Cell type-dependent modulation of senescence features using Weo electrolyzed water

Weo electrolyzed water (WEW) reduces cellular senescence in normal fibroblasts and cancer cells

To investigate the effects of WEW at different charges and time courses on normal human BJ fibroblasts and human T47D breast cancer cells that were subjected to oxidative stress, we first assessed the expression of some senescence-associated biomarkers. As expected, exposure to H₂O₂ in both non WEW-treated proliferating cell types led to a change in cell morphology, enhanced senescence-associated-β-galactosidase staining (SA-β-gal), decreased rate of DNA synthesis, inhibition of cell proliferation, and over-expression of the cell cycle inhibitor p21, when compared to cells that were not induced to senesce.

Figure 1A shows representative phase contrast microscope images of BJ and T47D cells confirming an enlarged and flattened cell morphology characteristic of cellular senescence. H₂O₂-induced cells in non-electrolyzed water were used as controls for both cell types in this study. When comparing WEW-treated and tap water-treated H₂O₂-induced senescent BJ and T47D cells, microscopic analysis of SA–β–gal staining revealed a decreased SA-β-gal activity in both cell types (Figure 1B, 1C). Moreover, immunofluorescence analysis of the incorporation of 5-ethynyl-2’-deoxyuridine (EdU) showed that a low percentage of BJ and T47D cells incorporated EdU after senescence induction (Figure 2A, 2B).

Elevated p21, one of the most well-established senescence markers, and reduced Ki67, a cell proliferation marker, are associated with the induction of senescence. To analyze the expression of both markers, cDNAs were generated from RNA extracted from H₂O₂-treated BJ and T47D cells, further treated or not with WEW, and RT-PCR was performed. Our results revealed that WEW significantly downregulated p21 gene expression in both cell types (Figure 3A), particularly in normal fibroblasts, which was confirmed at the protein level (Figure 3B). However, even though WEW increased the expression of Ki67 in normal fibroblasts, WEW didn’t have any significant effect on its expression in cancer cells as shown in Figure 3C.

WEW modulates the expression of a SASP factor in a cell type-dependent fashion

A common characteristic of cellular senescence is the upregulation of the senescence-associated secretory phenotype (SASP), which includes interleukins, chemokines, and extracellular matrix components [17]. Depending on the cell type investigated, some SASP factors will be more highly expressed than others. We examined the expression of CXCL-1, a chemokine that plays an important role in regulation of immune and inflammatory responses [18]. We assessed if WEW could modulate the expression of this SASP factor after oxidative stress induction, and we observed a substantial difference in the cell-specific regulation of this gene (Figure 4). The expression of CXCL-1 in senescence-induced BJ cells treated with WEW was significantly downregulated compared with tap water-treated control cells. However, for the T47D cancer cells, even though WEW did not increase senescence per se, a significant upregulation of the SASP factor CXCL-1 was observed.

WEW modulates the expression of stress response genes in a cell type-dependent manner

We previously obtained some RNA-Seq results to screen for genes potentially regulated by WEW, and we determined that several stress response genes, such as GDF-15, DDIT3 and TRIB3, were significantly modulated (data not shown). GDF-15 has recently been described as an aging marker, and the expression of DDIT3 and TRIB3 genes is similarly modulated during stress response [19–23]. The expression of these stress response genes in BJ fibroblasts was downregulated in cells induced to senescence/stress with H₂O₂ and then consequently treated with WEW, compared with tap water-treated cells (Figure 5). On the other hand, an upregulation of the same genes was observed in stress-induced breast cancer T47D cells treated with WEW. These results suggest that WEW triggers gene modulation in stressed cells, which is cell strain-specific.

Weo electrolyzed water preparation

Weo Electrolyzed Water (WEW) was produced using boron doped diamond-based water electrolysis technology (Weo LLC, Miami, FL, USA). The technology consists of two silicone electrodes with 2-3 microns of diamond layered on a single side. The electrodes are enclosed in an electrolytic cell where the diamond sides of the electrodes face one another, separated by 1 mm of distance. The other 5 sides of the electrode are sealed in thermoplastic housing and silicone rubber gaskets. The opposite side of the diamond side is electrically connected to a DC power source supplying 0-48VDC. Supplying a DC voltage induces a positive or negative polarity in the respective electrode, and the polarity is alternated every 30 seconds.

Unlike typical Hydrogen Water Bottles (HWB) in the marketplace, Weo’s technology does not separate the anodic and cathodic compartments with a Proton Exchange Membrane (PEM). Instead, both oxidative and reductive chemical species are present in the water during electrolysis. Two different sized electrode pairs were tested, Unit 1 having an active electrode area of 5.76 cm² x 2 (micro unit) and Unit 2 having an area of 12.5 cm² x 2 (mini unit). With all other conditions held constant (water conductivity, voltage, volume, duration), a larger electrode area can generate more chemical reactions, a higher charge, and different concentrations of oxygen and hydrogen species generating a different palette of bioactive species. A total of 500 ml of 37° C filtered tap water solution was prepared just before use for all experiments, and water was circulated through the electrolytic cell at a flow rate of 0.5LPM. During the experiments the current was kept between 4-8A by adjusting the voltage.

To prepare the WEW based media, DMEM or RPMI-1640 powdered media (GIBCO) and sodium bicarbonate (NaHCO₃; MediaTech) were weighed according to manufacturer’s instructions and added to 100 ml of electrolyzed water. WEW sample’s pH was adjusted to 7.0-7.4 by adding 1N HCl (Fisher) and filtered through a 0.2 μm membrane filter. Electrolyzed water samples were used immediately after preparation.

Cells

Normal human BJ fibroblasts and human T47D breast cancer cells were obtained from ATCC. Fibroblasts were cultured in Dulbecco’s Modified Eagle Medium (GIBCO) with 10% heat inactivated fetal bovine serum (FBS) (GIBCO) in the absence of any added antibiotic. Cancer cells were cultured in RPMI-1640 medium (GIBCO) with 10% FBS in the absence of any added antibiotic as well. Both cell types were maintained in T75 flasks (CytoOne) at 37° C in a humidified incubator with 5% CO₂. To induce senescence by oxidative stress, cells were harvested when ~80% confluent using 0.25% Trypsin-EDTA (GIBCO). Following resuspension in their respective media, 1 ml of the cell suspension was counted and measured using a Scepter 3.0 cell counter (Millipore SIGMA).

BJ cells were seeded at 1x10⁵ cells per well on 6-well plates or at 1x10⁴ cells per well on 24-well plates, and T47D cells were seeded at 0.5x10⁵ cells per well on 6-well plates or at 0.5x10⁴ cells per well on 24-well plates. After 24h incubation, DMEM was replaced with WEW (or tap water) and fibroblasts were induced to senescence with 25 μM H₂O₂ for 2h, medium was replaced with WEW (or tap water) without hydrogen peroxide and cells incubated for 48h; an induction for 2h was repeated and assays were performed after 48h. After replacing RPMI-1640 and treatment with freshly made WEW media (or tap water) for 3d, cancer cells were induced to senescence with 100 μM H₂O₂ and assays were performed after three days of induction.

SA-β-Gal staining

Fibroblasts and cancer cells were seeded on 6-well plates, treated with WEW (or tap water) and induced to senescence with H₂O₂ as described above. SA-β-gal activity was determined using the senescence β-galactosidase staining kit (Cell Signaling Technology) according to manufacturer’s instructions. Cells were incubated at 37° C without CO₂ and monitored using a phase contrast light microscope (Leica) until the staining became visible. A minimum of 500 cells were counted to determine the percentage of SA-β-gal-positive cells.

EdU incorporation

Cells were treated with WEW (or tap water) and induced to senescence on 24-well plates containing coverslips as described above. Cells were then washed twice with PBS (GIBCO), fixed with 3.7% formaldehyde (VWR), and permeabilized with 0.5% Triton X-100 (Millipore). To assess the rate of DNA synthesis, the Click-iT chemistry was performed by reacting EdU with Alexa Fluor 488 (ThermoFisher Scientific) for 30 min at dark. Coverslips were washed twice in PBS prior to mounting them with ProLong™ Gold Antifade Mountant with DAPI (Molecular Probes). Images were obtained using a phase contrast fluorescence microscope (Leica).

Quantitative real time polymerase chain reaction (RT-PCR)

Both cell types were seeded, treated with WEW or tap water, and induced to senescence in triplicates onto 6-well plates prior to trypsinization with 0.25% Trypsin-EDTA (GIBCO) and centrifugation at 300 g for 5 min. Cell pellets were suspended in cell lysis buffer (RLT Plus buffer; QIAGEN) containing 10:1000 β-mercaptoethanol and homogenized by vortexing. Total RNA was isolated using the RNeasy Plus Mini kit (QIAGEN) according to the manufacturer’s protocol. RNA concentrations from each sample were quantified using the NanoDrop One^C spectrophotometer, and samples were normalized to ~1 μg prior to reverse transcription. cDNA was synthesized using the qScript Ultra SuperMix (Quantabio). Quantitative reverse transcription reactions were done in triplicate using FastSYBR Mixture - Low ROX (CWBIO) and analyzed using the QuantStudio 3 sequence detector. Primer sets (Integrated DNA Technologies) for human genes used are listed in Table 1. Fold changes were calculated by the delta-delta Ct method (fold = 2ddCt). Samples were normalized to the cycle threshold value obtained during exponential amplification of actin.

Western blotting

Cells were seeded, treated with WEW or tap water, and induced to senescence in 6-well plates. Whole cell lysates from each triplicate sample were prepared by washing in cold buffered 1X PBS (GIBCO) and then lysed in RIPA buffer (Invitrogen) containing 1:100 protease inhibitors. After centrifugation (12,000 rpm, 15 min) at 4° C, protein supernatants were transferred into new tubes. Protein concentrations of the samples were determined with a bicinchoninic acid protein assay (Pierce). Lysates were normalized to 25 μg and then heated for 5 min (95° C) in Tris-Glycine SDS sample buffer and sample reducing agent (Invitrogen) prior to loading onto XCell4 SureLock™ Midi-Cell 10% SDS polyacrylamide gel electrophoresis system (Invitrogen) and blotted onto nitrocellulose using a Trans-Blot Cell Blotter (BioRad).

Membranes were then blocked with 5% non-fat milk in TBST (20 mM Tris, 150 mM NaCl, 0.05% Tween-20) for 30 min and incubated at 4° C overnight with primary antibodies p21 and α-Tubulin (both from Cell Signaling) at a 1:1000 dilution. Subsequently, the membranes were washed with TBST three times for 5 min and incubated with secondary antibodies (HRP-anti-rabbit or HRP-anti-mouse, both from Cell Signaling) at 1:2000 dilution for 1h at room temperature. Bound antibody was visualized by incubation with ECL solution (Pierce) for 1 min at room temperature and then imaged using the Azure 280 Western Blot Imaging System (Azure Biosystems). Protein bands were analyzed and quantified using the ImageJ software.

Statistical analysis

Control and treated groups were compared using the analysis of variance (ANOVA) test. Results are presented as the mean value ± SEM of three independent experiments performed in triplicates. Statistical significance was determined by an unpaired two-tailed Student’s t-test in which a P < 0.05 was considered to indicate statistical significance. Data was processed using Microsoft Excel software.