Medical Technology SA
Volume 26 No. 1 | June 2012
Peer reviewed ORIGINAL ARTICLE
Laser irradiation with 648 nm light stimulates autophagic
human skin keratinocytes
D Evans1 (D.BioMed), M Maskew2 (MBBCH, MSc), H Abrahamse1 (PhD)
Laser Research Centre, Faculty of Health Sciences, University of Johannesburg, Johannesburg, South Africa
Health Economics and Epidemiology Research Office, Department of Medicine, University of the Witwatersrand, Johannesburg, South
Africa
1
2
Correspondence to: D Evans | [email protected]
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)
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 several human conditions or pathologies including bacterial and
viral infections, ageing, diabetes, cancer, atherosclerosis, neurodegenerative disorders and cardiovascular disease [1, 5]. This
catabolic process, also termed Type II programmed cell death,
involves self-digestion of intracellular organelles by doublemembraned 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 inhibition 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-
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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 (phagocytosis), resulting in the removal of dead cells. Defects in apoptosis
perturb development, promote tumorigenesis and impair chemotherapy, 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 survival. 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 permeability 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 preventing conditions such as ageing, diabetes, cancer, atherosclerosis,
neurodegenerative disorders and cardiovascular disease where
autophagy has been implicated [1, 5].
Low level lasers have been used to promote pain relief, wound
Medical Technology SA
Volume 26 No. 1 | June 2012
healing, immune modulation and to strengthen the regenerative 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 antibacterial, 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 formation 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 flavins 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 disorders and chronic renal failure [16].
Since little is known about the effect of laser irradiation on autophagic 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 A powerful oxidizer and major
contributor of oxidative damage
200 uM H2O2
– causes lipid peroxidation of
membrane and hydroxylation of
proteins and DNA.
Sigma 31642 200 uM Add to 3 ml culture medium
500 ml
(in EtOH) 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. Culture medium was not replaced
during the recovery phase.
18
Oligomycin
0.4 μg/ml Oligo
Sigma 75352 0.4 ug/ml Add to 3 ml culture medium
10 mg/ml
(in EtOH) 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
An F0-ATP synthase inhibitor and
disrupts mitochondrial membrane potential.
19
Apoptosis
Absolute Ethanol
5% EtOH
Induces cellular damage with
vacuolization of cells and
necrosis
Sigma
BCR656
Cytostatic and Cytotoxic
Tert-hydroperoxide Chemically induces oxidative
12 mM tBHP
stress
Sigma B2633
100 ml
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. Culture medium was not replaced
during the recovery phase.
21
12 mM
(in H20)
22
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.
[ ] concentration
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Volume 26 No. 1 | June 2012
Figure 1: Summary of the methods used to measure the effect of 648 nm laser irradiation on apoptotic or autophagic human skin keratinocytes. 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 cells 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.
Materials and methods
Cell culture
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].
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 pituitary extract. Upon reaching 60-75% confluency, the cells were
trypsinized and 6 X 105 cells (in 3 ml culture media) were seeded in 3.4 cm diameter culture plates and incubated overnight to
allow the cells to attach.
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 autophagy however an apoptotic control was incubated for the
same duration. Apoptosis was confirmed by caspase 3/7 activity
[16, 23]
.
In vitro models
648 nm Diode laser irradiation
Oxidative stress has been shown to induce autophagy or apoptotic 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-deoxy-D-glucose (2-DOG), 500 μM hydrogen peroxide (H2O2),
12 μM tert-butylhydroperoxide (tBHP) and 5% absolute etha-
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
calculated as follows: Irradiance (J/cm2) = Time (s) X [power
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Volume 26 No. 1 | June 2012
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 30 min at room temperadehydrogenase (LDH), a
ture and protected from
stable cytosolic enzyme,
light.
released into culture
medium upon cell lysis.
96-well flat bottom plate.
50 μl culture medium and
50 μl substrate in 96-well
flat bottom plate.
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 96well 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 metabolically 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 protected 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 temperature 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
(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 energy 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 before 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 assay 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 presented. Experiments were independently repeated (n=3 to 6).
Statistical analysis
Differences in cellular functions/parameters (viability, proliferation, 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 viability, 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 irradiation 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 percentage (%). 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 translation initiation complexes accumulate [16]. Cells treated with 5%
EtOH showed changes consistent with autophagy however the
presence of cytoplasmic stress granules indicates severe damage 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
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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 fragmentation, 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 consistent 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).
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 membranes 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 intracellular 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 between irradiated and un-irradiated normal keratinocytes. Irradiated 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
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(-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 unirradiated cells.
The protein content using the Quanti-IT protein assay (Invitrogen, 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
(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-
Medical Technology SA
Volume 26 No. 1 | June 2012
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).
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) compared 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.
decrease in fluorescence (indicator of cell number and metabolic 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 significant 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).
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 12 mM tBHP
treated cells was calculated and compared to the change between irradiated and un-irradiated normal keratinocytes. Irradiated 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 proliferation after 96 h (-9% vs. 18%; p = 0.902) compared to irradiated 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.
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 intracellular 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 intracellular 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 (Invitrogen, 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-irradiated cells (0.848 mg/ml) however the NADH CellTiter-Blue®
Cell Viability Assay (Promega WI, G8080) showed a significant
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 irradiation 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
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 programmed cell death, it has been proposed that apoptosis-like
mitochondrial impairment triggers keratinocyte differentiation.
In particular, it has been shown that the treatment of keratinoISSN 1011 5528 | www.smltsa.org.za
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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 calciumdependent secondary messenger systems. (bars are mean with standard deviation; *p < 0.05; n = 3 to 6).
cytes with inducers of mitochondrial dysfunction (rotenone,
staurosporine and protoporphyrin) leads to differentiationrelated changes, including flattened morphology, stratification
and expression of keratin 10. Reports have suggested that the
activation of the apoptotic pathway is necessary for keratinocyte differentiation. A decrease in mitochondrial membrane
potential and the release of cytochrome c for transcription factor activation and regulation of gene expression have been reported 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, viable 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 important role during proliferation and differentiation [39]. A possible
explanation may also be that the over-expression of the antiapoptotic protein Bcl-2 decreases the endoplasmic reticulum
(ER) Ca2+ load and protects cells from death. Alternatively buffering 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 cytoplasm 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
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their function (ATP viability and cell proliferation) is similar or
greater (although not statistically significant) than normal irradiated 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 oligomycin 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 maintaining 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 chemical 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 differentiation. 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 apoptosis, most likely by decreasing ROS production, down-regulating
pro-apoptotic proteins and activating anti-apoptotic proteins, as
well as by increasing energy metabolism.
Medical Technology SA
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 apoptotic 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 membrane and elevations in intracellular Ca2+ may mediate apoptosis. Irradiated 5% EtOH treated cells showed a decrease in intracellular 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 additional 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 transducing 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 contrary, 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 production 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 attenuates 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 (reversible) or repair oxidative stress to reduce apoptotic signals however 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 pathway. The irradiated tBHP treated cells showed a significant decrease in ATP viability, increase in cytotoxicity and modest decrease 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 apoptotic 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 coincidental 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
Volume 26 No. 1 | June 2012
maintained in the cytoplasm. Cell stress often results in highly
elevated calcium levels, depolarised mitochondrial membrane
potential, decreased cAMP levels and decreased ATP production. 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 intracellular ion concentration including calcium. Laser irradiation
has been shown to cause mitochondrial polarization which improves membrane permeability leading to increased Ca2+ flux,
pH value, cAMP level, ATP production, anti-apoptotic factors,
transcription factors, and DNA synthesis. This cascade of cellular 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 nutrient 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 theoretically 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-estimate 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 temporary indication of toxicity. Additional assays such as growth
curves, clonogenic assays and DNA synthesis (BrdU incorporation) [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 irradiation. 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. normal vs. oligomycin). Thirdly, true assays for apoptosis and autophagy 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 using fluorescent probes) and autophagy (by electron microscopy
ISSN 1011 5528 | www.smltsa.org.za
41
Medical Technology SA
assessing lipidation of the LC3 protein or using other methods
recently described [49-51]. Lastly, cellular responses were measured 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 cellular 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, apoptotic 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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