Human adenovirus type 3 restores pharmacologically inhibited exosomal cargo in lung carcinoma cells

Abstract

Introduction: Drug repurposing is fast growing and becoming an attractive approach for identifying novel targets, such as exosomes for cancer and antiviral therapy. Exosomes are a specialized class of extracellular vesicles that serve as functional mediators in intercellular communication and signaling that are important in normal physiological functions. A continuously growing body of evidence has established a correlation between the abnormal release of exosomes with various viral disease pathologies including cancer. Cells that are virus-infected release exosomes known to influence the process via the loading and transfer of viral components, such as miRNA, small (s) RNA, DNA, and proteins. Inhibition of exosome release may abate the spread and severity of viral infection, thus making exosomes an attractive target for antiviral therapies. We previously demonstrated the pharmacological inhibition of exosomes. Methods: Herein, we used a cell-based assay to determine the effect of Human adenovirus type 3 (HAdV3) on the exosome inhibition process by azole and Heparin derivatives. HAdV3-infected cells were treated with two concentrations of each inhibitor at different time points. Results: HAdV3 activities led to increased total sRNA, DNA, and exosome particle concentrations via particle tracking in the presence of Climbazole and Heparin relative to uninfected exosomes. In addition, there was an increased expression of classical markers such as ALG-2 interacting protein X (ALIX), and tetraspanin (CD63), (p < 0.05) and upregulated transcription factor interferon regulatory factor (IRF) 8 in the presence of HAdV3 after 24 hours (h) of treatment. Whereas higher concentrations of Climbazole and Heparin sodium salt were found to inhibit total exosome protein (p < 0.001) and exo-RNA (p < 0.01) content even in the presence of HAdV3 relative to infected exosomes only. Activities of HAdV3 in the presence of selected inhibitors resulted in the positive regulation of exosome related DNA damage/repair signaling proteins. Blocking exosome secretion partially obstructed viral entry. Immunological studies revealed that HAdV3 fiber protein levels in A549 cells were reduced at all concentrations of Climbazole and Heparin and both multiplicities of infections (p < 0.001). Discussion: Our findings suggest that while HAdV may bolster inhibited exosome content and release when modulating certain activities of the endosomal pathway mediators, HAdV entry might be constrained by the activities of these pharmacological agents.

Keywords: Climbazole; HAdV3; Heparin; cargo; drug repurposing; exosomes; inhibitors.

FIGURE 1

Graphical illustration of experimental workflow.

FIGURE 2

HAdV3 positively modulates exoDNA and RNA in the presence of Climbazole and Heparin. A549 cells were maintained overnight in exosome-free DMEM media and then treated with HAdV3-Climbazole, 5 µM (C5) and 10 µM (C10) or HAdV3-Heparin mixture 0.176 µM (H.176) and 0.88 µM (H.88) and MOIs (750 and 1500) of HAdV3 (n = 4). Infected cells only and uninfected cells (UNINF) served as control for exo RNA (A) 6, (B) 24, and (C) 48 h, and exoDNA at (D) 6, (E) 24, and (F) 48 h. ExoDNA showed a yield increase after 24 h HAdV3 infection in the presence of both Climbazole concentrations relative to uninfected cell-derived exoDNA, on the other hand, HAdV3 decreased exoRNA yield after 24 h of infection in the presence of Climbazole and Heparin. Mean values and SD were derived from four independent experiments. *Denotes significance at different levels of significance compared to controls and was calculated by one-way ANOVA using GraphPad Prism, Significance levels (*p < 0.05, *11 *0.01 and ***0.001).

FIGURE 3

HAdV3 positively modulates total exosome protein in the presence of Climbazole and Heparin. A549 cells were maintained overnight in exosome-free DMEM media and then treated with HAdV3-Climbazole, 5 µM (C5) and 10 µM (C10) or HAdV3-Heparin mixture 0.176 µM (H.176) and 0.88 µM (H.88) and MOIs (750 and 1500) of HAdV3 (n = 4). Infected cells only and non-infected cells (CNT) served as control for (A) 6, (B) 24, and (C) 48 h. Total exosome protein derived from HAdV3 infected cells was augmented relative the control in the presence of both concentrations of Climbazole and Heparin but it decreased after 48 h of infection. Mean values and SD were derived from four independent experiments. *Denotes significance at different levels of significance compared to controls and was calculated by one-way ANOVA using GraphPad Prism, Significance levels (*p < 0.05, **0.01 and ***0.001).

FIGURE 4

HAdV3 reduced inhibitor-treated exosome particle sizes but increased particle concentration. A549 cells were cultured in exo-free DMEM media and then treated with HAdV3-Climbazole, 5 µM (C5) and 10 µM (C10) or HAdV3-Heparin mixture 0.176 µM (H.176) and 0.88 µM (H.88) and MOIs (750 and 1500) of HAdV3 (n = 4). Infected cells only served as control. Zeta-particle tracking analysis showing exosome data at (A) 6, (B) 24, and (C) 48 h. At each time point, we determined i) mean size, ii) particle scatterplot, and iii) concentration of particle distribution after virus infection and inhibitor treatments. There were no changes in exosome particle sizes after 6 h of infection in the presence of Climbazole and Heparin but particle concentration significantly increased relative to that of untreated cells, 24 h treated cell-derived exosomes exhibited reduced particle sizes at lower concentration of inhibitors relative to untreated, however, particle concentration increased at the same concentration. Mean values and SD were derived from four independent experiments. *Denotes significance at different levels of significance compared to controls and was calculated by one-way ANOVA using GraphPad Prism, Significance levels (*p < 0.05, **0.01 and ***0.001).

FIGURE 5

HAdV3 positively modulates CD63 and Alix in the presence of Heparin. (A) A549 cells were maintained overnight in exosome-free DMEM media and then treated with HAdV3-Climbazole, 5 µM (C5) and 10 µM (C10) or HAdV3-Heparin mixture 0.176 µM (H.176) and 0.88 µM (H.88) and MOIs (750 and 1500) of HAdV3 (n = 4). Infected cells only served as control for CD63 at (A) 6, (B) 24, and (C) 48 h, and for Alix at (D) 6, (E) 24, and (F) 48 h. HAdV3 increased CD63 and Alix levels after 24 h of infection in the presence of Climbazole and Heparin. Mean values and SD were derived from four independent experiments. *Denotes significance at different levels of significance compared to controls and was calculated by one-way ANOVA using GraphPad Prism, Significance levels (*p < 0.05, **0.01 and ***0.001). Zeta-particle tracking analysis showing exosome A.) mean size B.) total particle concentration and C.) scatterplot of particle distribution after 6 h virus infection and inhibitor treatments. Significance (*p < 0.05, **0.01 and ***0.001).

FIGURE 6

Caspase 1 and IL-1β declined in the presence of HAdV3. (A) A549 cells were maintained overnight in exosome-free DMEM media and then treated HAdV3-Climbazole, 5 µM (C5) and 10 µM (C10) or HAdV3-Heparin mixture 0.176 µM (H.176) and 0.88 µM (H.88) and MOIs (750 and 1500) of HAdV3 (n = 4). Infected cells only served as control for Caspase 1 at (A) 6, (B) 24, and (C) 48 h, and for IL-1β at (D) 6, (E) 24, and (F) 48 h. Caspase 1 level was not significantly altered, however, IL-1β level significantly increased at 6 h with HAdV3 treatment, then declined at 24 h and 48 h of infection in the presence of Climbazole and Heparin relative to uninfected. Mean values and standard deviations SD were derived from four independent experiments. *Denotes significance at different levels of significance compared to controls and was calculated by one-way ANOVA using GraphPad Prism, Significance levels (*p < 0.05, **0.01 and ***0.001).

FIGURE 7

HAdV3 positively modulates TLR7 and Histone-X in the presence of Climbazole and Heparin. (A) A549 cells were maintained overnight in exosome-free DMEM media and then treated with HAdV3-Climbazole, 5 µM (C5) and 10 µM (C10) or HAdV3-Heparin mixture 0.176 µM (H.176) and 0.88 µM (H.88) and MOIs (750 and 1500) of HAdV3 (n = 4). Infected cells only served as control for TLR7 (A) 6, (B) 24, and (C) 48 h. HAdV3 significantly increased the expression of TLR7 and Histone-X proteins at (D) 6, (E) 24, and (F) 48 h of HAdV3 infection in the presence of Climbazole and Heparin, TLR7 was significantly in Heparin treated A549-exo relative to Climbazole treated A549-exo. *Denotes significance at different levels of significance compared to controls and was calculated by one-way ANOVA using GraphPad Prism, Significance levels (*p < 0.05, **0.01 and ***0.001).

FIGURE 8

Climbazole and Heparin inhibit expression HAdV3 fiber protein. (A) A549 cells were maintained overnight in exosome-free DMEM media and then treated with HAdV3-Climbazole, 5 µM (C5) and 10 µM (C10) or HAdV3-Heparin mixture 0.176 µM (H.176) and 0.88 µM (H.88) and MOIs (750 and 1500) of HAdV3 (n = 4). Infected cells only served as control for 6, 24 and 48 h. Western blot shows fiber protein expression after (A) 6 h, and (B) 48 h infection of A549 the presence inhibitors. Mean values and SD were derived from four independent experiments. *Denotes significance at different levels of significance compared to controls and was calculated by one-way ANOVA using GraphPad Prism, Significance levels (*p < 0.05, **0.01 and ***0.001).