Peer reviewed ORIGINAL ARTICLEheroza.org/wp-content/uploads/2012/06/Evans-and-Maskew...ISSN 1011 5528 | 31 Medical Technology SA Volume 26 No. 1 | June 2012 Peer reviewed ORIGINAL

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Volume 26 No. 1 | June 2012Medical Technology SA

Peer reviewed ORIGINAL ARTICLE

Abstract

Background Laser phototherapy promotes cell viability, cell proliferation and migration. This study aimed to determine if laser irradiation could stimulate cellular responses of stressed cells to promote cell survival.

Materials and Methods Human keratinocytes were treated with 200 μM hydrogen peroxide (H2O2) or 0.4 μg/ml oligomycin to induce autophagy and 5% absolute ethanol (EtOH) or 12 μM tertbutylhydroperoxide (tBHP) to induce apoptosis (control). Cells were irradiated using 1.5 J/cm2 with 648 nm and cellular responses were measured after 1 h or after 24 h and 96 h at 37°C.

Results Irradiated cells treated with 200 μM H2O2 showed an increase in cell proliferation and decrease in intracellular calcium. Irradiated oligomycin treated cells showed a significant increase in intracellular calcium. Irradiated apoptotic (control) cells showed a decrease in ATP viability, an increase in cytotoxicity, decrease in intracellular Ca2+ and decrease in cell proliferation.

Conclusion Irradiated 200 μM H2O2 cells reverted to metabolically active, viable cells capable of proliferating within 96 h of laser irradiation. Changes in intracellular calcium following laser irradiation appear to influence cell survival and proliferation of stressed keratinocytes.

Keywords

Cell stress, keratinocytes, laser biostimulation, laser phototherapy, Low-Level Laser Therapy (LLLT)

Laser irradiation with 648 nm Light stimuLates autophagic human skin keratinocytesD Evans1, M Maskew2, H Abrahamse1

1Laser Research Centre, Faculty of Health Sciences, University of Johannesburg, Johannesburg, South Africa2Health Economics and Epidemiology Research Office, Department of Medicine, University of the Witwatersrand, Johannesburg, South Africa

Corresponding Author: D Evans | [email protected]

Introduction

Autophagy, a lysosomal process involved in the maintenance of cellular homeostasis, is responsible for the turnover of long-lived proteins and organelles that are either damaged or functionally redundant [1]. This process is important in normal development, differentiation and tissue remodelling but can be induced by a change in environmental conditions such as nutrient deprivation [2-4]. Autophagy has been implicated in sev-eral human conditions or pathologies including bacterial and viral infections, ageing, diabetes, cancer, atherosclerosis, neu-rodegenerative disorders and cardiovascular disease [1, 5]. This catabolic process, also termed Type II programmed cell death, involves self-digestion of intracellular organelles by double-membraned vesicles or vacuoles that encircle the components to be recycled [6]. There is a loss of cell viability with a highly vacuolated morphology however mitochondrial membrane potential remains unaffected. The mitochondria are believed to be the main target for laser therapy. Mitochondria are involved in a range of cellular processes such as generating adenosine triphosphate (ATP), supplying cellular energy, signaling, cellular differentiation, cell death, as well as the control of the cell cycle and cell growth.

Autophagy acts as a pro-survival or pro-death mechanism in different physiological and pathological conditions [7]. It has a cytoprotective role in response to many stresses since the inhi-bition of autophagy leads to enhanced cell death [8]. It remains to be determined if autophagic death following prolonged stress is due to cellular exhaustion and/or depletion of cellular com-

ponents or if it is due to the activation of a specific cell death mechanism [8].

Type I programmed cell death or apoptosis is an irreversible, strongly conserved route to death and results in activation of endonucleases, cleavage of DNA into fragments and activation of other proteases (caspases) that lead to cell fragmentation into particles that are then ingested by adjoining cells (phagocyto-sis), resulting in the removal of dead cells. Defects in apoptosis perturb development, promote tumorigenesis and impair chem-otherapy, suggesting that diversion to an alternative cell death pathway such as autophagy or necrosis in these circumstances may be therapeutically beneficial [9]. Apoptosis is cytotoxic while autophagy is cytomodulatory.

It is not clearly established whether the autophagic response can be precisely modulated or regulated to prevent disease or promote health [6]. Manipulation of autophagy may provide a useful way to prevent disease progression or promote cell sur-vival. Since low level lasers do not produce heat but are able to penetrate the interior of cells in a non-destructive manner with highly focused light in the 10-250mW range – stimulating ATP energy production, cell metabolism and membrane permeabil-ity to promote healing, reduce pain and stimulate physiological processes – laser therapy may be a safe alternative method to promote cell survival in damaged or stressed cells or prevent-ing conditions such as ageing, diabetes, cancer, atherosclerosis, neurodegenerative disorders and cardiovascular disease where autophagy has been implicated [1, 5].

32 www.smltsa.org.za | ISSN 1011 5528


Low level lasers have been used to promote pain relief, wound healing, immune modulation and to strengthen the regenera-tive forces of body tissues. Molecularly it is known to stimulate mitochondrial membrane potential (MMP), cytokine secretion and cell proliferation [10]. Laser irradiation has been shown to stimulate the immune system (immuno-corrective) and has anti-bacterial, anti-viral, anti-allergic, anti-toxic, anti-cancer and anti-inflammatory effects [11]. It increases energy and normalizes tissue metabolism, activates ATP-synthesis and energy forma-tion in cells, increases oxidation of energy-carrying molecules and normalises the parameters of the hormonal, immune and reproductive system. Various mechanisms for the effect have been proposed, including absorption of light by mitochondrial enzymes with localized heating [12], photon absorption by fla-vins and cytochromes in the mitochondrial respiratory chain affecting electron transport [13], production of singlet oxygen by excitation of endogenous porphyrins [14], and photoactivation of

calcium channels resulting in increased intracellular calcium concentration and cellular proliferation [15]. In vitro studies have shown that 648 nm diode laser irradiation (1.5 J/cm2; 3.3 mW/cm2) stimulates cell viability, proliferation and cell signalling of stress induced premature senescent cells indicating that laser irradiation may be beneficial for conditions such as immune senescence, skin ageing, muscle atrophy, premature ageing in patients with advanced heart disease, neurodegenerative disor-ders and chronic renal failure [16].

Since little is known about the effect of laser irradiation on au-tophagic cells, this study aimed to determine if laser irradiation with 1.5 J/cm2 using 648 nm could stimulate cellular functions of autophagic cells and promote cell survival. Potential benefits of laser therapy on autophagic cells may include: (i) added cell survival, (ii) preventing the onset of apoptosis during nutrient deprivation or (iii) preventing or delaying the onset of several conditions where autophagy has been implicated.

Table 1. Summary of cell stress conditions induced in human keratinocyte cell cultures.

Action Details [ ] Incubation Ref

Autophagy

Hydrogen peroxide 200 uM H2O2

A powerful oxidizer and major contributor of oxidative damage – causes lipid peroxidation of membrane and hydroxylation of proteins and DNA.

Sigma 31642500 ml

200 uM (in EtOH)

Add to 3 ml culture medium without rEGF and BPE. Incubate at 37°C for 2 h. Wash with 3 washes of 2 ml warmed PBS. Add fresh medium and replace in incubator (37°C, 5% CO2, 80% humidity). Everyday for 4 days with 2 days (72 h) recovery. Cul-ture medium was not replaced during the recovery phase.

18

19

Oligomycin0.4 μg/ml Oligo

An F0-ATP synthase inhibitor and disrupts mitochondrial mem-brane potential.

Sigma 7535210 mg/ml

0.4 ug/ml(in EtOH)

Add to 3 ml culture medium without rEGF and BPE. Wash with 3 washes of 2 ml warmed PBS. Add fresh medium and replace in incubator (37°C, 5% CO2, 80% humidity). Everyday for 2 days with 30 min recovery.

20

Apoptosis

Absolute Ethanol5% EtOH

Induces cellular damage with vacuolization of cells and necrosis

Cytostatic and Cytotoxic

SigmaBCR656

5% EtOH Add to 3 ml culture medium without rEGF and BPE. Incubate at 37°C for 2 h. Wash with 3 washes of 2 ml warmed PBS. Add fresh medium and replace in incubator (37°C, 5% CO2, 80% humidity). Everyday for 4 days with 2 days (72 h) recovery. Cul-ture medium was not replaced during the recovery phase.

21

Tert-hydroperoxide 12 mM tBHP

Chemically induces oxidative stress

Sigma B2633 100 ml

12 mM(in H20)

Add to 3 ml culture medium without rEGF and BPE. Wash with 3 washes of 2 ml warmed PBS. Add fresh medium and replace in incubator (37°C, 5% CO2, 80% humidity). Everyday for 2 days with 30 min recovery.

22

[ ] concentration



Materials and methods

Cell culture

Human keratinocyte cell cultures designated CCD-1102 KERTr (ATCC CRL-2310) were grown in keratinocyte-serum free media (SFM, Invitrogen 17005075) containing 35 ng/ml recombinant epidermal growth factor (rEGF) and 0.05 mg/ml bovine pitui-tary extract. Upon reaching 60-75% confluency, the cells were trypsinized and 6 X 105 cells (in 3 ml culture media) were seed-ed in 3.4 cm diameter culture plates and incubated overnight to allow the cells to attach.

In vitro models

Oxidative stress has been shown to induce autophagy or ap-optotic signalling pathways [17]. The use of chemicals to induce apoptosis or autophagy in in vitro cell culture models has been described elsewhere [16-23]. Several studies have shown that apoptosis can be chemically induced using 33 mM 2-de-oxy-D-glucose (2-DOG), 500 μM hydrogen peroxide (H2O2),

12 μM tert-butylhydroperoxide (tBHP) and 5% absolute etha-nol (EtOH) whereas autophagy can be induced using 15 μM carbonylcyanide ρ-trifluoromethoxyphenylhydrazone (FCCP), 0.4 μg/ml oligomycin, oridonin, arsenic trioxide (As2O3) and 200 μM H2O2 [16-23].

Apoptosis (control) or autophagy was induced by exposing keratinocytes to different chemicals to induce cell stress [16-23] (Table 1). Different incubation times were used to induce au-tophagy however an apoptotic control was incubated for the same duration. Apoptosis was confirmed by caspase 3/7 activity [16, 23].

648 nm Diode laser irradiation

Digital photographs of the cells were taken everyday to record changes in cell morphology. After the recovery phase cells were irradiated with visible red laser light. A 648 nm diode laser with a power output of 30 mW, power density of 3.3 mW/cm2 and spot size of 3.4 cm (area 9.1 cm2) was used to irradiate culture dishes with a dose of 1.5 J/cm2 (Figure 2). Dosage was

Figure 1: Summary of the methods used to measure the effect of 648 nm laser irradiation on apoptotic or autophagic human skin keratinoc-ytes. Cells were exposed to EtOH or H2O2 everyday for two days with a 30 minute recovery period on the last day prior to irradiation while cells were exposed to tBHP or oligomycin everyday for four days with 72 h recovery period on the last day prior to laser irradiation. Apoptotic or autophagic keratinocytes were irradiated with 1.5 J/cm2 and cell were then incubated for 1 h at 37°C with humidified air containing 5% CO2 before the cellular responses were measured. Cells were incubated for 24 h and 96 h before changes in cell number were measured.



calculated as follows: Irradiance (J/cm2) = Time (s) X [power (W)/surface (cm2)] [24]. Studies have shown that 1.5 J/cm2 results in a significant increase in migration [25] and proliferation of keratinocytes [16, 23, 26]. Cell culture dishes were irradiated from a fiber optic positioned at a distance of 8 cm above the cell monolayer with the culture dish lid off at room temperature (21°C) in the dark on a dark surface. Cells were irradiated in SFM media without phenol red to minimize the loss of laser en-ergy through absorption by colored culture media. The laser tip (±5 mm) was expanded so that the spot size area was the same area as the culture dish. The entire dish was irradiated with a homogenous beam for approximately 7 min 35 sec duration to deliver 1.5 J/cm2 (Figure 1).

Cellular responses

After laser irradiation, cells were incubated at 37°C for 1 h be-fore they were trypsinized and resuspended at a concentration of 8 X 104 cells/100 μl in supplemented SFM media [16, 23]. The cell culture media was removed for the LDH cytotoxicity as-say while the cell suspension was used for the ATP luminescent viability assay, WST-1 proliferation assay (24 h and 96 h) and intracellular calcium (Ca2+) (Table 2) [27-30]. Changes between the un-irradiated and irradiated samples were graphically pre-sented. Experiments were independently repeated (n=3 to 6).

Statistical analysis

Differences in cellular functions/parameters (viability, prolif-eration, intracellular calcium and cytotoxicity) between the

irradiated and un-irradiated keratinocytes for each model were estimated using the Student’s t-test or One Way ANOVA for parametric or normally distributed data and the Mann-Whitney Rank Sum Test and Kruskal-Wallis test for non-parametric or non-normally distributed data. Alpha was set at the level of 0.05 (95%). The effect of laser irradiation on a change in vi-ability, proliferation, intracellular calcium or cytotoxicity was estimated for each model (EtOH, H2O2, tBHP or oligomycin) compared to normal irradiated keratinocytes. We used normal irradiated human keratinocytes as the reference group – where other variables (i.e. wavelength, dose/fluence, duration of ir-radiation and irradiation conditions) were kept constant [13, 23,

31]. The change between irradiated and un-irradiated cells for each cellular parameter was calculated and expressed as a per-centage (%). Analysis was done using SigmaPlot 8.2 (SYSTAT software) and SAS 9.1 (SAS Institute Inc., Cary, NC).

Results

Morphology: Un-irradiated and irradiated normal keratinocytes (CCD-1102 KERTr) showed a typical cobblestone appearance consistent with normal keratinocyte morphology. Cells treated with 5% EtOH showed a highly vacuolated morphology with evidence of cytoplamic granules which may be mRNA stress granules or dynamic cytoplasmic foci in which stalled transla-tion initiation complexes accumulate [16]. Cells treated with 5% EtOH showed changes consistent with autophagy however the presence of cytoplasmic stress granules indicates severe dam-

Table 2. Summary of the methods used to assess laser induced cellular responses.

Summary of in vitro tests used to assess cellular responses

Action Incubation Detection Ref

LDH membrane integrity

Promega CytoTox 96

non-radioactive cytotoxicity

assay (G1780)

The assay measures lactate dehydrogenase (LDH), a stable cytosolic enzyme, released into culture medium upon cell lysis.

30 min at room tempera-ture and protected from light.

96-well flat bottom plate. 50 μl culture medium and 50 μl substrate in Absorb-ance at 490 nm.

27

WST-1 cell proliferation

Roche WST-1 cell proliferation

reagent (11644807 001)

A colorimetric assay for the non-radioactive quantification of cell proliferation, cell viability and cytotoxicity in a 96-well flat bottom plate.

Incubate cell suspension at 37°C and 5% CO2 for 24 h and 96 h after laser irradiation then add WST-1 reagent. Incubate for 4 h at 37°C and 5% CO2 with 10 μl WST-1 reagent.

100 μl (8 X 104 cells) cell suspension. Intensity of the formazen dye, at 450 nm, is directly proportional to the number of metaboli-cally active cells.

28

Adenosine triphosphate

(ATP)

Promega CellTiter Glo luminescent

assay (G7570)

Addition of reagent results in cell lysis and generation of luminescent signal proportional to the amount of ATP present which is proportional to the number of cells present in culture.

Incubate reagent and cells for 2 min at room temperature on an orbital shaker to induce lysis. Incubate for 10 min at room temperature and pro-tected from light. Measure luminescence.

100 μl ATP CellTiter Glo reagent and 100 μl cell suspension (8 X 104 cells). Luminescent signal generated is proportional to ATP [ ].

29

Intracellular calcium (Ca2+)

BioAssay QuantiChromTM Calcium assay kit (DICA-500)

Phenolsulphonephthalein dye forms a very stable blue coloured complex specifically with free calcium.

Incubate reagent and cells for 3 min at room tem-perature in 96-well clear bottom plate.

200 μl reagent and 50 μl of cell suspension (4 X 104 cells). Intensity of the colour, at 612 nm, is directly proportional to [Ca+].

30

[ ] concentration



age which suggests apoptosis (cytotoxic) rather than autophagy (cytomodulatory) [16]. Cell morphology of tBHP showed changes characteristic of apoptosis (i.e. cell shrinkage, loss of membrane asymmetry and attachment and controlled disintegration of the cell into apoptotic bodies). Cells treated with 200 uM H2O2 showed a highly vacuolated morphology with double mem-branes consistent with autophagy. Oligomycin treated cells showed evidence of cloudy swelling or autophagy, a reversible process characterized by large cytoplasmic vacuoles (Figure 2) [23]. Cells treated with 200 uM H2O2 (<5 X 105 cells/3ml) and 12 mM tBHP (<3 X 105 cells/3 ml) showed a decrease in cell number when compared to normal keratinocytes (>1 X 106

cells/3 ml).

Autophagy – 200 uM H2O2: Cells treated with 200 uM H2O2 and irradiated showed an increase in ATP cell viability (p = 0.11), a decrease in LDH cytotoxicity (p = 0.672), a decrease in intracel-lular calcium (p = 0.024) and an increase in cell proliferation after 96 h (p = 0.014) compared to 200 uM H2O2 un-irradiated cells (Figure 3 and 4), however only intracellular calcium and cell proliferation were statistically significant.

The change between irradiated and un-irradiated 200 uM H2O2 treated cells was calculated and compared to the change be-

tween irradiated and un-irradiated normal keratinocytes. Irradi-ated cells treated with 200 uM H2O2 showed an increase in ATP viability (5% vs. 2%; p = 0.823), a decrease in LDH cytotoxicity (-13% vs. 3%; p = 0.134), a decrease in intracellular calcium (-30% vs. -5%; p = 0.02) and an increase in cell proliferation after 96 h (29% vs. 18%; p = 0.775) compared to irradiated normal keratinocytes, however only a decrease in intracellular calcium was statistically significant.

Autophagy – 0.4 μg/ml oligomycin: Cells treated with 0.4 μg/ml oligomycin and irradiated with 1.5 J/cm2 did not show an increase in ATP cell viability (p = 0.981), showed a modest decrease in LDH cytotoxicity (p = 0.22), an 11.2% increase in intracellular Ca2+ (p = 0.04) and a 4.7% increase in cell proliferation after 96 h compared to 0.4 μg/ml oligomycin un-irradiated cells.

The protein content using the Quanti-IT protein assay (Invitro-gen, Paisley UK, Q33211) confirmed the anti-proliferative effect as irradiated cells treated with oligomycin showed a -2.88% decrease (1.39 mg/ml) in protein content or cell volume when compared to un-irradiated cells (1.43 mg/ml). Irradiated cells also showed a decrease in caspase 3/7 activity (-9.33%), a decrease in cytochrome c (-10.07%) and an increase in JC-1

Figure 2: Morphological responses of human keratinocytes incubated in sub-cytotoxic conditions. Normal keratinocytes displayed the normal typical cobblestone appearance. While autophagy is a reversible process that can be both a survival and death pathway, apoptosis is irreversible, leading only to cell death [43]. 25 μg/ml Actinomycin D (19 h 37°C and 5% CO2) at was added to induce apoptosis and served as a positive control. Apoptosis is marked by cell shrinkage, loss of membrane asymmetry and attachment, blebbing, DNA frag-mentation, chromatin condensation leading to the appearance of pyknotic nuclei and controlled disintegration of the cell into so-called apoptotic bodies (arrows). Cells treated with 12 mM tBHP showed changes consist-ent with apoptosis. Keratinocytes treated with 5% EtOH showed a highly vacuolated morphology suggesting autophagy however the evidence of cytoplasmic stress granules (arrow) and a highly vacuolated morphology together with the cellular responses indicated severe damage preceding apoptosis [24]. Cells treated with 200 μM H2O2 or 0.4 μg/ml oligomycin showed evidence of autophagy. In autophagy, there is a loss of cell viability with a highly vacuolated morphology (arrows) presumed to present extensive recycling of damaged organelles (200 X magnification).



(ΔΨmt) mitochondrial membrane potential (18.45%) (results not shown).

The change between irradiated and un-irradiated 0.4 μg/ml oligomycin treated cells was calculated and compared to the change between irradiated and un-irradiated normal keratinoc-ytes. Irradiated cells treated with 0.4 μg/ml oligomycin showed an increase in ATP viability (6% vs. 2%; p = 0.426), a decrease in LDH cytotoxicity (-40% vs. 3%; p = 0.184), an increase in intracellular calcium (9% vs. -5%; p = 0.05) and an increase in cell proliferation after 96 h (14% vs. 18%; p = 0.386) com-pared to irradiated normal keratinocytes. From the results only an increase in intracellular calcium was statistically significant when comparing irradiated 0.4 μg/ml oligomycin treated cells to un-irradiated or normal keratinocytes.

Apoptosis – 5% EtOH: Irradiated cells treated with 5% EtOH showed a decrease in ATP cell viability (p = 0.82), an increase in LDH cytotoxicity (p<0.001), a decrease in intracellular Ca2+ (p = 0.05) and a decrease in cell proliferation after 96 h (p = 0.04) compared to 5% EtOH un-irradiated cells.

The change between irradiated and un-irradiated 5% EtOH treated cells was calculated and compared to the change between irradiated and un-irradiated normal keratinocytes. Irradiated cells treated with 5% EtOH showed a decrease in ATP viability (-11% vs. 2%; p = 0.181), an increase in LDH cytotoxicity (28% vs. 3%; p = 0.02), a decrease in intracellu-lar calcium (-28% vs. -5%; p = 0.03) and a decrease in cell proliferation after 96 h (-70% vs. 18%; p = 0.04) compared to irradiated normal keratinocytes. Significant changes in LDH cytotoxicity, intracellular calcium and cell proliferation were observed between irradiated cells treated with 5% EtOH and the un-irradiated cells and the normal keratinocytes.

Apoptosis – 12 mM tBHP: Cells treated with 12 mM tBHP and irradiated showed a decrease in ATP cell viability (p = 0.04), an increase in LDH cytotoxicity (p = 0.012), a decrease in intracel-lular Ca2+ (p = 0.576) and a 3.17% increase in cell proliferation after 96 h (p = 0.853) compared to 12 mM tBHP un-irradiated cells.

The protein content using the Quanti-IT protein assay (Invitro-gen, Paisley UK, Q33211) confirmed the results as irradiated cells treated with tBHP showed an 11.9% increase (0.963 mg/ml) in protein content or cell volume when compared to un-ir-radiated cells (0.848 mg/ml) however the NADH CellTiter-Blue® Cell Viability Assay (Promega WI, G8080) showed a significant decrease in fluorescence (indicator of cell number and meta-bolic activity or viability) in irradiated cells (p = 0.044). The protein content and WST-1 confirms an increase in cell number however the NADH and ATP luminescence confirms a signifi-cant decrease in cell viability. Irradiated cells treated with tBHP showed an increase in JC-1 (ΔΨmt) mitochondrial membrane potential (5.4%), a decrease in cytochrome c (-12.5%) and a decrease in caspase 3/7 activity (-11.0%) which indicates an anti-apoptotic effect (results not shown).

The change between irradiated and un-irradiated 12 mM tBHP treated cells was calculated and compared to the change be-tween irradiated and un-irradiated normal keratinocytes. Irra-diated cells treated with 12 mM tBHP showed a decrease in ATP viability (-52%% vs. 2%; p = 0.04), an increase in LDH cytotoxicity (17% vs. 3%; p = 0.014), a decrease in intracellular calcium (-6% vs. -5%; p = 0.915) and a decrease in cell prolif-eration after 96 h (-9% vs. 18%; p = 0.902) compared to irradi-ated normal keratinocytes. Only the ATP cell viability and LDH cytotoxicity showed statistically significant differences between the irradiated 12 mM tBHP treated cells and the un-irradiated cells or the normal keratinocyte cells.

Discussion

We demonstrate that in vitro cell stress models using 5% EtOH, tBHP, 200 uM H2O2 or oligomycin induces cellular damage. We demonstrate that laser irradiation with 1.5 J/cm2 promotes cell survival of 200 uM H2O2 treated cells by increasing cell proliferation and decreasing intracellular calcium. Oligomycin and H2O2 autophagic cells respond differently to laser irradia-tion as the in vitro models use different mechanisms of action to induce cell stress which results in different levels of cellular damage. Laser irradiation may contribute to cell survival by

Figure 3: Cellular responses such as ATP luminescent cell viability (A) and LDH cytotoxicity (B) were measured after laser irradiation with 1.5 J/cm2 using 648 nm. Groups included irradiated and un-irradiated normal (N) keratinocytes and keratinocytes treated with 200 μM H2O2 or 0.4 μg/ml oligomycin to induce autophagy or keratinocytes treated with 5% EtOH or 12 mM tBHP to induce apoptosis. Irradiated cells treated with 5% EtOH showed a significant increase in LDH cytotoxicity while irradiated cells treated with tBHP showed a significant decrease in ATP viability and a significant increase in LDH cytotoxicity (bars are mean with standard deviation; *p<0.05; n=3 to 6).



Figure 4: Cellular responses such as intracellular Ca2+ (A) and percentage change in proliferation after 96 h using the WST-1 assy (B) were measured after laser irradiation with 1.5 J/cm2 using 648 nm. Groups included irradiated and un-irradiated normal (N) keratinocytes and keratinocytes treated with 200 uM H2O2 or 0.4 μg/ml oligomycin to induce autophagy or keratinocytes treated with 5% OH or 12 mM tBHP to induce apoptosis. Irradiated cells treated with H2O2 showed a significant decrease in intracellular Ca2+ which is consistent with the significant increase in cell proliferation after 96 h. Irradiated cells treated with oligomycin showed a significant increase in intracellular Ca2+ with only a minor increase (4.7%) in cell proliferation after 96 h. Irradiated cells treated with 5% EtOH showed a significant decrease in intracellular Ca2+ and a significant decrease in cell proliferation indicating a decreased cell proliferation and increased apoptosis by interfering with calcium-dependent secondary messenger systems. (bars are mean with standard deviation; *p < 0.05; n = 3 to 6).

conserving cellular components until damaged organelles can be recycled or the cellular function of the compromised cells can be restored to maintain homeostasis.

As differentiation of keratinocytes share similarities with pro-grammed cell death, it has been proposed that apoptosis-like mitochondrial impairment triggers keratinocyte differentiation. In particular, it has been shown that the treatment of keratino-cytes with inducers of mitochondrial dysfunction (rotenone, staurosporine and protoporphyrin) leads to differentiation-related changes, including flattened morphology, stratification and expression of keratin 10. Reports have suggested that the activation of the apoptotic pathway is necessary for keratino-cyte differentiation. A decrease in mitochondrial membrane potential and the release of cytochrome c for transcription fac-tor activation and regulation of gene expression have been re-ported during in vitro keratinocyte terminal differentiation [32-35]. Studies have reported that a rise in intracellular free calcium is associated with an increase in Ca2+ transport across the plasma membrane and is a common mechanism controlling in vitro differentiation in mouse keratinocytes [36, 37]. Both oligomycin and tBHP treated cells showed an increase in mitochondrial membrane potential and decrease in cytochrome c which does not indicate keratinocyte differentiation in these models.

Elevated intracellular Ca2+ ([Ca2+]i) levels trigger growth arrest (anti-proliferative) and induce differentiation of keratinocytes [38]. In H2O2 irradiated cells, the decrease in intracellular Ca2+ is consistent with an increase in the growth rate. Results from this study suggest that laser irradiation using 1.5 J/cm2 promotes cell survival since the H2O2 irradiated cells revert to active, vi-able cells that are capable of actively proliferating within 96 h of laser irradiation. Some studies have suggested that core machinery for autophagy is conserved which plays an impor-tant role during proliferation and differentiation [39]. A possible explanation may also be that the over-expression of the anti-apoptotic protein Bcl-2 decreases the endoplasmic reticulum

(ER) Ca2+ load and protects cells from death. Alternatively buff-ering of intracellular free calcium can, in some cases, inhibits cell death. The decrease in LDH cytotoxicity in H2O2 irradiated cells supports an anti-apoptotic effect as damage to the cyto-plasm and the plasma membrane would result in an increase in the LDH activity. When comparing the change between H2O2 irradiated and normal irradiated cells results suggest that laser irradiation can stimulate H2O2 treated cells so that their cellular their function (ATP viability and cell proliferation) is similar or greater (although not statistically significant) than normal irradi-ated human keratinocytes. The decrease in intracellular calcium is consistent with cell growth and cell survival as increased intracellular calcium concentration can initiate intracellular apoptotic signalling.

Oligomycin itself inhibits ATP synthase, disrupts mitochondrial membrane potential (ΔΨmt) and may be similar to that of p-(tri-fluoromethoxy) phenyl-hydrazone (FCCP) which uncouples oxidative phosphorylation [23]. Studies have reported that oli-gomycin results in mitochondrial breakdown with concomitant Ca2+ influx. Modestly elevating [Ca2+]i can inhibit apoptosis since [Ca2+]i mediated multiple signalling cascades (i.e. Ca2+/calmodulin- dependent protein kinases kinase, protein kinase B and the phosphorylation of BAD) are critical for cell survival. Results from this study indicate that irradiation with 1.5 J/cm2 results in a modest increase in cell number (<20%) while main-taining membrane integrity with a pro-survival or anti-apoptotic effect. The effect of laser irradiation on ATP viability may be limited in oligomycin treated and irradiated cells as the chemi-cal itself inhibits oxidative phosphorylation and ATP synthesis. Increased [Ca2+]i in oligomycin treated and irradiated cells may be responsible for an anti-apoptotic or pro-survival effect which protects cells from death and maintains cell numbers. Higher levels of [Ca2+]i would result in the activation of the apoptotic pathway, trigger growth arrest and induce keratinocyte differ-entiation. The LDH cytotoxicty, caspase 3/7, cytochrome c and



JC-1 results confirmed an autophagic and not apoptotic cell death pathway. The increase in intracellular calcium is most likely related to the action of oligomycin itself and not due to apoptosis or keratinocyte differentiation. It is well known that laser irradiation protects cells against caspase-mediated apopto-sis, most likely by decreasing ROS production, down-regulating pro-apoptotic proteins and activating anti-apoptotic proteins, as well as by increasing energy metabolism.

5% EtOH is both cytotoxic and cytostatic which explains the decrease in cell number observed in the morphology results. The significant increase in LDH cytotoxicity indicates late ap-optotic changes since the cytoplasm and the plasma membrane become seriously damaged which would result in an increase in the LDH activity. Cell death can also be induced when Ca2+ gains entry from the extracellular medium via the plasma mem-brane and elevations in intracellular Ca2+ may mediate apopto-sis. Irradiated 5% EtOH treated cells showed a decrease in intra-cellular calcium which indicates another mechanism, possibly the lowering of extracellular Ca2+, blocking membrane Ca2+ channels or by interfering with calcium-dependent secondary messenger systems, which by themselves can trigger apoptosis in vitro [40, 41]. Irradiated 5% EtOH treated cells showed an ad-ditional decrease in cell number and increase in cytotoxicity suggesting that laser irradiation may contribute to or precipitate apoptosis in these cells. Laser irradiation may affect apoptotic signalling, enzymes or regulatory proteins which are essential to initiating the apoptotic pathway. These proteins function by either targeting mitochondria functionality or directly transduc-ing the signal via adaptor proteins to the apoptotic mechanisms. Irradiated EtOH treated apoptotic cells showed poor cellular function (significant increase in LDH cytotoxicity, decrease in intracellular calcium and decrease in proliferation) compared to irradiated normal human keratinocytes suggesting that laser irradiation had no beneficial effect on these cells – on the con-trary, laser irradiation appears to precipitate apoptosis possibly by interfering with mitochondria functionality however further studies are needed to confirm this.

tBHP uncouples cellular respiration and induces the produc-tion of ROS, lipid peroxidation, mitochondrial dysfunction, ATP depletion and impaired active transport. Therefore tBHP itself may be responsible for limiting the effect of laser irradiation on ATP synthesis. Studies have shown that laser therapy attenu-ates reactive oxygen species (ROS) production [42] or blocks the effect of ROS released [43]. Results from this study suggest that laser irradiation may reduce ROS and exert an anti-apoptotic, protective or pro-survival effect on pre-apoptotic cells (revers-ible) or repair oxidative stress to reduce apoptotic signals how-ever the significant decrease in viability and small increase in cell number indicates that laser irradiation can not affect cells that are already committed to apoptosis. Again, laser irradiation may affect apoptotic signalling, enzymes or regulatory proteins which are essential to initiating or stopping the apoptosis path-way. The irradiated tBHP treated cells showed a significant de-crease in ATP viability, increase in cytotoxicity and modest de-crease in intracellular calcium consistent with a minor increase in cell number (<5%). An increase in [Ca2+]i would indicate growth arrest or mediate apoptosis, both at early stages and late

steps (post commitment point). Irradiated tBHP treated apop-totic control cells showed poor cellular function (decreased ATP viability, increased LDH cytotoxicity while intracellular calcium and proliferation after 96 h were similar) compared to normal irradiated human keratinocytes.

The switch from a life to a death signal involves the coinci-dental detection of Ca2+ and pro-apoptotic stimuli and depends on the amplitude of the mitochondrial Ca2+ signal. Because of the toxicity of Ca2+ ions, a low Ca2+ concentration must be maintained in the cytoplasm. Cell stress often results in highly elevated calcium levels, depolarised mitochondrial membrane potential, decreased cAMP levels and decreased ATP produc-tion. When low ATP production persists, DNA synthesis reduces significantly and pro-apoptotic factors increase heralding cell death [44]. Laser irradiation is believed to stimulate the redox activity in the respiratory chain with subsequent effects on intra-cellular ion concentration including calcium. Laser irradiation has been shown to cause mitochondrial polarization which im-proves membrane permeability leading to increased Ca2+ flux, pH value, cAMP level, ATP production, anti-apoptotic factors, transcription factors, and DNA synthesis. This cascade of cel-lular events accelerates cell proliferation [44].

Generation of reactive oxygen species (ROS) through oxidative stress causes cell death or compromises the long-term survival of cells [17]. The role of ROS in the induction of autophagic cell death is not clear however it has been shown that under nutri-ent starvation, ROS induces autophagy. It has been suggested that ROS could be involved in caspase-independent cell death [45]. ROS has many effects on cells including DNA damage, mitochondrial dysfunction, activation of signalling pathways and activation of transcription factors leading to upregulation of genes [46]. Studies have confirmed that laser phototherapy (gallium-arsenide GaAs, λ = 904 nm, 45 mW average power, 5 J/cm2 for 35 s) reduces oxidative stress [47]. Laser phototherapy increases levels of superoxide dismutase (SOD), which is key in the process of clearing ROS, so phototherapy should theo-retically help to prevent or even reduce some of the damaging effects of ROS. Studies using visible (634 nm) red laser light have shown that laser irradiation increases nitric oxide (NO) production but decreases intracellular ROS so laser irradiation could reduce the effect of ROS and promote cell survival.

Limitations: These findings should be considered in light of the study limitations. Firstly, cell viability was assessed using the ATP luminescent assay which can under-estimate or over-esti-mate the metabolic ability of cells to proliferate. However, the assay used is a homogenous method of determining the number of viable cells in culture based on the quantification of ATP, which signals the presence of metabolically active cells. Cells may exhibit delayed death or survival effects that cause a tem-porary indication of toxicity. Additional assays such as growth curves, clonogenic assays and DNA synthesis (BrdU incorpora-tion) [48] will be explored. Secondly, despite seeding a constant cell number at the beginning of the experiment each chemical (H2O2, tBHP, 5% EtOH or oligomycin) affected the cell number differently resulting a different final cell number prior to laser ir-radiation. To reduce this effect, un-irradiated controls were used for each stress condition while normal keratinocytes were used



as the reference to estimate the effect of cell type on an increase in viability, proliferation, intracellular calcium and cytotoxicity – changes between the irradiated and un-irradiated cells were calculated and then compared between the models (i.e. nor-mal vs. oligomycin). Thirdly, true assays for apoptosis and au-tophagy were not reported – although caspase 3/7 results were discussed for oligomycin and tBHP. Future work will include assays to assess apoptosis (by activation of executioner caspases or by observing chromatin condensation and fragmentation us-ing fluorescent probes) and autophagy (by electron microscopy assessing lipidation of the LC3 protein or using other methods recently described [49-51]. Lastly, cellular responses were meas-ured within 1 hr, which is sufficient to measure the direct effect of laser irradiation [52, 53] however a longer incubation period (> 24 h) is required to demonstrate cell proliferation (24 and 96 h) and protein expression. Cellular responses were observed after a 1 h incubation which suggests the mechanism by which laser irradiation with 648 nm reverses autophagy does not require gene transcription however this still needs to be confirmed.

Conclusion

Cells treated with 200 μM H2O2 and irradiated showed changes in intracellular calcium consistent with growth when compared to un-irradiated cells or when compared to irradiated normal keratinocytes. Irradiated 200 μM H2O2 treated cells reverted to metabolically active, viable cells capable of proliferating within 96 h of laser irradiation. Results suggest that laser irradiation at 648 nm stimulated 200 μM H2O2 treated cells so that their cel-lular function was similar or even greater than that of irradiated normal keratinocytes. The role of intracellular calcium in cell death is controversial – further investigations are warranted to determine the effect of laser irradiation on calcium-dependent secondary messenger systems, calcium channel blockers, apop-totic signalling and regulatory proteins and the combined effect on cell signalling and mitochondrial functionality.

Results from this study indicate that visible red laser light can exert different responses in cells as they respond to stress. These include (i) anti-differentiating (ii), stimulating cells to revert to viable actively proliferating cells (iii) promoting cell survival by potentially reducing ROS, and (iv) promoting cell proliferation. Results suggest that laser irradiation may potentially promote a pro-survival or anti-apoptotic effect after cell stress. This points to the potential benefit of irradiating autophagic keratinocytes to prevent disease progression or promote health.

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