High Fluence Low-power Laser Irradiation Research Articles

In vitro anti-tumor effect of high-fluence low-power laser light on apoptosis of human colorectal cancer cells.

Colorectal cancer is the third most common malignancy all over the world, along with high morbidity and mortality. As a treatment, high-fluence low-power laser irradiation (HF-LPLI) has reported that its biostimulatory activity can suppress or even destruct tumor growth in neoplastic diseases. The aim of the present study is to examine a therapeutic capacity of HF-LPLI for colorectal cancer treatment by using human colon cancer cell (HT29) model. The in vitro cancer cell model was used to analyze the underlying mechanism of laser-induced apoptosis. Laser irradiation was performed five times (once a day for five consecutive days) with 635nm laser light for 8 and 16min (fluence = 128 and 256J/cm2), respectively. The efficiency of the HF-LPLI treatment was evaluated by MTT, fluorescence staining, cell wound healing, and western blot test during the 5-day period. Experiment data showed that HF-LPLI had a dose-dependent stimulating effect on cell viability, migration, and apoptosis of HT29 cells. The inhibition effect of laser treatment at 256J/cm2 on cell viability was statistically significant. Meanwhile, the wound healing and western blot tests also confirmed that HF-LPLI could inhibit cell migration and induce cell apoptosis. The current research results demonstrate that 635nm HF-LPLI can be an alternative treatment option for colorectal cancer by increasing the expression of caspase-3 and inducing HT29 tumor cell apoptosis through activation of the mitochondrial pathway.

High-fluence low-power laser irradiation promotes odontogenesis and inflammation resolution in periodontitis by enhancing stem cell proliferation and differentiation.

Periodontitis can exert a severe impact on the life of patients, and the use of stem cell therapy for this disease is promising. The inflammatory response consequent to periodontitis can promote stem cell proliferation. Activated inflammation triggers inhibitory cytokine secretion, thus reducing inflammation subsequent to stem cell activation. High-fluence low-power laser irradiation (HF-LPLI) has the ability to regulate stem cell function through its effect on inflammation. Thus, the aim of the present study was to examine whether HF-LPLI is able to activate stem cells to promote regeneration in periodontitis by promoting inflammation resolution, as well as to evaluate the underlying mechanism of action if an effect is observed. Stem cells were treated with HF-LPLI following inflammation activation. Reverse transcription-quantitative polymerase chain reaction and EdU assay were used to evaluate cell proliferation and differentiation. Flow cytometry and immunofluorescence were also used to detect the ability of HF-LPLI to regulate the surrounding inflammatory environment. Animal models of periodontal disease were treated with stem cells and HF-LPLI, and regeneration was detected by hematoxylin and eosin staining and in vivo imaging. It was observed that HF-LPLI promoted inflammation resolution by reducing the excessive inflammatory response, and finally stimulated stem cell proliferation and differentiation. Furthermore, in vivo results revealed that stem cells treated with HF-LPLI induced bone regeneration. HF-LPLI stimulated stem cell proliferation and differentiation by promoting inflammation resolution subsequent to stem cell activation, providing a new strategy for the clinical treatment of periodontitis.

Phototherapy-Induced Antitumor Immunity: Long-Term Tumor Suppression EffectsviaPhotoinactivation of Respiratory Chain Oxidase-Triggered Superoxide Anion Burst

Our previous studies have demonstrated that as a mitochondria-targeting cancer phototherapy, high-fluence, low-power laser irradiation (HF-LPLI) results in oxidative damage that induces tumor cell apoptosis. In this study, we focused on the immunological effects of HF-LPLI phototherapy and explored its antitumor immune regulatory mechanism. We found not only that HF-LPLI treatment induced tumor cell apoptosis but also that HF-LPLI-treated apoptotic tumor cells activated macrophages. Due to mitochondrial superoxide anion burst after HF-LPLI treatment, tumor cells displayed a high level of phosphatidylserine oxidation, which mediated the recognition and uptake by macrophages with the subsequent secretion of cytokines and generation of cytotoxic T lymphocytes. In addition, in vivo results showed that HF-LPLI treatment caused leukocyte infiltration into the tumor and efficaciously inhibited tumor growth in an EMT6 tumor model. These phenomena were absent in the respiration-deficient EMT6 tumor model, implying that the HF-LPLI-elicited immunological effects were dependent on the mitochondrial superoxide anion burst. In this study, for the first time, we show that HF-LPLI mediates tumor-killing effects via targeting photoinactivation of respiratory chain oxidase to trigger a superoxide anion burst, leading to a high level of oxidatively modified moieties, which contributes to the phenotypic changes in macrophages and mediates the antitumor immune response. Our results suggest that HF-LPLI may be an effective cancer treatment modality that both eradicates the treated primary tumors and induces an antitumor immune response via photoinactivation of respiratory chain oxidase to trigger superoxide anion burst.

Cancer PhototherapyviaSelective Photoinactivation of Respiratory Chain Oxidase to Trigger a Fatal Superoxide Anion Burst

Here, we develop a novel cancer treatment modality using mitochondria-targeting, high-fluence, low-power laser irradiation (HF-LPLI) in mouse tumor models and explore the mechanism of mitochondrial injury by HF-LPLI. We demonstrated that the initial reaction after photon absorption was photosensitization of cytochrome c oxidase (COX), to inhibit enzymatic activity of COX in situ and cause respiratory chain superoxide anion (O2(-•)) burst. We also found that HF-LPLI exerted its main tumor killing effect through mitochondrial O2(-•) burst via electron transport chain (ETC). These phenomena were completely absent in the respiration-deficient cells and COX knockdown cells. With a carefully selected irradiation protocol, HF-LPLI could efficaciously destroy tumors. The inhibition of enzymatic activity of COX and generation of O2(-•) by HF-LPLI in vivo were also detected. It is the first time that the mechanism involved in the interaction between light and its photoacceptor under HF-LPLI treatment is clarified. Our results clearly indicate that HF-LPLI initiates its effects via targeted COX photoinactivation and that the tumor-killing efficacy is dependent of the subsequent mitochondrial O2(-•) burst via ETC. Based on both in vitro and in vivo results, we conclude that HF-LPLI can selectively photoinactivate respiratory chain oxidase to trigger a fatal mitochondrial O2(-•) burst, producing oxidative damage on cancer cells. This study opens up the possibilities of applications of HF-LPLI as a mitochondria-targeting cancer phototherapy.

Mitochondrial oxidative stress causes mitochondrial fragmentation via differential modulation of mitochondrial fission–fusion proteins

Mitochondria are dynamic organelles that undergo continual fusion and fission to maintain their morphology and functions, but the mechanism involved is still not clear. Here, we investigated the effect of mitochondrial oxidative stress triggered by high-fluence low-power laser irradiation (HF-LPLI) on mitochondrial dynamics in human lung adenocarcinoma cells (ASTC-a-1) and African green monkey SV40-transformed kidney fibroblast cells (COS-7). Upon HF-LPLI-triggered oxidative stress, mitochondria displayed a fragmented structure, which was abolished by exposure to dehydroascorbic acid, a reactive oxygen species scavenger, indicating that oxidative stress can induce mitochondrial fragmentation. Further study revealed that HF-LPLI caused mitochondrial fragmentation by inhibiting fusion and enhancing fission. Mitochondrial translocation of the profission protein dynamin-related protein 1 (Drp1) was observed following HF-LPLI, demonstrating apoptosis-related activation of Drp1. Notably, overexpression of Drp1 increased mitochondrial fragmentation and promoted HF-LPLI-induced apoptosis through promoting cytochrome c release and caspase-9 activation, whereas overexpression of mitofusin 2 (Mfn2), a profusion protein, caused the opposite effects. Also, neither Drp1 overexpression nor Mfn2 overexpression affected mitochondrial reactive oxygen species generation, mitochondrial depolarization, or Bax activation. We conclude that mitochondrial oxidative stress mediated through Drp1 and Mfn2 causes an imbalance in mitochondrial fission-fusion, resulting in mitochondrial fragmentation, which contributes to mitochondrial and cell dysfunction.

High fluence low‐power laser irradiation induces apoptosis via inactivation of Akt/GSK3β signaling pathway

High fluence low-power laser irradiation (HF-LPLI) is a newly discovered stimulus through generating reactive oxygen species (ROS) to trigger cell apoptosis. Activation of glycogen synthase kinase 3β (GSK3β) is proved to be involved in intrinsic apoptotic pathways under various stimuli. However, whether the proapoptotic factor GSK3β participates in HF-LPLI-induced apoptosis has not been elucidated. Therefore, in the present study, we investigated the involvement of GSK3β in apoptosis under HF-LPLI treatment (120 J/cm2, 633 nm). We found that GSK3β activation could promote HF-LPLI-induced apoptosis, which could be prevented by lithium chloride (a selective inhibitor of GSK3β) exposure or by GSK3β-KD (a dominant-negative GSK3β) overexpression. We also found that the activation of GSK3β by HF-LPLI was due to the inactivation of protein kinase B (Akt), a widely reported and important upstream negative regulator of GSK3β, indicating the existence and inactivation of Akt/GSK3β signaling pathway. Moreover, the inactivation of Akt/GSK3β pathway depended on the fluence of HF-LPLI treatment. Furthermore, vitamin c, a ROS scavenger, completely prevented the inactivation of Akt/GSK3β pathway, indicating ROS generation was crucial for the inactivation. In addition, GSK3β promoted Bax activation by down-regulating Mcl-1 upon HF-LPLI treatment. Taken together, we have identified a new and important proapoptotic signaling pathway that is consisted of Akt/GSK3β inactivation for HF-LPLI stimulation. Our research will extend the knowledge into the biological mechanisms induced by LPLI.

The reactive oxygen species–Src–Stat3 pathway provokes negative feedback inhibition of apoptosis induced by high‐fluence low‐power laser irradiation

High-fluence low-power laser irradiation (HF-LPLI) can induce apoptosis by triggering mitochondrial oxidative stress. Signal transducer and activator of transcription 3 (Stat3) is an important transcription factor in the modulation of cell proliferation and apoptosis. Here, using real-time single-cell analysis and western blotting analysis, we investigated the changes in activities of Stat3 in COS-7 cells upon HF-LPLI (633 nm, 80 and 120 J·cm(-2)) and the underlying mechanisms involved. We found that Stat3 was significantly activated by HF-LPLI in a time-dependent and dose-dependent manner. Stat3 activation attenuated HF-LPLI-induced apoptosis, as shown by the fact that both dominant negative Stat3 (Y705F) and Stat3 small interfering RNA expression enhanced cellular apoptosis induced by HF-LPLI. Moreover, we also found that Src kinase was the major positive regulator of Stat3 activation induced by HF-LPLI. Reactive oxygen species (ROS) generation was essential for Stat3 and Src activation upon HF-LPLI, because scavenging of ROS by vitamin C or N-acetylcysteine totally abrogated the activation of Stat3 and Src. Taken together, these findings show that the ROS-Src-Stat3 pathway mediates a negative feedback inhibition of apoptosis induced by HF-LPLI in COS-7 cells. Our research will provide new insights into the mechanism of apoptosis caused by HF-LPLI, and also extend the functional study of Stat3.

Survivin mediates self-protection through ROS/cdc25c/CDK1 signaling pathway during tumor cell apoptosis induced by high fluence low-power laser irradiation

Survivin, an important member of inhibitor-of-apoptosis (IAP) family, can be up-regulated by various pro-apoptotic stimuli, such as UV, photodynamic therapy (PDT) and cisplatin. High fluence low-power laser irradiation (HF-LPLI) is a newly discovered pro-apoptotic stimulator. The anti-apoptotic mechanism of survivin during HF-LPLI-induced apoptosis is still not investigated. Here, we report that HF-LPLI up-regulates survivin activity through reactive oxygen species (ROS)/cdc25c protein phosphatase (cdc25c)/cyclin-dependent kinase (CDK1) signaling pathway in human lung adenocarcinoma cells (ASTC-a-1). The up-regulation of survivin activity can reduce HF-LPLI-induced apoptosis, while down-regulation of the activity can promote the apoptosis. In addition, activated survivin delays mitochondrial depolarization, cytochrome c release, caspase-9 and Bax activation, all of which are typical pro-apoptotic events of cell apoptosis induced by HF-LPLI. On the basis of the present studies, we conclude that survivin can mediate self-protection during tumor cell apoptosis caused by HF-LPLI.

High fluence low‐power laser irradiation induces mitochondrial permeability transition mediated by reactive oxygen species

High fluence low-power laser irradiation (HF-LPLI) can induce cell apoptosis via the mitochondria/caspase-3 pathway. Here, we further investigated the mechanism involved in the apoptotic process in human lung adenocarcinoma cells (ASTC-a-1) at a laser irradiation fluence of 120 J/cm(2) (633 nm). Cytochrome c release was ascribed to mitochondrial permeability transition (MPT) because the release was prevented by cyclosporine (CsA), a specific inhibitor of MPT. Furthermore, mitochondrial permeability for calcein (approximately 620 Da) was another evidence for the MPT induction under HF-LPLI treatment. A high-level intracellular reactive oxygen species (ROS) generation was observed after irradiation. The photodynamically produced ROS caused onset of MPT, as the ROS scavenger docosahexaenoic acid (DHA) prevented the MPT. However, CsA failed to prevented cell death induced by HF-LPLI, indicating the existence of other signaling pathways. Following laser irradiation, Bax activation occurred after mitochondrial depolarization and cytochrome c release, indicating Bax activation was a downstream event. In the presence of CsA, Bax was still activated at the end-stage of apoptotic process caused by HF-LPLI, suggesting that Bax was involved in an alternative-signaling pathway, which was independent of MPT. Under HF-LPLI treatment, cell viabilities due to pre-treatment with DHA, CsA, or Bax small interfering RNA (siRNA) demonstrated that the MPT signaling pathway was dominant, while Bax signaling pathway was secondary, and more importantly ROS mediated both pathways. Taken together, these results showed that HF-LPLI induced cell apoptosis via the CsA-sensitive MPT, which was ROS-dependent. Furthermore, there existed a secondary signaling pathway through Bax activation. The observed link between MPT and triggering ROS could be a fundamental phenomenon in HF-LPLI-induced cell apoptosis.

Mechanistic study of apoptosis induced by high-fluence low-power laser irradiation using fluorescence imaging techniques

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/cm(2) and 80 J/cm(2), respectively. The dynamics of reactive oxygen species (ROS) generation was determined by measuring changes in fluorescence resulting from oxidation of intracellular dichlorodihydrofluorescein diacetate (H(2)DCFDA) to (DCF). The changes of mitochondrial membrane potential, DeltaPsim, 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/cm(2), ASTC-a-1 cells): (1) immediate generation of mitochondrial ROS following laser irradiation, reaching a maximum level 60 min after irradiation; (2) onset of DeltaPsim 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 DeltaPsim decrease, independent of the caspase-8 activation.

Single cell analysis of PKC activation during proliferation and apoptosis induced by laser irradiation

Laser irradiation has been shown to trigger cellular proliferation and apoptosis in various cell types. Studying the signaling pathways involved in the laser irradiation is important for understanding these processes. In present study, to monitor the protein kinase Cs (PKCs) activity in living cells in real time, we transfected and screened human lung adenocarcinoma cells (ASTC-a-1) stably expressing C kinase activity reporter (CKAR) constructed based on fluorescence resonance energy transfer (FRET) technique. The CKAR is a specific, reversible reporter of phosphorylation by PKCs and it can monitor the ongoing balance between PKCs and phosphatases. The increasing dynamics of PKCs activity is monitored during cell proliferation induced by low-power laser irradiation (LPLI) (0.8 J/cm2) in serum-starved ASTC-a-1 cells stably expressing CKAR reporter using FRET imaging on laser scanning confocal microscope and using spectrofluorometric analysis on a luminescence spectrometer, respectively. However, the decreasing dynamics of PKCs activity has been monitored in real time using FRET imaging for the cells treated with high fluence LPLI (60 J/cm2), which was previously found to induce cell apoptosis. Taken together, LPLI induces the ASTC-a-1 cell proliferation by specifically activating PKCs. However, PKCs activity decreases during cell apoptosis induced by high fluence LPLI. Our results indicate that PKCs play an important role in the laser irradiation-induced biological effects.

Measuring dynamics of caspase-3 activity in living cells using FRET technique during apoptosis induced by high fluence low-power laser irradiation

Low-power laser irradiation (LPLI) has been used for therapies such as curing spinal cord injury, healing wound etc. Yet, the mechanism of LPLI remains unclear. In order to determine the effects of high fluence LPLI on cell growth and caspase-3 activity, we have measured the dynamics of caspase-3 activity during cell apoptosis induced by high fluence LPLI treatment. He-Ne laser was used to irradiate human lung adenocarcinoma cells (ASTC-a-1). Cell Counting Kit-8 was used for cytotoxicity assay. A fluorescent microscope was used to perform fluorescence resonance energy transfer (FRET) imaging. A luminescence spectrometer was used to acquire the fluorescent emission spectrum. Statistical analysis was performed with Student's paired t-test. Cytotoxicity assay showed that when light irradiation fluence exceeded 60 J/cm2, LPLI treatment induced ASTC-a-1 cell apoptosis in a fluence-dependent manner. FRET imaging and spectrofluorometric analysis demonstrated that caspase-3 was activated during high fluence LPLI-induced cell apoptosis. Using FRET technique, we have reported that high fluence LPLI can induce human lung adenocarcinoma cells (ASTC-a-1) apoptosis. The activation of caspase-3 plays an important role in the apoptotic process.