2-Bromopalmitate treatment attenuates senescence phenotype in human adult cells - possible role of palmitoylation

2-BP slows proliferation without inducing markers of senescence

Since protein palmitoylation has been shown to play a role in the regulation of the DNA damage response [8], we sought to determine whether the potent palmitoylation inhibitor 2-BP could affect the senescence phenotype of human VSMCs. We first determined the influence of 2-BP on non-senescent VSMCs. To analyze the effects of palmitoylation inhibition in our model, we treated proliferating VSMCs with increasing doses of 2-BP and analyzed the protein level of palmitoylation, cellular proliferation, DNA synthesis, DNA damage and SA-β-gal activity.

As expected, 2-BP treatment was associated with a dose-dependent decrease in the total protein palmitoylation level in whole-cell lysates compared to the control, as detected by an acyl-biotinyl exchange assay (ABE) followed by Western blotting (Figure 1A and Supplementary Figure 1).

VSMCs are relatively fast proliferating cells that exhibit a gradually slowing of their proliferation rate within a few days of culture, which may result in the acquisition of some features of a senescent phenotype, such as the nonspecific activation of SA-β-gal. Therefore, we compared the results obtained in treated cells to those in untreated cells (CTRL) 48 h after seeding, when the cells retained a high proliferation rate.

Treatment of VSMCs with 2-BP decreased the proliferation rate of the cells (Figure 1B, 1C). However, we did not observe any changes in the number of BrdU-incorporating cells (Figure 1D, 1H), the expression of senescence markers, such as SA-β-gal activity (Figure 1E, 1I) or the number of 53BP1 or γH2AX foci, (Figure 1F, 1G, 1I), in 2-BP-treated cells. These results suggest that palmitoylation inhibition in growing cells does not induce permanent growth arrest.

2-BP treatment affects the development of a senescent phenotype in VSMCs treated with doxorubicin

The inhibition of protein palmitoylation with 2-BP has been shown to affect DNA damage-induced focus assembly/disassembly and to override cell cycle arrest in primary mouse embryonic fibroblasts [8]. Therefore, we hypothesized that 2-BP could influence the phenotypic transition from proliferating to senescent cells upon DNA damage caused by doxorubicin treatment. To prevent cytotoxicity in cultures co-treated with doxorubicin and 2-BP [8], we pretreated VSMCs with 2-BP for 24 h. The 2-BP was removed by washing, and fresh medium containing 1 μM doxorubicin (DOX) was added and left for 2 h to induce senescence according to our previously established protocol [9]. Three days after the initial 2-BP treatment, the levels of senescence markers were analyzed. We found that 2-BP partially released the inhibition of proliferation caused by doxorubicin (Figure 2A, 2B). Notably, the observed effect was dose dependent but not dose proportional. At least 24 palmitoyltransferases might be differentially affected by 2-BP treatment, which may result in an activity configuration-promoting response that is not necessarily proportional to the dose.

The changes in proliferation were reflected by an increase in the number of BrdU-positive cells compared to doxorubicin-treated cells (Figure 2C). Treatment with 2-BP was also associated with decreased activity of SA-β-gal in DOX-treated cells (Figure 2D). The expression of DNA damage markers, such as 53BP1 and γH2AX, quantified by the number of foci per nucleus (Figure 2E, 2F), decreased significantly in 2-BP-treated cells compared to the number in cells treated with doxorubicin alone, regardless of the concentration of the inhibitor. Representative images of SA-β-gal activity analysis and 53BP1, γH2AX, and BrdU immunostaining are shown in Figure 2G. We observed similar trends in the changes in the proliferation rate of doxorubicin-treated human fibroblasts upon 2-BP treatment, but these changes were not significant, which suggests that our findings are cell/dose specific (Supplementary Figure 2).

The senescence-modifying activity of 2-BP is at least partially mediated by disruption of the DNA damage response

It has been previously shown that 2-BP may affect the DNA damage response by blocking p53 activation and translocation to the nucleus [3, 9]. To determine whether the inhibition of senescence phenotype development by 2-BP is dependent on this mechanism, we treated cells with 50 μM 2-BP for 24 h before (pretreatment) or after (posttreatment) the induction of DNA damage with doxorubicin (Figure 3A). We hypothesized that cultured cells treated with 2-BP after the induction of DNA damage would not respond to phenotypic changes in the same way as the pretreatment variants because the DNA damage response pathway would already be initiated and p53 would be activated. We did not observe significant changes in the levels of DNA damage markers, such as the number of γH2AX and 53BP1 foci in the nuclei, or in the percentage of BrdU-positive cells in the posttreatment variant compared to doxorubicin-treated cells on day 3 or day 7. Cells pretreated with 2-BP before DOX treatment showed significantly fewer 53BP1 foci (day 7) and more BrdU-positive cells (day 3) compared to DOX-treated cells (Figure 3B–3D, 3F). Notably, the level of activity of SA-β-gal decreased significantly in pretreated and posttreated cells on day 3 regardless of differences in the levels of DNA damage markers and BrdU incorporation in pre- and posttreated variants. However, the initial decrease in SA-β-gal activity was no longer observed on day 7 (Figure 3E, 3F). Western blot (WB) analysis revealed a significant decrease in the p-p53 protein level caused by 2-BP pretreatment of DOX-treated cells (Figure 4A, 4D), but culturing the cells with 2-BP after DOX treatment (posttreatment variant) did not affect p-53 phosphorylation (Supplementary Figure 3).

As expected, we observed a significant decrease in HMGB1 and lamin B1 levels upon induction of senescence by doxorubicin treatment. However, the levels of these proteins remained unchanged upon treatment with 2-BP and doxorubicin (Figure 4B–4D). Similarly, 2-BP alone did not influence HMGB1 or lamin B1 levels in posttreated cells (Supplementary Figure 3).

Of note, doxorubicin treatment alone was associated with a decrease in the total protein palmitoylation level after 3 days. Notably, 2-BP and doxorubicin did not act synergistically in this regard (Supplementary Figure 4).

Cell culture

Human vascular smooth muscle cells (VSMCs) or human fibroblasts were purchased from ATCC (Manassas, VA, USA) (normal diploid cells derived from young males, at least from three different donors). VSMC were cultured in vascular cell basal medium (ATCC) supplemented as defined by the manufacturer and kept in a humidified atmosphere (37° C and 5% CO₂ in the air). Cells were seeded at a density of 3000/cm² and passaged every 3-4 days. To induce senescence, 24 h after seeding, the cells were treated with 1 μM doxorubicin for 2 h. After doxorubicin treatment, the cells were washed with warm PBS and cultured in fresh medium for 3 or 7 days. For experiments with 2-bromopalmitate, cells were treated for 24 h with 10, 25 or 50 μM 2-BP. 2-BP stock solutions, at concentrations of 10, 25 and 50 mM, were prepared in EtOH immediately before use and diluted to working solutions in the culture medium. Depending on the experimental variant, the cells were incubated with 2-BP before (pretreatment) or after (posttreatment) doxorubicin treatment. The 2-BP-treated cells were washed with warm PBS and cultured in fresh culture medium.

Monitoring of proliferation

We monitored the proliferation of unstained VSMCs or fibroblasts in different experimental groups using the IncuCyte SX1 Live-Cell Analysis System (Sartorius, Göttingen, Germany). The system allows bright field and fluorescence imaging of live cells in culture plates inside a standard incubator (37° C, 5% CO₂ and 100% humidity). The system is equipped with algorithms allowing the stain-free analysis of cell proliferation by automatic cell counting. BF images of a 96-well plate were collected every 2 h. Integrated IncuCyte software was used to analyze the results.

SA-β-gal staining and analysis

The detection of SA-β-gal activity was performed according to Dimri et al. [24]. Briefly, cells were fixed with 2% formaldehyde and 0.2% glutaraldehyde in PBS, washed, and exposed overnight at 37° C to a solution containing 1 mg/ml 5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside, 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, 150 mM NaCl, 2 mM MgCl2, and 0.02 M phosphate buffer, pH 6.0. All of the agents used were purchased from Sigma Aldrich (St. Louis, MO, USA). For imaging, the cells were embedded in 4 μl of mounting medium supplemented with DAPI (Abcam, Cambridge, UK) and mounted on glass slides. Images were taken at least 10 min after embedding the cells in the medium with transmitted light or fluorescence (excitation 340-380 nm, emission 435-485 nm), using a Nikon Eclipse Ti-U fluorescence microscope and Nikon Digital Sight DS-U3 camera (Nikon, Tokyo, Japan) with a 20x objective at a resolution of 2560 x 1920 pixels and exposure set at 15 ms (BF images) with 30% of the maximum lamp intensity. For SA-β-gal staining, we analyzed at least 100 cells from 3-5 independent biological replicates using a previously described automatic method [25]. Briefly, BF (SA-β-gal) and fluorescent (DAPI) images were analyzed using a Fiji-based platform developed and validated by us, which automatically selects areas of the image with blue, green, and red color intensities above the experimentally determined color threshold (Hue = 117-185; Saturation = 80-255; Brightness = 0-255). The area and intensity of the SA-β-gal signal were used to calculate the integrated density.

BrdU incorporation and staining

The cells were treated with 10 μM BrdU for at least 18 h of culture then washed with PBS and fixed in ice-cold 70% ethanol. Fixed cells were washed with 0.5% Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA) in PBS, incubated in 2 N HCl for 30 min, washed twice with PBS, incubated for 1 min in 0.1 M borax solution (Sigma-Aldrich), washed twice in PBS again and incubated with primary anti-BrdU antibody (Becton Dickinson, Franklin Lakes, NJ, USA) diluted 1:120 in 1% BSA 0.5% Tween-20 in PBS for 1 h. After incubation, the cells were washed twice with 0.5% Tween-20 in PBS and incubated with a secondary antibody conjugated to a fluorochrome (Thermo Fisher Scientific, Waltham, MA, USA). The stained cells were mounted on a microscope slide with mounting medium supplemented with DAPI (Abcam) and imaged under an Eclipse fluorescence microscope (Nikon, Tokyo, Japan). The number of BrdU-positive cells and the total cell number (based on DAPI staining) were calculated using ImageJ (National Institutes of Health, Bethesda, MD, USA).

Immunostaining of γH2AX and 53BP1

The cells were washed with PBS, fixed in 4% paraformaldehyde, permeabilized in 0.3% Triton X-100 in PBS then blocked in PBS supplemented with 2% BSA, 1.5% normal goat serum and 0.1% Triton X-100 for 1 hour. Permeabilized cells were incubated with the following primary antibodies: anti-53BP1 (NB100-304, Novus, USA), anti-γH2AX (ab26350, Abcam) diluted in blocking solution for 2 hours. The sections were then washed with PBS supplemented with 0.1% Triton X-100 and incubated with Alexa Fluor 488-conjugated anti-rabbit, Alexa Fluor 555-conjugated anti-mouse or Alexa Fluor 594-conjugated anti-guinea pig secondary antibody in blocking solution for 1 h. The cells were mounted on a microscope slide with mounting medium supplemented with DAPI (Abcam). Images of cells stained for γH2AX and 53BP1 were obtained with an Eclipse fluorescence microscope (Nikon, Tokyo, Japan). Images of cells stained for p62 were obtained using an Sp8 confocal (Leica, Wetzlar, Germany) microscope with an HPPL APO CS2 63x/1.4° oil objective, a laser-405 diode (Pico Quant, Berlin, Germany) and white light (NKT Photonics, Birkerød, Denmark). The interval of the optical section was 0.5 μm, and the pixel section was 0.180 μm. The γH2AX and 53BP1 foci were analyzed in each cell nucleus (based on DAPI staining). Analysis was performed using ImageJ (National Institutes of Health, Bethesda, MD, USA).

Acyl-biotinyl exchange assay

To analyze changes in protein palmitoylation levels, an acyl-biotinyl exchange (ABE) assay [26] was used. Cells were lysed and homogenized using a Dounce homogenizer in buffer containing 50 mM Tris HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 4% SDS and 1% Triton X-100. Proteins were reduced with 10 mM TCEP (tris(2-carboxyethyl)phosphine), and the samples were incubated for 16 h at 4° C with 50 mM N-ethylmaleimide (NEM) to block free thiol groups. Proteins were precipitated using ice-cold ethanol to remove unreacted NEM. The pellets were resuspended in the same buffer, and the samples were divided in half. Both halves were treated with 400 μM thiol-reactive biotinylation reagent HPDP-biotin (N-[6-(biotinamido)hexyl]-3’-(2’-pyridyldithio)propionamide). One half was treated with 1 M hydroxylamine to cleave thioester-linked palmitoyl moieties and expose newly formed thiols to HPDP-biotin. The other half was treated with Tris buffer (pH 7.5) as a control to nonspecific binding of HPDP-biotin. Biotinylated proteins were analyzed with Western blotting.

Western blotting

Cultured cells were lysed in reducing sample buffer containing 125 mM Tris–HCl (pH 6.8), 4% SDS, 20% glycerol, 100 mM DTT, and 0.2% bromophenol blue and denatured for 10 min at 95° C. The total protein concentration was estimated using a bicinchoninic acid (BCA) protein assay kit, and 20 μg of each sample was loaded on a gel. Protein lysates were separated by SDS-PAGE using 4-12% (w/v) Bis-Tris gels. The separated proteins were transferred to nitrocellulose membranes (Amersham GE Healthcare, UK) and blocked in 5% non-fat powdered milk in TBS containing 0.1% Tween-20 (TBST) for 1 h. The membranes were incubated overnight with primary antibodies against p-p53, p-ATM, HMGB1, LMB-1 and GAPDH, washed in TBST, incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (anti-mouse or anti-rabbit, Dako Denmark A/S) at a 1:2000 dilution in 5% milk, washed and visualized with enhanced chemiluminescence (ECL) (Thermo Fisher Scientific). Signals were detected using X-ray film or ChemiDoc (Bio-Rad, Hercules, CA, USA) and analyzed with ImageJ (National Institutes of Health, Bethesda, MD, USA).

Statistical analysis

Statistical significance was analyzed using one-way ANOVA or two-way ANOVA, as indicated in the figure legends. All statistical tests were performed using GraphPad Prism 9 (GraphPad, San Diego, CA, USA). The data are presented as the means +/− SEMs.