Ozone at low concentrations does not affect motility and proliferation of cancer cells in vitro.

Exposure to low ozone concentrations is used in medicine as an adjuvant/complementary treatment for a variety of diseases. The therapeutic potential of low ozone concentrations relies on their capability to increase the nuclear translocation of the Nuclear factor erythroid 2-related factor 2 (Nrf2), thus inducing the transcription of Antioxidant Response Elements (ARE)-driven genes and, through a cascade of events, a general cytoprotective response. However, based on the controversial role of Nrf2 in cancer initiation, progression and resistance to therapies, possible negative effects of ozone therapy may be hypothesised in oncological patients. With the aim to elucidate the possible changes in morphology, migration capability and proliferation of cancer cells following mild ozone exposure, we performed wound healing experiments in vitro on HeLa cells treated with low ozone concentrations currently used in the clinical practice. By combining a multimodal microscopy approach (light and fluorescence microscopy, scanning electron microscopy, atomic force microscopy) with morphometric analyses, we demonstrated that, under our experimental conditions, exposure to low ozone concentrations does not alter cytomorphology, motility and proliferation features, thus supporting the notion that ozone therapy should not positively affect tumour cell growth and metastasis.

Introduction

Oxygen-ozone (O2-O3) therapy is a modestly invasive practice used in medicine as an adjuvant/complementary treatment for a variety of diseases, and is based on the administration of low O3 concentrations (reviews in[1,2]). Although the biological mechanisms accounting for the therapeutic effects of O3 administration have only partially been elucidated, it is known that this highly oxidizing gas acts in a dose-dependent manner.[3,4] High O3 doses induce a severe oxidative stress resulting in tissue inflammation and damage through the activation of the nuclear factor kappa-light-chainenhancer of activated B cells (NF-kB); on the contrary, low doses initiate a hormetic response (i.e., “the beneficial effect of a low level exposure to an agent that is harmful at high levels”[5]) leading to a cytoprotective antioxidant response. In particular, the Nuclear factor erythroid 2-related factor 2 (Nrf2), that promotes the transcription of Antioxidant Response Elements (ARE) and the consequent production of antioxidant enzymes,[6] proved to be stimulated by the low O3 concentrations used for various therapeutic purposes. [7-13] Recently, conclusive evidence has been provided that low O3 concentration increases the nuclear translocation of Nrf2, thus inducing the Nrf2-mediated Keap1-dependent transcription of ARE-driven genes.[14]

Thus, robust clinical indication and growing scientific data support the efficacy of O3 therapy in various medical fields such as e.g., orthopedics,[15-17] gastroenterology,[18-20] pneumology,[21,22] dentistry. [23-25] However, doubts persist about potential negative side effects of O3 administration.

In particular, O3 therapy is frequently applied to oncological patients due to its efficacy in reducing some adverse side effects of the anti-cancer treatments.[26-30] Possible undesired consequences of O3 therapy in oncological patients may be hypothesized based on recent findings on the controversial role of Nrf2 in cancer initiation, progression and resistance to therapies. Hyperactivation of Nrf2 may promote tumorigenesis by multiple ways: by helping incipient tumour cells to overcome the oxidative stress, which represents a barrier against neoplastic transformation and cancer initiation; [31] by supporting aberrant cell proliferation through both the induction of a metabolic switch towards anabolic pathways[32] and the modulation of mRNA translation;[33] by promoting angiogenesis[34] and drug resistance[35] through its potent cytoprotective effect.

In fact, no negative effects have so far been reported in oncological patients following O3 therapy (recent reviews in[36,37]); however, to our knowledge, no experimental data are presently available on the possible changes in morphology, migration capability and proliferation of cancer cells following exposure to low O3 concentrations.

With the aim to elucidate this issue, we performed wound healing experiments in vitro[38] on HeLa cells exposed to the low O3 concentrations currently used in the clinical practice. A multimodal microscopy and morphometry approach was used to investigate the cytomorphological changes, proliferation and motility of this established cancer cell line that has previously been used as a suitable experimental model to study the effects of the exposure to low O3 concentrations at the cellular level.[14,39]

Materials and Methods

Cell culture, wound healing assay and ozone treatment

HeLa cells were grown in Dulbecco’s modified Eagle medium with 10% (v/v) foetal bovine serum (FBS), 1% (w/v) glutamine, 0.5% (v/v) amphotericin B, 100 units/mL of penicillin and 100 μg/mL of streptomycin (Gibco), at 37°C in a 5% CO2 humidified atmosphere. Cells were trypsinized (0.25% trypsin in PBS containing 0.05% EDTA) when subconfluent, and then seeded on glass coverslips in 6-multi-well microplates, for atomic force microscopy (AFM) and scanning electron microscopy (SEM), or in 24-multiwell microplates (for wound healing assay and fluorescence microscopy). For wound healing assay, 5x105 cells per well were seeded on 24x24 mm slides and, after 48 h, when the cells were confluent, the cell monolayers were scratched with a sterile 200 μL pipette tips and immediately exposed to O2-O3 gas mixtures at two different O3 concentrations (10 and 35 μg O3/mL O2) according to Costanzo et al.[40] A single treatment was performed since, in the clinical practice, the O3 concentrations used for our experiments are administered once or twice a week; however, HeLa cell are rapidly dividing (their cell cycle lasts about 20 h), thus making it impossible to submit the same cells to repeated treatments without causing excessive oxidative stress. The gas was produced by an OZO2 FUTURA apparatus (Alnitec s.r.l., Cremosano, CR, Italy) which generates O3 from medical-grade O2, and allows photometric realtime control of gas flow rate and O3 concentration. Cells exposed to air under the same experimental conditions served as control. To evaluate cell migration during wound healing, images at 4x magnification were taken at 0 h, 2 h, 6 h and 24 h post-treatment using an inverted microscope (Leica DMIL) equipped with an Optika Microscopes (Ponteranica, BG, Italy) camera: the cell-free area was measured in a total of 12 randomly selected microscope fields per sample (4 fields in 3 independent experiments). The progressive reduction of the cell-free area was expressed as percentage, taking as 100 % the value at time 0.

Actin staining, and evaluation of the S-phase and mitotic cell fraction

To visualize actin microfilaments, 24 h after scratching the cell monolayers the slides were fixed with 4% (v/v) formaldehyde in PBS (30 min at room temperature, RT) and 70% (v/v) ethanol in water (30 min at -20°C); after rehydration with PBS for 5 min at RT, the slides were incubated with Alexa 488-conjugated phalloidin (Molecular Probes, Invitrogen, Monza, Italy) diluted 1:40 in PBS for 1 h at RT, stained for DNA with Hoechst 33342 (0.1 μg/mL in PBS for 10 min), rinsed in PBS, and finally mounted in 1:1 PBS:glycerol.

The percentage of S-phase cells was also assessed 24 h after scratching the cell monolayers: the cells were pulse-labelled with 20 μM Bromodeoxyuridine (BrdU, Sigma-Aldrich, St. Louis, MO, USA) for 30 min at 37°C, then fixed with 70% ethanol and incubated for 20 min at RT in 2 N HCl, to denature DNA partially; after neutralization with 0.1 M sodium tetraborate (pH 8.2) for 3 min, samples were washed in PBS, permeabilized for 15 min in PBS containing 0.1 % bovine serum albumin and 0.05 % Tween-20, and incubated for 1 h with a mouse monoclonal antibody recognizing BrdU (BD, Franklin Lakes, NJ, USA) diluted 1:20 in PBS. After two washes with PBS, samples were incubated for 1 h with an Alexafluor 488-conjugated anti-mouse secondary antibody (Molecular Probes, Invitrogen, Milan), diluted 1:200. The cell samples were washed with PBS, stained for DNA with 0.1 μg/mL Hoechst 33342 in PBS for 10 min, and finally mounted in PBS:glycerol (1:1). The percentage of BrdU-positive cells was evaluated in the region located within 100 μm of the wound edge in 30 randomly-selected fields (40x) per experimental condition. The same microscopic fields were used to evaluate the mitotic index. For observation of all samples, we used an Olympus BX51 microscope equipped with a 100W mercury lamp, under the following conditions: 450-480 nm excitation filter (excf), 500 nm dichroic mirror (dm), and 515 nm barrier filter (bf) for Alexa 488; 330-385 nm excf, 400 nm dm, and 420 nm bf, for Hoechst 33342. Images were recorded with a QICAM Fast 1394 Digital Camera (QImaging, Surrey, BC, Canada) and processed with Image-Pro Plus software (Media Cybernetics, Inc., Rockville, MD, USA).

The mean ± standard error (SE) values of the analysed variables were calculated for each experimental condition, and statistical comparisons were performed by the one-way Anova test (statistical significance was set at p≤0.05).

Scanning electron microscopy

For SEM analysis, 7x104 cells per well were seeded on slides of 12 mm in diameter. After 2 h and 24 h from the scratching, the cells were fixed in 4% paraformaldehyde in PBS for 2 h at 4°C, washed in the same buffer, post-fixed with 1% OsO4 at 4°C for 1 h, and dehydrated with acetone (Sigma-Aldrich). The samples were then treated by critical point dryer (CPD 030, Balzers), mounted on metallic specimen stubs and sputter-coated with gold (MED 010 Balzers). SEM imaging was performed by an XL30 ESEM (FEI-Philips). SEM images were used to perform quantitative evaluation of cell size and roughness. By using ImageJ software (NIH), the surface area of 50 cells per sample was measured; moreover, the cell surface facing the wound edge was measured both including and excluding cell protrusions in 10 cells per sample, the ratio between the two values was then calculated in order to obtain an index of cell surface irregularity (the higher the value the rougher the cell). Statistical evaluation of the results was performed by the one-way Anova.

Atomic force microscopy

For AFM analysis, 7x104 cells per well were seeded on slides of 12 mm in diameter. For topographic images at AFM, 2 h and 24 h post-treatment the cells were fixed in 2% glutaraldehyde in phosphate buffer for 20 min, washed in the same buffer and deposited onto 20 mm mica discs (Ted Pella Inc., Redding, CA, USA), after having dried the surfaces of the slides. The mica discs were mounted on suited metallic specimen discs in order to be analysed with a NT-MDT Solver-pro equipped with a 60x60-μm scanner and gold-coated NSG-01 silicon probes, from the same company, with a curvature radius of 10 nm. To evaluate the cell thickness, the height profile was measured in 5 points (2 in the peripheral and 3 in the central region of the cell) along a line (n=25 for each experimental condition). Height values were pooled according to the different experimental conditions, the mean ± SE values were calculated and statistical comparison performed by the one-way Anova.

Results and Discussion

Cell movement plays a basic role in cell and tissue homeostasis as well as in many physiological and pathophysiological processes.[41] Altered regulation of cell motility is involved in several disorders, and migration of cancer cells is considered as a prerequisite for tumour metastasis.[42] Under our experimental conditions, the wound healing assay (Figure 1a) showed that the migration rates in control and O3-treated HeLa cells were similar at each time point considered (2 h, 6 h and 24 h) after gas exposure (Figure 1b).

Figure 1.

a) Inverted microscope images and (b) mean ± SE values of cell-free areas of control (CTRL) and O3-treated HeLa cells at different steps of the wound healing assays. Scale bars: 100 μm.

In eukaryotic cells, reorganization of the cytoskeletal actin is the main responsible for cell shape modification and movement by driving the protrusions that push the membrane forward and lead to pseudopod extension.[43] After fluorescent phalloidin labelling, no evident changes in actin organization were observed in migrating cells at the wound edge after O3 exposure compared to the controls (Figure 2 a-c). This is consistent with the observations at SEM that demonstrated an unchanged morphological pattern in control and O3-treated cells (Figure 3 a-c): in all samples, HeLa cells were flattened and irregularly polygonal in shape (apart from the sphereshaped mitotic cells), and showed numerous filopodia and lamellipodia. The mean cell areas measured in SEM images were similar in the three experimental conditions at both 2 h and 24 h post-treatment (Figure 3d). Also the cell thickness at the peripheral and central region, as evaluated by AFM, was similar in control and O3-treated cells (with the only exception of the cells treated with O3 35 μg/mL after 2 h from gas exposure, that were transiently thicker than the controls in their central region) (Figure 4). Thus, O3 treatment did not affect the volume of the cells along the wound edge, and quantitative evaluation of their surface roughness confirmed that also the surface protrusions did not significantly change (Figure 3e).

Figure 2.

Fluorescence microscopy images of control (a), O3 10 μg/mL (b) and O3 35 μg/mL (c) treated HeLa cells labelled for actin filaments (green). Control (d), O3 10 μg/mL (e) and O3 35 μg/mL (f ) treated HeLa cells immunolabelled for BrdU (green). DNA was counterstained with Hoechst 33342 (blue). Scale bars: 20 μm.

Figure 3.

SEM images of control (a) O3 10 μg/mL (b) and O3 35 μg/mL (c) treated HeLa cells 24 h post wounding; insets: high magnification images of the dashed-line boxed areas; cell in interphase are flat with protruding microvilli, while mitotic cells (arrows) detach from the growth surface and become spherical in shape; scale bars: 25 μm; inset bars: 5 μm. Mean ± SE values of (d) cell area and (e) cell irregularity index at 2 h and 24 h from treatment; no statistical difference was found for both the cell area (2 h, P=0.605; 24 h, P=0.687) and irregularity index (2 h, P=0.850; 24 h, P=0.673).

Figure 4.

Representative AFM images of control (a) O3 10 μg/mL (b) and O3 35 μg/mL (c) treated HeLa cells 2 h post treatment. d,e) Mean ± SE values of cell height measured at AFM in control (CTRL) and in O3-treated cells. The asterisk indicates statistical significance (P=0.002), whereas no significant difference was found among samples for the peripheral region both after 2 h and 24 h (P=0.241 and P=0.522, respectively), and for the central region after 24 h (P=0.565).

O3 is a highly oxidizing gas and reactive oxygen species (ROS) are known to regulate cytoplasmic protrusions by controlling actin dynamics in a dose-dependent manner, high levels inducing protein depolymerization while low levels promoting polymerization. [44-46] Previous experiments[39] demonstrated that exposure to low O3 concentrations causes a minimal ROS production, unable to induce structural or functional alterations in cell organelles including mitochondria. However, the O3 effect on the polymerization of cytoskeletal proteins may depend on small local changes in ROS amounts. It could be therefore hypothesized that the higher oxidative stress caused by exposure to O3 35 μg/mL may induce a transient cytoskeletal remodelling that undergoes rapid restoration without affecting the cell periphery that is mainly involved in cell movement.

Recent findings[47] suggest that cell motility is affected also by Nrf2; in fact, its repressor factor Keap1 is involved in the disassembly of podosomes, known to promote cell motility and interactions; [48] however, Keap1 is unable to play its role when tethered to Nrf2. It is worth recalling that low O3 concentrations induce an antioxidant response by the Keap1/Nrf2 dependent pathway,[14] thus releasing free Keap1 molecules in the cytoplasm. This could explain why Nrf2 was found to play an antitumour role by reducing cell migration.[49-52]

Tumour invasion not only depends on the cell migration ability, but also on the cell proliferation rate. In previous studies, we demonstrated that exposure to low O3 concentrations does not change both death rate[14,39] and proliferation rate of subconfluent HeLa cells[39] as well as of other cancer cell lines.[53] The present study shows that mild exposure to O3 does not alter the percentage of BrdU-positive cells (26.92±1.47%, n=1915 in controls; 27.59±1.32%, n=1928 in cells treated with O3 10 μg/mL 3; 25.81±1.36%, n=1871 in cells treated with O3 35 μg/mL ; P=0.690) or the mitotic index (5.13±1.17% in controls, 5.81±1.05% in cells treated with O3 10 μg/mL, and 4.62±1.23% in cells treated with O3 35 μg/mL ; P=0.657) along the wound edge (Figure 2 d-f). These findings provide evidence that the exposure to low O3 concentrations leaves unchanged the in vitro cell proliferation not only in sub-confluent populations but also during wound healing, i.e., in a growth-stimulating condition.

In conclusion, the low O3 concentrations used under in vitro conditions in our study proved not to alter cytomorphology, migration features or cell proliferation of HeLa cells during wound healing, thus supporting the notion that O3 therapy should not positively affect tumour cell growth and metastasis. Especially due to the multiple indirect effects of O3 in a living organism, the in vivo conditions are obviously much more complex than those of an in vitro model; thus, further studies are needed to clarify the consequences of the exposure to low O3 concentrations in tumour-bearing organisms, with particular reference to the possible interference with chemotherapeutics.[36]