Low-power laser irradiation (LPLI) can cause cell proliferation, differentiation, or death; however, the cellular mechanisms of these effects of LPLI, at high or low fluences, are not well known. To investigate the mechanism of high-fluence LPLI-induced apoptosis, both human lung adenocarcinoma cells (ASTC-a-1) and African green monkey SV40-transformed kidney fibroblast cells (COS-7) were irradiated with a He-Ne laser for 10 min under a fluence of 120 J/cm2 and 80 J/cm2, respectively. The dynamics of reactive oxygen species (ROS) generation was determined by measuring changes in fluorescence resulting from oxidation of intracellular dichlorodihydrofluorescein diacetate (H2DCFDA) to (DCF). The changes of mitochondrial membrane potential, m, were studied by measuring the reduction of cellular fluorescence of Rhodamine 123 dyes using confocal laser scanning microscopy. The activation of caspase-3 in cells transfected by [SCAT3] reporters was observed using fluorescence resonance energy transfer (FRET) imaging. The activity of caspase-8 during high-fluence LPLI-induced apoptosis was studied by monitoring the cellular distribution of [Bid-CFP] reporters using fluorescence imaging. The following temporal sequence of cellular events was observed during apoptosis induced by high-fluence LPLI (120 J/cm2, ASTC-a-1 cells): (1) immediate generation of mitochondrial ROS following laser irradiation, reaching a maximum level 60 min after irradiation; (2) onset of m decrease 15 min after laser irradiation, reaching a minimum level 50 min after irradiation; and (3) activation of caspase-3 between 30 min and 180 min after laser irradiation. Our results also show that the high-fluence LPLI does not activate caspase-8, indicating that the induced apoptosis was initiated directly from mitochondrial ROS generation and m decrease, independent of the caspase-8 activation.
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.
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.
Low-power laser irradiation (LPLI) has been used for therapies such as curing spinal cord injury, healing
wound et al. Yet, the mechanism of LPLI remains unclear. Our previous study showed that low fluences laser irradiation
induces human lung adenocarcinoma cells (ASTC-a-1) proliferation, but high fluences induced apoptosis and caspase-3
activation. In order to study the mechanism of apoptosis induced by high fluences LPLI further, we have measured the
dynamics of generation of reactive oxygen species (ROS) using H2DCFDA fluorescence probes during this process.
ASTC-a-1 cells apoptosis was induced by He-Ne laser irradiation at high fluence of 120J/cm2. A confocal laser scanning
microscope was used to perform fluorescence imaging. The results demonstrated that high fluence LPLI induced the
increase of mitochondria ROS. Our studies contribute to clarify the biological mechanism of high fluence LPLI-induced
cell 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.
Ras/Raf signaling pathway is an important signaling pathway that governs cell proliferation, differential and apoptosis. Low-energy laser irradiation (LELI) was found to modulate various processes. Generally, cell proliferation is induced by low doses LELI and apoptosis is induced by high doses LELI. Mechanism of biological effect of LELI has not been clear. Recently, activation of MEK (mitogen-activated protein kinase) and ERK (extracellular-signal-regulated kinase), which are downstream protein kinases of Ras/Raf, are observed during LELI-induced cell proliferation by immunoprecipitation and western blot analysis. RaichuRas reporter consisting of fusions of H-ras, the Ras-binding domain of Raf (RafRBD), a cyan fluorescent protein (CFP) and a yellow fluorescent protein (YFP). Therefore, intramolecular binding of GTP-Ras to RafRBD brings CFP close to YFP and increases FRET between CFP and YFP. Human lung adenocarcinoma cell line (ASTC-a-1) was transfected with the plasmid (pRaichuRas) and then treated with LELI at dose of 60J/cm2. Effect of LELI on Ras/Raf in physiological condition of living cells was observed by fluorescence resonance energy transfer (FRET) technique during lung adenocarcinoma cell apoptosis induced by high dose (60J/cm2) LELI. Experimental results showed that after high dose LELI treatment, the binding of Ras and Raf decreases obviously, Ras/Raf signaling pathway deregulates and cell apoptosis occurs.
Caspase8 is activated and cleaves Bid into two fragments when cells are exposed to death-inducing molecules such as tumor necrosis factor-α (TNF-α). Then the C-terminal fragment relocates from cytosol to mitochondria and promotes the release of cytochrome c, in the final cellular apoptosis is induced. Despite recent progress in the study of Bid during apoptosis induction, it remains unclear how C-terminal fragment of Bid cleaved moves to mitochondria and then induces the release of cytochrome c and so on. The 14-3-3 proteins are known to sequester certain pro-apoptotic members of Bcl-2 family. In order to further study the biological action of Bid during apoptosis, especially under physiological condition of living cell, the plasmids pBid-CFP and pYFP-14-3-3 were constructed. By the transient transfection of pBid-CFP and pYFP-14-3-3, the dynamic process of interaction of Bid and 14-3-3 protein in individual living cell during the apoptosis was primarily investigated with FRET (fluorescent resonance energy transfer) technique by the use of fluorescence microscopy.
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.
Low-energy laser irradiation (LELI) has been shown to promote cell proliferation in various cell types, yet the mechanism of which has not been fully clarified. The Ras/Raf/MEK (mitogen-activated protein kinase)ERK kinase)/ERK (extracellular-signal-regulated kinase) signaling pathway is a network that govern proliferation, differentiation and cell survival. Recent studies suggested that Ras/Raf/MEK/ERK pathway is involved in the LELI-induced cell proliferation. Here, we utilized fluorescence resonance energy transfer (FRET) technique to investigate the effect of LELI on Ras/Raf signaling pathway in living cells. Raichu-Ras reporter plasmid was utilized which consisted of fusions of H-ras, the Ras-binding domain of Raf(RafRBD), a cyan fluorescent protein (CFP) and a yellow fluorescent protein (YFP), so that intramolecular binding of GTP-Ras to RafRBD brings CFP close to YFP and increases FRET between CFP and YFP. Human lung adenocarcinoma cell line (ASTC-a-1) were transfected with the plasmid (pRaichu-Ras) and then were treated by LELI. The living cell imaging showed the increase of FRET at different time points after LELI at the dose of 1.8 J/cm2, which corresponds to the Ras/Raf activation assayed by Western Blotting. Furthermore, this dose of LELI enhanced the proliferation of ASTC-a-1 cells. Taken together, these in vivo imaging data provide direct evidences with temporal or spatial resolution that Ras/Raf/MEK/ pathway plays an important role in LELI-promoted cell proliferation.
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