Introduction

Nowadays, laser technology is widely used for cancer treatment. In tumor treatment, lasers could be utilized independently and in combination with chemicals. In particular, surface carcinoma treatment based on solitary laser operation in many cases employs thermal effect resulted from interaction of laser radiation with biological tissues causing their ablation. Such therapy, mainly used for basalioma, has serious drawbacks associated with necrotic lesions of biological tissues. Photodynamic therapy (PDT) is a more promising and sparing approach that employs laser interaction with photoactive chemicals—photosensitizers to generate singlet oxygen, superoxide anion radical, hydrogen peroxide, and other reactive oxygen species (ROS). PDT driven ROS can damage cellular components directly or via induction of free-radical chain reactions. Overproducing of ROS during PDT induces cell death via necrosis, apoptosis, or autophagy [1]. A widespread use of PDT is due to its advantages over ablative therapy: low invasiveness, high selectivity, relatively low toxicity of photosensitizers (PSs), short duration treatments, possible refresher courses of treatment with rare complications [1]. However, PSs are absorbed by both tumor cells and healthy tissues causing general photosensitization of organism and photodermatitis. Also, for a number of PDT applications, the PSs are required to achieve therapeutic effect, although producing undesirable toxic effect, for example, for removing skin benign tumors and treatment of skin diseases. Besides, high cost of PS administration and complications stimulate searching for efficient PDT without using PSs [1, 2].

To date, numerous reliable data have been reported highlighting that the laser light at the wavelength of 1262–1268 nm coincides with absorption band of oxygen molecule transforming it into singlet oxygen. It was considered that direct 3O2 → 1O2 transition is prohibited according to spin-orbital selection rules. However, recent experimental data allow new conception of direct 3O2 → 1O2 transition in solvents. Now, it is assumed that in solvents, O2 → O2 bi-molecular collisions mix electron orbital states by an intermolecular exchange interaction. So now, direct generation of singlet oxygen in solvents from triplet oxygen state seems to be theoretically explained [3].

Singlet oxygen is considered to trigger a cascade of reactions through different types of peroxide formation leading to further ROS generation in a cell and, consequently, to its death through apoptosis, necrosis, or autophagy [2, 4, 5]. Although singlet oxygen is a highly active compound, there is no clear evidence of its direct ability to induce intracellular oxidative stress [5]. Damaging effect of singlet oxygen is in strong dependence on the place of its generation [6]. Singlet oxygen is not able to displace over a distance longer 0.02 μm that is comparable with the size of a protein molecule [7]. Unlike other reactive oxygen species, singlet oxygen cannot initiate radical chain reactions since it has no extra unpaired electron. Thus, it could only chemically modify lipids, DNA, and protein molecules. One should note that the cells contain a large enough amount of endogenous compounds that are able to exhibit PS properties. Such compounds are mainly endogenous porphyrins represented by cytochrome molecules. Porphyrins exhibit absorption bands in the range of 400–900 nm and in the infrared (IR) spectrum of 1000–1550 nm [8–10].

Most porphyrins in a cell are a part of proteins localized in mitochondria. Mitochondria are main source of intracellular reactive oxygen species and can cause the intracellular oxidative stress [11]. The oxygen pressure in mitochondrion is about 30–40 mmHg [12], while in the cytoplasm, it is about 3–7 mmHg only [13]. This makes mitochondrial components very sensitive to singlet oxygen inducers.

According to these data, one can assume that besides a direct damaging effect from singlet oxygen, there could be other mechanisms inducing cell death after laser irradiation at the wavelength of 1262–1268 nm. Mitochondria are the most likely triggers of damaging effect induced by laser irradiation at the wavelength of 1262–1268 nm. In our opinion, mitochondria could be damaged either via laser interaction with endogenous porphyrins or due to overproduction of singlet oxygen in the mitochondrial membrane. Here, we report experiments that allow to estimate mitochondria contribution to oxidative stress induced by laser irradiation at 1265 nm. We study dynamics of oxidative stress, change of mitochondrial potential, cell viability, DNA damages, and reduced glutathione (GSH) in the culture of tumor and normal cells exposed to 1265 nm laser irradiation.

Materials and methods

1265 laser parameters

Raman conversion to 1264–1270 nm of the radiation emitted by double-clad Yb-doped fiber laser is among the most efficient methods of laser generation in this spectrum range. Raman laser is a coherent light source based on amplification due to Raman effect (stimulated Raman scattering, scattering of high power optical radiation on molecular vibrations in medium) but not due to stimulated emission from excited atoms or ions [14, 15]. The laser generation can be obtained at any wavelengths within IR range (1–2.1 μm). The use of phosphate-silicate fibers as a Raman converter allows significant simplification of the laser system due to high Raman shift in these fibers (1330 cm−1) in comparison with the standard silica fibers demonstrating Raman shift lower than 440 cm−1 [5].

The Raman laser (convertor) used in this study is pumped by the ytterbium fiber laser operating at 1087 nm. The Ytterbium laser, in its turn, is pumped by a set of semiconductor laser diodes operating at λ = 978 with the total power of about 12 W. The average output power of the ytterbium laser is about 10 W.

A large difference between the refractive indices of the inner and outer claddings (of about 0.015) ensures pumping efficiency over 95 % for the ytterbium fiber laser pumped through a standard conic shape taper. Concentration of ytterbium ions in the active fiber core is 7 × 1019 cm−3, while the absorption coefficient of the pump is not higher than 1.5 dB/m.

The Raman converter cavity based on the phosphate fiber is formed by a pair of standard fiber Bragg gratings spliced with the fiber ends. Fiber Bragg grating [14, 15] is a short segment of optical fiber (waveguide) comprising a structure with periodically varying refractive index. Areas with the modified refractive index are usually perpendicular to the fiber axis. The first fiber Bragg grating is with the peak reflectivity higher than 99 % and the second of 30 %, both inscribed in a standard Flexcore-1060 fiber. Concentration of P2O5 is 13 % per mole, the refractive index difference of core/inner cladding Δn is about 0.01. The optical losses at pump and operation wavelengths are about 2 dB/km and less than 1 dB/km, respectively. The pump absorption in the converter is better than 20 dB. The maximal power of the Raman laser operating at 1265 ± 3 nm is about 4 W. The total efficiency of the laser system is above 33 % [5].

In general, the radiation power on the tissue surface (with size r) can be defined as

$$ P={\displaystyle \underset{0}{\overset{r}{\int }}I(r)dS=2\pi {\displaystyle \underset{0}{\overset{r}{\int }}I(r)rdr}}. $$
(1)

Intensity of the used laser source could be assumed axially symmetric (and independent of the angle) with Gaussian radial distribution. So, the intensity distribution of laser source is described by the expression

$$ I={I}_0 \exp \left(-{r}^2/{r}_0^2\right), $$
(2)

where I 0 = P/πr 20 is the maximal laser beam intensity and P is the laser source average power. The parameter r 0 is determined by the laser source parameters and a distance between the light source and tissue.

If (2) is fulfilled and coefficient of reflection from the irradiated tissue is negligible, the power absorbed by tissue is

$$ {P}_r=P\left(1- exp\left(-{r}^2/{r}_0^2\right)\right). $$
(3)

Assuming that the size of irradiated area is r ≥ r 0, the power absorbed by tissue is

$$ {P}_a=P\left(1- \exp \left(-{a}^2/{r}_0^2\right)\right). $$
(4)

If the size of irradiated area is r > r 0, as in our case, the energy absorbed by tissue is described as W = Pt, where P is the power of a continuous radiation source and t is the exposure time.

Accordingly, the surface dose of laser radiation absorbed by a biological tissue (E, J/cm2) can be calculated from the ratio:

$$ E=Pt/S, $$

where P is the average output power (W), t is the exposure time (sec), and S is the laser spot area on biological tissue (cm2).

Chemicals and cell cultures

Tetramethylrhodamine ethyl ester perchlorate (TMRE), 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA), acridine orange, ethidium bromide, monochlorobimane, rotenone, gentamycin, phenol-free Dulbecco’s Modified Eagle’s Medium/Nutrient F-12 Ham (DMEM/F12) medium, NaCl, and other salts and reagents have been purchased from Sigma-Aldrich. All chemicals have purity grade >99 % or higher unless otherwise stated. DMEM/F12 medium and fetal bovine serum have been obtained from Paneco Ltd (Russia, Moscow) and РАА Laboratories (Austria), respectively. Cell cultures have been purchased from American Type Culture Collection (USA).

Colorectal cancer HCT-116 cell line and ovarian epithelium CHO-K cell line are maintained in DMEM/F12 medium, supplemented with 10 % fetal bovine serum, gentamycin at 50 μg/ml in a fully humidified atmosphere containing 5 % carbon dioxide at 37 °C. Twenty-four hours before laser irradiation, the cells are trypsinized and made a new passage in 8-well slide chamber (SPL Lifesciences) at the concentration of 5 × 105 cells/ml in 0.5-ml phenol-free DMEM/F12 medium (to minimize heating effect during laser irradiation) supplemented with 10 % fetal bovine serum and gentamycin at 50 μg/ml.

Laser treatment of cell cultures

Cells are irradiated in an incubation medium by using stage top chamber of the microscope incubator UNO (OkoLab) maintaining a constant temperature in a humidified atmosphere 95 and 5 % CO2 for 5–30 min. The temperature in a stage top chamber is selected assuming heating of the cell culture plate under laser radiation. In our case, it is 30 °С. The temperature at the cell bottom controlled by a thermocouple is 37.5 ± 0.5 °C. The laser is mounted at the bottom side of the plate at a distance of 0.5 cm. During one irradiation session, two wells with cell culture are used in 8-well slide chamber. One well is exposed to irradiation while another, shielded from laser irradiation with a steel plate, is used as a control. For cell viability assay, the irradiation dose is 66.6, 200, and 400 J/cm2. The laser irradiation dose varies with irradiation time. For other experiments, the dose is 400 J/cm2. The dose of 400 J/cm2 allows a profound effect on cells and avoidance of cell bottom heating above 38 °C. At least three irradiation sessions have been performed for each time and irradiation dose for all experiments.

Fluorescent microscopy

Cell viability is evaluated 24 h after irradiation by staining cells with a mixture of fluorescent dyes such as acridine orange and ethidium bromide. Cells are washed twice with phosphate-buffered saline (PBS), pH = 7.5 and a mixture of acridine orange (3 μg/ml) and ethidium bromide (5 μg/ml) in PBS (pH = 7.5) is added to the well. Cells are immediately studied under Nikon Тi-S fluorescence microscope, dead (red and orange fluorescence) and live (green or yellow fluorescence) cells are counted [16].

Cellular ROS content is determined using DCFH-DA. One, 3, and 6 h after cell irradiation (400 J/cm2), cells are labeled by adding DCFH-DA (10 μM in ethanol) to the incubation medium with the subsequent incubation for 30 min at 37 °C in dark conditions. Then, cells are washed twice with PBS (pH = 7.5), resuspended in PBS and kept for 15 min at 4 °C. During all manipulations, cells are attached at the bottom of 8-well slide chamber. Cell fluorescence images are captured immediately within 5 min after incubation at 4 °C by using Nikon Тi-S microscope coupled with DS-Qi1MC camera, using Nikon S Plan Fluor ELWD 20 × 0.45 lens and filter 480/529, supported by NIS-elements 4.0 software [17]. More than 300 cells have been analyzed for DCFH-DA fluorescence.

To assay the mitochondrial potential, cells are irradiated at 400 J/cm2, and 1, 3, 6 h after irradiation, they are incubated in a growth medium with 50 nM TMRE for 20 min at 37 °C. During fluorescence assay, cells are kept in 8-well slide chamber placed in the stage top chamber of the microscope incubator UNO (OkoLab) at 37 °C in a humidified atmosphere and 5 % CO2 [18]. Images are captured using Nikon Тi-S microscope coupled with DS-Qi1MC camera, using Nikon S Plan Fluor ELWD 20 × 0.45 lens and filter 480/529, supported by NIS-elements 4.0 software. More than 300 cells are analyzed for TMRE fluorescence.

To study contribution of mitochondria to ROS generation, rotenone is added to the cells 20 min before adding DCFH-DA and TMRE at the final concentration of 50 nM [19].

To assay the reduced glutathione, cells are irradiated at 400 J/cm2, and 1, 3, 6 h after irradiation, they are incubated in a growth medium with 5 μM monochlorobimane at 37 ° C for 20 min [20]. During fluorescence assay, cells are kept in 8-well slide chamber placed in the stage top chamber of the microscope incubator UNO (OkoLab) at 37 °C in a humidified atmosphere and 5 % CO2. Images are captured using Nikon Тi-S microscope coupled with DS-Qi1MC camera, using Nikon S Plan Fluor ELWD 20 × 0.45 lens and filter 380/450, supported by NIS-elements 4.0 software.

Quantitative image analysis is performed using Image J software. The region is drawn around each cell to be measured, and the identical region is placed in an area without fluorescent objects to be used for background subtraction. Corrected total cell fluorescence (CTCF) = Integrated Density—(area of selected cell × mean fluorescence of background readings) [21].

DNA damage assay

The kinetics of rejoining of DNA strand breaks is quantified by Comet assay according to the international recommendations. Briefly ~4 × 104 cells are mixed with two volumes of low-melting agarose, immediately transferred onto microscopic slides and placed on ice. After the agarose has polymerized, slides are incubated in lysis solution (2.5 M NaCl, 0.1 M EDTA, 10 mM Tris–HCl, 1 % Triton X-100, pH 10 at 4 °C) for 60 min and in alkaline solution (300 mM NaOH, 1 mM EDTA, pH 13) for 30 min. After electrophoresis in alkaline solution at 0.75 V/cm and 300 mA for 20 min at 4 °C, slides are washed twice with 0.5 M Tris, pH 7.4, fixed in 96 % ethanol and stained with ethidium bromide. The tail moment (TM; percentage DNA in tail × distance between centers of head and tail) is calculated for ~100 cells per slide using comet scoring software (Comet Score) and expressed as the mean value [22].

Statistical analysis

Each test has been performed in triplicate and results have been expressed as mean ± SD. Differences between irradiated and control cells are regarded as statistically significant when p calculated by the two-sided Student t test is <0.05.

Results

Cell mortality after 1265 nm laser exposure

Figure 1 shows mortality of HCT-116 and CHO-K cells under laser irradiation 1265 nm. The diagram demonstrates that the laser irradiation at the wavelength of 1265 nm induces a dose-dependent increase of mortality of HCT-116 and CHO-K cells. However, no difference is registered in the sensitivity of HCT-116 cancer cells and CHO-K normal cells to laser radiation at the wavelength of 1265 nm.

Fig. 1
figure 1

HCT-116 and CHO-K cell mortality 24 h after laser irradiation. Results are presented as mean values ± S.D. Asterisk denotes statistically significant differences between control and other groups (p < 0.05)

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Effect of 1265 nm laser irradiation on intracellular ROS concentration

The level of intracellular ROS in HCT-116 and CHO-K rotenone treated cells exposed to 1265 laser is shown in Fig. 2. The irradiation dose in all experiments is 400 J/cm2. An increase of ROS concentration has been registered in HCT-116 cells 1 h after irradiation session. Further, during the experiment, ROS concentration is maintained high and reaches its maximum 6 h after irradiation (Fig. 2a). Six hours after irradiation, the ROS concentration in HCT-116 cells is 5 times higher than in the control group. Figure 2b shows the effect of laser radiation on ROS intracellular concentration in CHO-K normal cells. The laser-induced ROS concentration increases 3 h after exposure. After 6 h, the concentration of ROS reaches its maximum demonstrating tenfold increase compared with the control group. Maximal intracellular ROS concentrations after laser irradiation have been registered for CHO-K cells. Rotenone treated HCT-116 and CHO-K cells demonstrate a decreasing ROS concentration after laser irradiation. Figure 3a shows that at any time of registration rotenone reduces ROS concentration in HCT-116 cells. In CHO-K cells, rotenone decreases ROS concentration 3 h after irradiation only. In other time intervals, ROS concentration in CHO-K cells after irradiation in groups with and without rotenone (Fig. 2b) exhibits no difference. The evaluation of ROS concentration 6 h after irradiation cannot be considered reliable due to increase of oxidative stress induced by apoptosis and necrosis.

Fig. 2
figure 2

Level of intracellular reactive oxygen species (ROS) in HCT-116 (a) and CHO-K (b) cells exposed to 1265 laser with or without rotenone (Rt). Control group is not irradiated by laser and is not treated with rotenone. Irradiation dose is 400 J/cm2. ROS level expressed as DCFH-DA corrected total cell fluorescence (CTCF). Results are presented as mean values ± S.D. Asterisk denotes statistically significant differences between control and other groups. Number sign denotes statistically significant difference between laser irradiated rotenone treated cells and laser irradiated cells (p < 0.05)

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Fig. 3
figure 3

Changes of mitochondrial potential in HCT-116 (a) and CHO-K (b) cells exposed to 1265 laser with or without rotenone (Rt). Irradiation dose is 400 J/cm2. Control group is not irradiated by laser and is not treated with rotenone. Mitochondrial potential is expressed as TMRE corrected total cell fluorescence (CTCF). Results are presented as mean values ± S.D. Asterisk denotes statistically significant differences between control and other groups. Number sign denotes statistically significant difference between laser irradiated and rotenone treated cells (p < 0.05)

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Changes in mitochondrial potential and reduced glutathione level after laser irradiation

Mitochondrial potential is an important indicator of metabolic activity in a cell. Also, it demonstrates mitochondria ability to generate ROS. Figure 3 shows changes of mitochondrial potential in HCT-116 and CHO-K cells exposed to 1265 laser at the irradiation dose of 400 J/cm2. As shown in Fig. 3a, no changes of the total cellular mitochondrial potential have been registered in HCT-116 cells exposed to laser irradiation in the experiment. Rotenone treated HCT-116 cells exposed to laser irradiation exhibit a decrease of mitochondrial potential in all time intervals. Irradiated СНО-К cells show an increase of mitochondrial potential (Fig. 3b). The maximal increase of mitochondrial potential in СНО-К cells is recorded 6 h after exposure, and it is 1.5 times higher than in the control group. Rotenone causes a decrease of mitochondrial potential in CHO-K cells after laser irradiation in any time interval. Mitochondrial potential in rotenone treated CHO-K cells is 1.2–2.2 times less compared with both the cells exposed to irradiation and the control group (Fig. 3b). The experiments on evaluation of mitochondrial potential after laser irradiation in the presence of rotenone demonstrate that mitochondria are the source of ROS in irradiated cells, since a decrease of mitochondrial potential in the presence of rotenone (Fig. 3) is accompanied by ROS decrease in the presence of rotenone (Fig. 2).

Figure 4 shows changes of intracellular reduced GSH level in HCT-116 and CHO-K cells exposed to 1265 laser at the irradiation dose of 400 J/cm2. Intracellular GSH concentration is an important indicator of a cell metabolism. GSH is a source of hydrogen atoms in intracellular chemical reactions and contributes to cell resistance to ROS. Figure 4 shows that the laser radiation causes a decrease of intracellular GSH in HCT-116 cells in all time intervals. The maximal decrease of GSH concentration in HCT-116 cells has been observed 1 h after laser irradiation, and it is 2.5 times less than in the control group. Three and 6 h after laser irradiation GSH concentration in HCT-116 cells is 2.1 and 1.4 times less than in the control group. In CHO-K cells, GSH concentration at 1 and 3 h is 1.18 and 1.37 times higher than in the control group, while 6 h after exposure, it is 1.65 times lower compared with the control group.

Fig. 4
figure 4

Changes of intracellular reduced glutathione level in HCT-116 and CHO-K cells exposed to 1265 laser. Irradiation dose is 400 J/cm2. Intracellular reduced glutathione level expressed as monochlorobimane corrected total cell fluorescence (CTCF). Results are presented as mean values ± S.D. Asterisk denotes statistically significant differences between control and other groups (p < 0.05)

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DNA damage after laser irradiation

Figure 5 shows the dynamics of the level of DNA strand breaks expressed as a tail moment after irradiation in HCT-116 and CHO-K cells. One can see that after exposure, DNA damage in both cell lines exhibits complex dynamics demonstrating similar behavior. Five minutes after exposure, the first increase of a tail moment has been registered. At the moment of 15 min after irradiation, DNA damage in HCT-116 and CHO-K cells is 2.0 and 4.5 times, respectively, higher than in the control group. At the moments of 30 min and 1 h, DNA damage in the experimental group is the same as in the control group. At 2 h after irradiation, DNA damage in HCT-116 and CHO-K cells is 4.5–7.6 times higher than in the control group. Further, the level of DNA strand breaks is significantly higher in the test group than in the control group at all the time points and reaches the maximal value at 6 h after irradiation. Study of the laser irradiation effect on DNA damage in HCT-116 and CHO-K cells demonstrates complex dynamics of the level of DNA strand breaks. The three stages have been distinguished in this dynamics: Stage 1 is associated with an increase of DNA damage; it starts at 5 min after irradiation and achieves the maximum 15 min after the exposure. Stage 2 is characterized by a decrease of the level of DNA damage down to the values registered for the control group during the time interval 30 min-1 h. Stage 3 is associated with an increase of DNA damage of the irradiated cells.

Fig. 5
figure 5

Level of DNA strand breaks expressed as tail moment from comet assays. Irradiation dose is 400 J/cm2. Values for 1256 nm laser irradiated HCT-116 and CHO-K cells are presented as mean ± SD. Asterisk denotes statistically significant differences between control and laser irradiated cells (p < 0.05)

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Discussion

A number of studies report generation of singlet oxygen in cells under laser irradiation at the wavelength of 1260–1270 nm [2, 4, 5]. Singlet oxygen is an excited state of molecular oxygen. It is responsible for many chemical and biological processes like respiration and photo-oxidation. High oxidation activity of singlet oxygen makes it attractive for therapeutic use, in particular, to kill tumor cells. Singlet oxygen is recognized to be the primary cytotoxic agent in PDT. Also, during PDT, some other ROS types, such as superoxide anion radical and hydrogen peroxide, are generated in the presence of PSs providing energy transfer from photons to molecular oxygen [1]. The singlet oxygen efficiency depends on the place of its generation. Experimental studies report that singlet oxygen generated in mitochondria is more efficient for tumor cell killing compared with singlet oxygen generated in a cytoplasm [6]. Due to very short lifetime of about 10−11–10−9 s, singlet oxygen is able to affect a very small area of about 0.01–0.02 μm [7]. Generally, singlet oxygen oxidizes the amino acid residues of proteins. The efficiency of oxidation of the amino acid residues of proteins by singlet oxygen is twice higher than that of the unsaturated fatty acids [23]. The damaging effect of singlet oxygen in the presence PSs is associated with the damage of lipids and proteins and depends strongly on their localization. The strongest damaging effect by singlet oxygen is observed for PS localized in the mitochondrial membrane [24, 25]. In this case, it damages mitochondrial permeability pores and mitochondrial membranes causing functioning disorder of mitochondria and, consequently, cell death [26].

In the experimental group, laser irradiation of HCT-116 and CHO-K cells at 1265 nm induces higher dose-dependent cell mortality compared with the control group (Fig. 1), thereby, indicating generation of damaging agent. Analysis of the dynamics of intracellular ROS concentration (Fig. 2) shows that laser irradiation causes a steady state increase of ROS concentration in HCT-116 and CHO-K cells during all the time of observation. Along with singlet oxygen, under normal conditions, ROS can be derived from various cell processes, such as by-products of the respiratory chain activity, NADPH oxidase, xanthine oxidase, and arachidonic acid oxygenase [23]. However, the mitochondrial respiratory chain is considered to be the main ROS source [11, 23, 27]. The content of ROS generated from a normal cell metabolism is comparable or even exceeds the content of ROS obtained after ultraviolet or gamma irradiation [27]. To check the sources of ROS, mitochondrial complex I inhibitor rotenone has been used [19]. Rotenone induces a decrease of intracellular ROS concentration (Fig. 2) and mitochondrial potential (Fig. 3) in HCT-116 and CHO-K cells irradiated at 1265 nm. In our opinion, ROS concentration is not reduced by rotenone in CHO-K cells 6 h after irradiation (Fig. 2b) due to increase of ROS production by other intracellular processes, such as apoptosis or necrosis.

Our experiments suggest that after laser irradiation at 1265 nm, ROS could be generated by mitochondria.

Mitochondria provide energy to the cells. The energy released as electrons flow through the respiratory chain is converted into H+ gradient through the inner mitochondrial membrane. This gradient, in its turn, dissipates through the ATP synthase complex (Complex V) and is responsible for the turning of a rotor-like protein complex required for ATP synthesis. In the absence of ADP or mitochondrial complex damage, the movement of H+ through ATP synthase ceases and H+ gradient builds up causing electron flow to slow down and the respiratory chain to become more reduced (State IV respiration). As a result, the physiological steady state concentration of ROS formation increases [23, 27]. In our experiments, mitochondrial potential reflexes the respiratory chain state. The higher the mitochondrial potential, the more reduced the respiratory chain is. Dynamics of mitochondrial potential shows that after laser exposure, the potential does not change (Fig. 3a) or increases (Fig. 3b). Absence of negative dynamics registered for the mitochondrial potential after laser treatment also supports assumption that mitochondria are the main ROS source in our experiments. A decrease of mitochondrial potential and ROS concentration in the presence of rotenone (Figs. 2 and 3) highlights an importance of mitochondria in ROS generation after irradiation of cells.

The dynamics of GSH concentration is similar to the dynamic of intracellular ROS concentration (Figs. 2 and 4). A decrease of GSH concentration could be explained by its use in reactions of ROS utilization [23, 28]. Besides, GSH concentration decrease is associated with its use in conjugation reactions with electrophilic groups of biomolecules catalyzed by glutathione-S-transferase produced from interaction with ROS and free radicals, in particular, with singlet oxygen [29]. In our experiments, laser irradiation at 1265 nm decreases GSH concentration in HCT-116 cells at all time points after irradiation. In CHO-K cells, laser irradiation decreases GSH concentration at 6-h time point only (Fig. 4). Difference in intracellular GSH concentration between normal (CHO-K) and cancer (HCT-116) cells could be attributed to some differences in cell metabolism of these cell types. A decrease of GSH concentration with increasing ROS concentration indicates the presence of oxidative stress that leads to DNA damage and cell death [23].

Dynamics of DNA damage (Fig. 5) in irradiated cells shows that immediately after exposure, the main damaging agent is singlet oxygen, but at further stages, the damage is provided by ROS generated by mitochondria. In our experiments, the two peaks of DNA damage have been registered. The first peak observed 15 min after irradiation is a result of increasing concentration of singlet oxygen under irradiation. But in the time interval of 30 min–1 h, the repair of DNA damage occurs and tail moment in the experimental group does not differ from that in the control group (Fig. 5). Further increase of tail moment within the interval 2–6 h is not associated with singlet oxygen and is a result of an increase of ROS concentration (Fig. 2) generated by another source that we believe to be mitochondria.

Mitochondria contain numerous molecules with a porphyrin structure, e.g., cytochromes а,b,c, р450, which could be used as PSs. Numerous absorption spectra of natural porphyrin molecules like cytochromes in the IR range between 1100 and 1700 nm have been reported in the literature [9, 10]. The synthetic porphyrins are reported to have absorption spectra in the IR range, in particular, around 1260 nm [8]. However, we believe that the damaging effect under laser irradiation at the wavelength of 1265 nm is due to singlet oxygen generated in a close vicinity of the respiratory chain components. The mechanism of accumulation of mitochondria-dependent ROS could be triggered by damage of the complex IV of respiratory chain containing cytochrome аа 3 . This cytochrome interacts directly with oxygen, thereby, laser-induced activation of oxygen to singlet state can seriously damage it. Cytochrome аа 3 damage could initiate the state when the respiratory chain becomes more reduced and ROS concentration is increased by mitochondrial complex I [30].

Note, this preliminary study is based on experiments on two cell lines and uses a limited number of methods for assay of intracellular parameters. Further studies are expected with various cancer cells and longer post-treatment periods to evaluate variations in treatment parameters and correlation with other antitumor therapies. To define precisely the mechanism of the 1265 nm laser irradiation effect, a number of further experiments are required to study contribution of endogenous mitochondrial porphyrins in ROS generation. Besides, the cytochrome activity and mitochondrial DNA damage should be evaluated after laser irradiation. These studies are believed to make possible efficient cancer treatments without photosensitizers. PDT without photosensitizers could be employed in treatment of skin benign tumors and other skin diseases where the use of photosensitizers is cost-ineffective or the therapeutic effect is lower than possible complications. In our opinion, PDT without photosensitizers can be useful for additional treatment of some benign tumors such as papilloma and keratosis and for malignant tumors such as basal and squamous cell carcinoma. IR lasers operating at 1100–1300 nm demonstrate deeper penetration into tissues compared with lasers involved in photodynamic therapy and so can reach the deep subcutaneous tissues. This advantage allows considering IR lasers as promising harmless supplementary method for treatments of benign and malignant skin tumors.

Conclusion

Laser irradiation of HCT-116 and CHO-K cells has induced a dose-dependent cell death via increasing intracellular ROS concentration, increase of DNA damage, decrease of mitochondrial potential, and reduced glutathione. It has been shown that, along with singlet oxygen generation, the increase of the intracellular ROS concentration induced by mitochondrial damage contributes to the damaging effect of the laser irradiation at 1265 nm.