Fig 1.
Treatment of HUVECs with archazolid for 24 h does not evoke cytotoxic effects.
Confluent HUVECs were treated either with archazolid (arch) or DMSO (co) for 24 h and 48 h. Both the metabolic activity (A) and the release of LDH (B) were determined. Treatment with lysis buffer served as positive control for LDH release. (A, B) Data are expressed as mean ± SEM (n = 3). *p ≤ 0.05 versus DMSO control.
Fig 2.
Archazolid alter the cell morphology and is functionally active in HUVECs.
Confluent HUVECs were treated with 1 nM archazolid (arch) or DMSO (co). (A) Microscopic images of fixed cells were taken. Scale bar represents 100 μm. (B) Fluorescence microscopic images of viable cells were taken. LysoTracker Red DND-99 was used to visualize the acidic compartments (lysosomes) and Hoechst 33342 (blue) staining was used to visualize nuclei. Scale bar represents 20 μm. (A, B) One representative image out of three independently performed experiments is shown.
Fig 3.
V-ATPase inhibition by archazolid increases the adhesion of MDA-MB-231 cells onto HUVECs and decreases their transendothelial migration.
(A, B) Confluent HUVECs were treated with archazolid (arch) or DMSO (co) for 24 h. Untreated MDA-MB-231 cells were stained with CellTracker Green CMFDA Dye and were added to the HUVEC monolayer. The cells were allowed to adhere for 10 min and 120 min. Non-adherent MDA-MB-231 cells were washed off. (A) The adhesion was quantified by measuring the fluorescence signal using a plate reader. Data are expressed as mean ± SEM (n = 7). *p ≤ 0.05 versus DMSO control. (B) Microscopic images show unlabeled HUVECs and CellTracker Green CMFDA Dye-labeled MDA-MB-231 cells after 10 min of adhesion. Scale bar represents 200 μm. One representative image out of three independently performed experiments is shown. (C) HUVECs were grown on a porous filter membrane and were treated with archazolid for 24 h. Untreated CellTracker Green-labeled MDA-MB-231 cells were allowed to transmigrate through the endothelial monolayer for 24 h. Transmigrated cells were quantified by measuring the fluorescence signal. Data are expressed as mean ± SEM (n = 5). *p ≤ 0.05 versus FCS control.
Fig 4.
V-ATPase inhibition by archazolid increases the adhesion of MDA-MB-231 cells onto HMEC-1 and the adhesion of PC-3 cells onto HUVECs.
Confluent endothelial cells were treated with archazolid (arch) or DMSO (co) for 24 h. (A) Untreated MDA-MB-231 cells were stained with CellTracker Green CMFDA Dye and added to an HMEC-1 monolayer. The cells were allowed to adhere for 10 and 120 min. Non-adherent MDA-MB-231 cells were washed off. (B) Untreated PC-3 cells were stained with CellTracker Green CMFDA Dye and added to the HUVEC monolayer. The cells were allowed to adhere for 10, 30 and 60 min. Non-adherent PC-3 cells were washed off. (A, B) The adhesion was quantified by measuring the fluorescence signal using a plate reader. Data are expressed as mean ± SEM (n = 3). *p ≤ 0.05 versus DMSO control.
Fig 5.
Archazolid does not upregulate the expression of cell adhesion molecules in HUVECs.
(A) Confluent HUVECs were treated with 1 nM archazolid (arch) or DMSO (co) for 12 h. The expression of ICAM-1, VCAM-1, E-selectin and N-cadherin was analyzed on the mRNA level by qPCR experiments. (B) Confluent HUVECs were treated with 1 nM archazolid (arch) or DMSO (co) for 24 h. The surface expression of ICAM-1, VCAM-1, E-selectin and N-cadherin was analyzed by flow cytometry. (A, B) Data are expressed as mean ± SEM (n = 3). *p ≤ 0.05 versus DMSO control. TNFα (TNF) served as positive control to induce both the mRNA (1 ng/ml TNF) and the cell surface expression (10 ng/ml TNF) of ICAM-1, VCAM-1, E-selectin and N-cadherin.
Fig 6.
Blocking integrin β1 subunits on MDA-MB-231 or PC-3 cells prevents the archazolid-mediated increase in tumor cell adhesion.
Confluent HUVECs were treated either with archazolid or DMSO for 24 h. MDA-MB-231 or PC-3 cells were stained with CellTracker Green CMFDA Dye. (A) The cell adhesion assay was performed after the integrin β1 subunit was blocked by a monoclonal antibody for 30 min on either MDA-MB-231 (n = 6) cells or HUVECs (n = 3). MDA-MB-231 cells were allowed to adhere for 10 min. (B) The cell adhesion assay was performed after the integrin β1 subunit was blocked by a monoclonal antibody for 30 min on either PC-3 cells (n = 5) or HUVECs (n = 3). PC-3 cells were allowed to adhere for 60 min. (A, B) Non-adherent tumor cells were washed off. The adhesion was quantified by measuring the fluorescence signal using a plate reader. Data are expressed as mean ± SEM. *p ≤ 0.05 versus DMSO control, #p ≤ 0.05 versus 1 nM archazolid.
Fig 7.
Collagen is the major ECM component mediating MDA-MB-231 and PC-3 cell adhesion.
Archazolid increases the amount of extracellular collagen on HUVECs. (A) 24-well plates were coated with 10 μg/ml collagen (col), fibronectin (fn) or laminin (ln) or were left uncoated (co). Untreated MDA-MB-231 or PC-3 cells were stained with CellTracker Green CMFDA Dye and added into ECM-coated or uncoated wells. After 10 min of incubation, non-adherent cells were washed off. Adhesion was quantified by measuring the fluorescence signal using a plate reader. Data are expressed as mean ± SEM (n = 3). *p ≤ 0.05 versus uncoated control. (B) A cell adhesion assay onto collagen was performed with untreated MDA-MB-231 or PC-3 cells (co) and MDA-MB-231 or PC-3 cells on which the integrin β1 subunit was blocked by a monoclonal antibody (ab). Data are expressed as mean ± SEM (n = 3). *,#p ≤ 0.05 versus control. (C) Confluent HUVECs were treated with 1 nM archazolid (arch) or DMSO (co) for 24 h. Surface collagen (green) was detected by immunofluorescence staining of viable cells and nuclei (blue) were visualized by Hoechst 33342 staining. Scale bar represents 20 μm. One representative image out of three independently performed experiments is shown. The increase in extracellular collagen was quantified. Data are expressed as mean ± SEM (n = 3). *p ≤ 0.05 versus DMSO control.
Fig 8.
Archazolid decreases the activity and expression of cathepsin B in endothelial cells.
Confluent HUVECs or HMEC-1 were treated with archazolid (arch) or DMSO (co) for 24 h. (A) Cathepsin B (catB) activity was measured in cell lysates of treated endothelial cells by using a fluorogenic cathepsin B substrate. Data are expressed as mean ± SEM (HUVECs n = 5; HMEC-1 n = 4). *p ≤ 0.05 versus DMSO control. (B) Western blot analysis of mature cathepsin B was performed and quantified densitometrically. Actin served as loading control. Data are expressed as mean ± SEM (n = 4). *p ≤ 0.05 versus DMSO control. One representative blot out of three independently performed experiments is shown.
Fig 9.
Overexpression of cathepsin B in endothelial cells attenuates both the basal and the archazolid-induced tumor cell adhesion.
HUVECs were transfected with a plasmid containing human cathepsin B (catB) or the empty vector (pcDNA3.1(-)delta MCS). After 48 h cells were treated with 1 nM archazolid (arch) or DMSO (co) for 24 h. (A) The expression of cathepsin B was determined by western blot analysis. One representative blot out of three independently performed experiments is shown. Actin served as loading control. (B) Untreated MDA-MB-231 cells were labeled with CellTracker Green CMFDA Dye, added to the HUVEC monolayer and were allowed to adhere for 10 min. Non-adherent MDA-MB-231 cells were washed off. The adhesion was quantified by measuring the fluorescence signal using a plate reader. Data are expressed as mean ± SEM (n = 7). *p ≤ 0.05 versus DMSO (control transfection). #p ≤ 0.05 versus 1 nM archazolid (control transfection).