KEYWORDS: Fluorescence resonance energy transfer, Luminescence, Laser irradiation, Proteins, Fluorescent proteins, Kidney, Life sciences, Cell mechanics, Molecules, Molecular energy transfer
Low-power laser irradiation (LPLI) has been regarded as playing a significant role in triggering cellular survival and
proliferation. However, the mechanism has not been fully understood. In this study, using real-time single-cell analysis,
we investigated the activity of Akt and its effects on cell proliferation induced by LPLI in african green monkey
SV40-transformed kidney fibroblast cells (COS-7). We utilized a recombinant fluorescence resonance energy transfer
(FRET) Akt probe (BKAR) to dynamically detect the activation of Akt after LPLI treatment. Our results show that
LPLI induced a gradual and continuous activation of Akt. Moreover, the activation of Akt can be completely abolished
by wortmannin, a specific inhibitor of PI3K, suggesting that the activation of Akt caused by LPLI is a PI3K-dependent
event. LPLI promotes cell proliferation through Akt activation since the cell viability was significantly inhibited by PI3K
inhibitor. We thus conclude that, Akt activation is well involved in LPLI triggered cell proliferation that acts as a time and dose-dependent manner.
Low-power laser irradiation (LPLI) leads to photochemical reaction and then activates intracellular several signaling
pathway. Reactive oxygen species (ROS) are considered to be the primary messengers produced by LPLI. Here, we
studied the signaling pathway mediated by ROS upon the stimulation of LPLI. Src tyrosine kinases are well-known
targets of ROS and can be activated by oxidative events. Using a Src reporter based on fluorescence resonance energy
transfer (FRET) technique, we visualized the dynamic Src activation in Hela cells immediately after LPLI. Moreover, Src
activity was enhanced by increasing the duration of LPLI. In addition, our results suggested that ROS were key
mediators of Src activation, as ROS scavenger, vitamin C decreased and exogenous H2O2 increased the activity of Src.
Meanwhile, Gö6983 loading did not block the effect of LPLI. CCK-8 experiments proved that cell vitality was
prominently improved by LPLI with all the doses we applied in our experiments ranging from 3 to 25J/cm2. The results
indicated that LPLI/ROS/Src pathway may be involved in the LPLI biostimulation effects.
Low-power laser irradiation (LPLI) has been shown to promote cell proliferation in various cell types, yet the
mechanism of which has not been fully clarified. Studying the signaling pathways involved in the laser irradiation is
important for understanding these processes. The Ras/Raf/MEK/ERK (extracellular-signal-regulated kinase) signaling
pathway is a network that governs proliferation, differentiation and cell survival. Recent studies suggest that Ras/Raf
signaling pathway is involved in the LPLI-induced cell proliferation, but the dynamic activation of Ras in living cells
induced by LPLI has not been reported. In present study, to monitor the dynamic activation of H-Ras after LPLI
treatment in living cells in real time, Raichu-Ras reporter was utilized, which was constructed based on fluorescence
resonance energy transfer (FRET) technique. Our results show that the dynamic activation of H-Ras at the cell edges of
the plasma membrane is monitored during cell proliferation induced by LPLI (0.8 J/cm2) in serum-starved COS-7 cells
expressing Raichu-Ras reporter using FRET imaging on laser scanning confocal microscope, and that LPLI causes the
redistribution of H-Ras from the cytoplasm to plasma membrane. The same results are observed in EGF treated COS-7
cells. Taken together, LPLI induces the COS-7 cells proliferation by activated Ras.
Low-power laser irradiation (LPLI) has been shown to promote cell proliferation in various cell types, yet the
mechanism of which has not been fully clarified. Investigating the signaling pathways involved in the laser irradiation is
important for understanding these processes. The small G protein Ras works as a binary switch in many important
intracellular signaling pathways and, therefore, has been one of the focal targets of signal-transduction investigations and
drug development. The Ras/Raf/MEK/ERK (extracellular-signal-regulated kinase) signaling pathway is a network that
governs proliferation, differentiation and cell survival. Recent studies suggest that Ras/Raf signaling pathway is involved
in the LPLI-induced cell proliferation. On the other hand, Protein kinase Cs (PKCs), the Ca2+ activated,
phospholipid-dependent serine/threonine protein kinases, have been recently presumed to be involved in the regulation
of cell proliferation induced by LPLI. In this report, to monitor the direct activations of Ras and PKCs after LPLI
treatment in living cells in real time, Raichu-Ras reporter and C kinase activity reporter (CKAR) were utilized, both of
which were constructed based on fluorescence resonance energy transfer (FRET) technique. The direct activation of Ras
is predominantly initiated from the different microdomains of the plasma membrane. The results are monitored during
cell proliferation induced by LPLI (0.8 J/cm2) in serum-starved COS-7 cells expressing Raichu-Ras reporter using FRET
imaging on laser scanning confocal microscope. Furthermore, the increasing activation of PKCs is also monitored during
cell proliferation induced by LPLI (0.8 J/cm2) in serum-starved human lung adenocarcinoma cells (ASTC-a-1)
expressing CKAR reporter using the similar way. Taken together, the dynamic increases of H-Ras and PKCs activities
are observed during the processes of cell proliferation induced by LPLI.
Bcl-2 family proteins (such as Bid and Bak/Bax) and 14-3-3 proteins play a key role in the mitochondria-mediated cell
apoptosis induced by cell death factors such as TNF-α and lower power laser irradiation (LPLI). In this report,
fluorescence resonance energy transfer (FRET) has been used to study the molecular mechanism of apoptosis in living
cells on a fluorescence scanning confocal microscope. Based on the genetic code technique and the green fluorescent
proteins (GFPs), single-cell dynamic analysis of caspase3 activation, caspase8 activation, and PKCs activation are
performed during apoptosis induced by laser irradiation in real-time. To investigate the cellular effect and mechanism of
laser irradiation, human lung adenocarcinoma cells (ASTC-a-1) transfected with plasmid SCAT3 (pSCAT3)/ CKAR
FRET reporter, were irradiated and monitored noninvasively with both FRET imaging. Our results show that high
fluence lower power laser irradiation (HFLPLI) can induce an increase of caspase3 activation and a decrease of PKCs
activation, and that LPLI induces the ASTC-a-1 cell proliferation by specifically activating PKCs.
Bax, a proapoptotic member of the Bcl-2 family, localizes largely in the cytoplasm but redistributes to mitochondria and
undergoes oligomerization to induce the release of apoptogenic factors such as cytochrome c in response to apoptotic
stimuli. Cytoplasmic protein 14-3-3zeta binds to Bax and, upon apoptotic stimulation, releases Bax by a
caspase-independent mechanism. However, the direct interaction of the cytoplasmic 14-3-3zeta and Bax in living cells
has not been observed. In present study, to monitor the dynamic interaction between 14-3-3zeta and Bax in living cells in
real time during apoptosis induced by tumor necrosis factor (TNF-α), DsRed-14-3-3zeta plasmid is constructed. By
cotransfecting DsRed- 14-3-3zeta and GFP-Bax plasmids into human lung adenocarcinoma cells (ASTC-a-1), we observe
the dynamic interaction between Bax and 14-3-3zeta using fluorescence resonance energy transfer (FRET) technique on
laser scanning confocal microscope. The results show that 14-3-3zeta remains in the cytoplasm but GFP-Bax translocates
to mitochondria completely after TNF-α stimulation. These results reveal that 14-3-3zeta binds directly to Bax in healthy
cells, and that 14-3-3zeta negatively regulates Bax translocation to mitochondria during TNF-α-induced apoptosis.
Low-power laser irradiation (LPLI) has been shown to promote cell proliferation in various cell types, yet the mechanism of which has not been fully clarified. Studying the signaling pathways involved in the laser irradiation is important for understanding these processes. The Ras/Raf/MEK/ERK (extracellular-signal-regulated kinase) signaling pathway is a network that governs proliferation, differentiation and cell survival. Recent studies suggest that Ras/Raf signaling pathway is involved in the LPLI-induced cell proliferation. Protein kinase Cs (PKCs) have been recently presumed to be involved in the regulation of cell proliferation induced by LPLI. In present study, to monitor the direct interaction between Ras and Raf and PKCs activation after LPLI treatment in living cells in real time, Raichu-Ras reporter and C kinase activity reporter (CKAR) were utilized, both of which were constructed based on fluorescence resonance energy transfer (FRET) technique. Our results show that the direct interaction between Ras and Raf is monitored during cell proliferation induced by LPLI (0.8 J/cm2) in serum-starved human lung adenocarcinoma cells (ASTC-a-1) expressing Raichu-Ras reporter using FRET imaging on laser scanning confocal microscope, and that the increasing dynamics of PKCs activity is also monitored during cell proliferation induced by LPLI (0.8 J/cm2) in serum-starved ASTC-a-1 cells expressing CKAR reporter using the similar way. Taken together, LPLI induces the ASTC-a-1 cell proliferation by activated Ras directly interacting with Raf and by specifically activating PKCs.
Light irradiation can modulate various biological processes. For instance, low-power laser irradiation (LPLI) can induce cell proliferation and differentiation. It has been used to treat diseases of regeneration limitation and to promote wound healing. The biological mechanism of light irradiation remains unclear. Our previous studies have shown that low fluence LPLI induced the proliferation of human lung adenocarcinoma cells (ASTC-a-1) through PKC channel, while high fluence LPLI induced caspase-3 activation and cell apoptosis. The mechanisms of the initiation and regulation of apoptosis are complex and diverse. There are two main pathways to initiate and regulate cell apoptosis, one is the death receptor pathway (receptor/caspase-8/caspase-3), and the other is the mitochondria pathway (mitochondria/ caspase-9/caspase-3). Using fluorescent imaging techniques, we observed a temporal sequence of events during apoptosis induced by high fluence LPLI and PDT. Both the high fluence LPLI and PDT triggers mitochondrial ROS production resulting in dissipation of ΔΨm and activation of caspase-3. Our results also show the two treatments do not activate caspase-8. These results suggest that caspase-3 activation induced by high fluence LPLI or PDT is initiated directly from mitochondria ROS generation and dissipation of ΔΨm, and independent of the cell death pathway involving caspase-8 activation. Because the progression of the apoptosis induced by high fluence LPLI is the same as that of PDT, we concluded that light is absorbed directly either by endogenous porphyrins or by the cytochromes in mitochondrion, resulting in initial ROS generation. During light irradiation induced apoptosis, apoptotic signals are initiated from mitochondrial ROS production due to photosensitization.
Protein kinase Cs (PKCs) play an important role in cellular proliferation, and low-energy laser irradiation (LELI) can enhance cellular proliferation. The present work contributes to the understanding of the mechanisms of action by studying effects of LELI at the dose of 0.8 J/cm2 on PKCs activities in the single lung adenocarcinoma cell (ASTC-a-1) and in real time by fluorescence resonance energy transfer (FRET) technique. C-kinase activity reporter (CKAR), consisting of a cyan fluorescent protein (CFP), the FHA2 phosphothreonine-binding domain, a PKC substrate sequence, and a yellow fluorescent protein (YFP), was utilized. The living cell imaging showed a decrease in FRET in the cytosol and nucleus after the cells were treated with LELI. These results suggest that PKCs could be activated by LELI throughout the cell, and the proliferation of ASTC-a-1 cells could be modulated by the activated PKCs.
Access to the requested content is limited to institutions that have purchased or subscribe to SPIE eBooks.
You are receiving this notice because your organization may not have SPIE eBooks access.*
*Shibboleth/Open Athens users─please
sign in
to access your institution's subscriptions.
To obtain this item, you may purchase the complete book in print or electronic format on
SPIE.org.
INSTITUTIONAL Select your institution to access the SPIE Digital Library.
PERSONAL Sign in with your SPIE account to access your personal subscriptions or to use specific features such as save to my library, sign up for alerts, save searches, etc.