean fluorescence intensity. (B) Graphs showing the response of A549 cells to ketoconazole (1, 5 μM), climbazole (5, 10 μM), and heparin (0.176, 0.88 μM) activities after (i) 6 h, (ii) 12 h, (iii) 24 h, and (iv) 48 h of treatment. Cells were cultured overnight in DMEM and replaced with 2% exosome-free media and then treated with the inhibitors or PBS (as control) for 6, 12, 24, and 48 h, cells were stained by using Calcein AM dye, where live cells appear as green fluorescence. A detailed description of this procedure is described in the “Materials and Methods” section. The graph depicts % viability change compared to untreated controls and represents the mean ± SD of 4 separate experiments. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3

Ketoconazole, heparin, and climbazole inhibit total exosome protein concentration in a dose-dependent approach after 24 and 48 h of treatment. (A) Exosome proteins were separated according to their molecular weight via SDS-PAGE (Also see Supplementary Fig. 3). Graphs showing exosome protein concentration after (B) ketoconazole, climbazole, and heparin treatments after 6 h, (C) 24 h, and (D) 48 h. Concentration of exosome protein was measured via Bradford Lowry Assay. Heparin significantly inhibited total exosome protein concentration at both concentrations and all time points relative to untreated cells. Statistical mean ± SD was derived from four independent experiments. Asterisks (*) denote significance levels at *p < 0.05, **p < 0.01, and ***p < 0.001 compared to controls and were computed using GraphPad Prism.

Fig. 4a

Evaluation of exosome particles size distribution and concentration using Zeta particle tracking analyzer and the quantitative particle analysis: Scatter plot of exosome distribution in response to activities of inhibitors ketoconazole (1 μM and 5 μM), climbazole (5 μM and 10 μM) and heparin (0.176 μM and 0.88 μM) after (A) 6 h, (See 24 and 48 h scatterplot data in Supplementary Fig. 1). Untreated A549 cell-derived exosomes were used as positive control while DMSO (0.1%) was used as a drug vehicle and thus serve as a control.

Figure 4bc

A graph showing exosome particle sizes and concentration/mL per dose inhibitory compound after (B) 6 h (C) 24 h. The exosome in the conditioned media was isolated, filtered through a 0.22 μM filter, and analyzed by the particle tracking analysis in 4 separate experiments. Each graph represents the mean ± SD of 11 frame positions obtained from 4 separate experiments. Data for exosome was captured using a particle metrix zeta-view analyzer (*p < 0.05, **p < 0.01, ***p < 0.001, and ***p < 0.0001).

Fig. 4d

A graph showing exosome particle sizes and concentration/mL per dose inhibitory compound after (D) 48 h. The exosome in the conditioned media was isolated, filtered through a 0.22 μM filter, and analyzed by the particle tracking analysis in 4 separate experiments. Each graph represents the mean ± SD of 11 frame positions obtained from 4 separate experiments. Data for exosome was captured using a particle metrix zeta-view analyzer (*p < 0.05, **p < 0.01, ***p < 0.001, and ***p < 0.0001).

Fig. 5a

Climbazole and heparin inhibit exosome biogenesis and secretion via alteration of Rab protein and exosome classical markers. A549 cells were plated overnight in exosome-free DMEM media and then treated with ketoconazole, climbazole, heparin, or DMSO (vehicle control) for (A) 6 h. Expression levels of all proteins were normalized to GAPDH. Ketoconazole and climbazole at 5 μM and 20 μM concentrations significantly inhibited the expression levels of CD63 in A549 cell-derived exosome after 6 h treatment. Flotillin and TSG101 levels were significantly downregulated in exosomes derived from cells treated with climbazole, ketoconazole, and heparin at all time points of treatment. Rab27a protein level was downregulated by ketoconazole and heparin treatments (Also see Supplementary Fig. 3). (*) Asterisk denotes significance at *p < 0.05, **p < 0.01, and ***p < 0.001 compared to controls and was computed using GraphPad Prism.

Fig. 5b

Climbazole and heparin inhibit exosome biogenesis and secretion via alteration of Rab protein and exosome classical markers. A549 cells were plated overnight in exosome-free DMEM media and then treated with ketoconazole, climbazole, heparin, or DMSO (vehicle control) for (B) 24 h. Expression levels of all proteins were normalized to GAPDH. Ketoconazole and climbazole at 5 μM and 20 μM concentrations significantly inhibited the expression levels of CD63 in A549 cell-derived exosome after 24 h treatment. Flotillin and TSG101 levels were significantly downregulated in exosomes derived from cells treated with climbazole, ketoconazole, and heparin at all time points of treatment. Rab27a protein level was downregulated by ketoconazole and heparin treatments (Also see Supplementary Fig. 3). (*) Asterisk denotes significance at *p < 0.05, **p < 0.01, and ***p < 0.001 compared to controls and was computed using GraphPad Prism.

Fig. 5c

Climbazole and heparin inhibit exosome biogenesis and secretion via alteration of Rab protein and exosome classical markers. A549 cells were plated overnight in exosome-free DMEM media and then treated with ketoconazole, climbazole, heparin, or DMSO (vehicle control) for (C) 48 h. Expression levels of all proteins were normalized to GAPDH. Ketoconazole and climbazole at 5 μM and 20 μM concentrations significantly inhibited the expression levels of CD63 in A549 cell-derived exosome after 48 h treatment. Flotillin and TSG101 levels were significantly downregulated in exosomes derived from cells treated with climbazole, ketoconazole, and heparin at all time points of treatment. Rab27a protein level was downregulated by ketoconazole and heparin treatments (Also see Supplementary Fig. 3). (*) Asterisk denotes significance at *p < 0.05, **p < 0.01, and ***p < 0.001 compared to controls and was computed using GraphPad Prism.

Fig. 5d

(D) Alix level was upregulated in ketoconazole-treated cell-derived exosome compared to untreated after 6 h but was significantly downregulated in ketoconazole, climbazole, and heparin treatment when compared to untreated after 24 and 48 h. Mean values ± SD were derived from four independent experiments. (*) Asterisk denotes significance at *p < 0.05, **p < 0.01, and ***p < 0.001 compared to controls and was computed using GraphPad Prism.

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