Photobiomodulation inhibits the activation of neurotoxic microglia and astrocytes by inhibiting Lcn2/JAK2-STAT3 crosstalk after spinal cord injury in male rats.

BACKGROUND: Neurotoxic microglia and astrocytes begin to activate and participate in pathological processes after spinal cord injury (SCI), subsequently causing severe secondary damage and affecting tissue repair. We have previously reported that photobiomodulation (PBM) can promote functional recovery by reducing neuroinflammation after SCI, but little is known about the underlying mechanism. Therefore, we aimed to investigate whether PBM ameliorates neuroinflammation by modulating the activation of microglia and astrocytes after SCI. METHODS: Male Sprague-Dawley rats were randomly divided into three groups: a sham control group, an SCI + vehicle group and an SCI + PBM group. PBM was performed for two consecutive weeks after clip-compression SCI models were established. The activation of neurotoxic microglia and astrocytes, the level of tissue apoptosis, the number of motor neurons and the recovery of motor function were evaluated at different days post-injury (1, 3, 7, 14, and 28 days post-injury, dpi). Lipocalin 2 (Lcn2) and Janus kinase-2 (JAK2)-signal transducer and activator of transcription-3 (STAT3) signaling were regarded as potential targets by which PBM affected neurotoxic microglia and astrocytes. In in vitro experiments, primary microglia and astrocytes were irradiated with PBM and cotreated with cucurbitacin I (a JAK2-STAT3 pathway inhibitor), an adenovirus (shRNA-Lcn2) and recombinant Lcn2 protein. RESULTS: PBM promoted the recovery of motor function, inhibited the activation of neurotoxic microglia and astrocytes, alleviated neuroinflammation and tissue apoptosis, and increased the number of neurons retained after SCI. The upregulation of Lcn2 and the activation of the JAK2-STAT3 pathway after SCI were suppressed by PBM. In vitro experiments also showed that Lcn2 and JAK2-STAT3 were mutually promoted and that PBM interfered with this interaction, inhibiting the activation of microglia and astrocytes. CONCLUSION: Lcn2/JAK2-STAT3 crosstalk is involved in the activation of neurotoxic microglia and astrocytes after SCI, and this process can be suppressed by PBM.

Thymoquinone Enhances the Effect of Gamma Knife in B16-F10 Melanoma Through Inhibition of Phosphorylated STAT3.

BACKGROUND: Patients with brain metastasis from melanoma have a dismal prognosis with poor survival time. Gamma Knife (GK) is an effective treatment to control brain metastasis from melanoma. Thymoquinone (TQ) has emerged as a potential therapeutic option due to its antiproliferative effects on various cancers. The purpose of the study was to assess the effect of GK on B16-F10 melanoma cells in vitro and intracerebral melanoma in vivo, and its synergistic effect in combination with TQ. METHODS: The effects of GK and combination treatment of GK and TQ were studied on B16-F10 melanoma cells by evaluating cytotoxicity with an adenosine triphosphate assay, apoptosis by acridine orange staining, and genotoxicity by comet assay. Western blot analysis was performed to investigate the expression of STAT3, p-STAT3 (Tyr705), JAK2, p-JAK2, caspase-3, Bax, Bcl-2, survivin, and ß-actin. Expression of inflammatory cytokines was assessed by enzyme-linked immunosorbent assay. GK alone and in combination with TQ was assessed in an established intracerebral melanoma tumor in mice. RESULTS: The effects of GK on cytotoxicity, genotoxicity, and apoptosis were enhanced by TQ in B16-F10 melanoma cells. GK induced apoptosis through inhibition of p-STAT3 expression, which in turn regulated pro- and antiapoptotic proteins such as caspase-3, Bax, Bcl-2, and survivin. Adding TQ to GK irradiation further enhanced this apoptotic effect of GK irradiation. GK was shown to reduce the levels of tumor-related inflammatory cytokines in B16-F10 melanoma cells. This effect was more pronounced when TQ was added to GK irradiation. GK with 15 Gy increased the survival of mice with intracerebral melanoma compared with untreated mice. However, despite the additive effect of TQ in addition to GK irradiation on B16-F10 melanoma cells in vitro, TQ did not add any significant survival benefit to GK treatment in mice with intracerebral melanoma. CONCLUSIONS: Our findings suggest that TQ would be a potential therapeutic agent in addition to GK to enhance the antitumor effect of irradiation. Further studies are required to support our findings.

Repeated exposure to far infrared ray attenuates acute restraint stress in mice via inhibition of JAK2/STAT3 signaling pathway by induction of glutathione peroxidase-1.

Exposure to far-infrared ray (FIR) has been shown to exert beneficial effects on cardiovascular and emotional disorders. However, the precise underlying mechanism mediated by FIR remains undetermined. Since restraint stress induces cardiovascular and emotional disorders, the present study investigated whether exposure to FIR affects acute restraint stress (ARS) in mice. c-Fos-immunoreactivity (IR) was significantly increased in the paraventricular hypothalamic nucleus (PVN) and dorsomedial hypothalamic nucleus (DMH) in response to ARS. The increase in c-Fos-IR parallels that in oxidative burdens in the hypothalamus against ARS. Exposure to FIR significantly attenuated increases in the c-Fos-IR, oxidative burdens and corticosterone level. ARS elicited decreases in GSH/GSSG ratio, cytosolic Cu/Zn-superoxide dismutase (SOD-1), glutathione peroxidase (GPx), and glutathione reductase (GR) activities. FIR-mediated attenuation was particularly observed in ARS-induced decrease in GPx, but not in SOD-1 or GR activity. Consistently, ARS-induced decreases in GPx-1-immunoreactivity in PVN and DMH, and decreases in GPx-1 expression in the hypothalamus were significantly attenuated by FIR. ARS-induced significant increases in phosphorylation of JAK2/STAT3, and nuclear translocation and DNA-binding activity of NFκB were observed in the hypothalamus. Exposure to FIR selectively attenuated phosphorylation of JAK2/STAT3, but did not diminish nuclear translocation and DNA-binding activity of NFκB, suggesting that JAK2/STAT3 constitutes a critical target for FIR-mediated pharmacological potential. ARS-induced increase in c-Fos-IR in the PVN and DMH of non-transgenic mice was significantly attenuated by FIR exposure or JAK2/STAT3 inhibitor AG490. GPx-1 overexpressing transgenic mice significantly protected increases in the c-Fos-IR and corticosterone level induced by ARS. However, neither FIR exposure nor AG490 significantly affected attenuations by genetic overexpression of GPx-1. Moreover, AG490 did not exhibit any additional positive effects against the attenuation by genetic overexpression of GPx-1 or FIR exposure. Our results indicate that exposure to FIR significantly protects ARS-induced increases in c-Fos-IR and oxidative burdens via inhibition of JAK2/STAT3 signaling by induction of GPx-1.

Photodynamic therapy activated STAT3 associated pathways: Targeting intrinsic apoptotic pathways to increase PDT efficacy in human squamous carcinoma cells.

5-Aminolaevulinic acid-based photodynamic therapy (ALA-PDT) has been used for part of squamous cell carcinoma (premalignant conditions or in situ cutaneous SCC-Bowen disease). However, mechanism of ALA-PDT is not fully understood yet on the cell apoptosis pathway. The aim of this study was to further investigate the effect and mechanism of 5-ALA-PDT on human squamous carcinoma A431cells. Apoptosis and cell viability after PDT were evaluated using Annexin V-FITC apoptosis detection kit and MTT assay. The mRNA and protein levels were detected by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot. Our data showed that 5-ALA-PDT significantly inhibited cell proliferation (p<0.05), but there was no significant difference when the photosensitizer reached to 4.8mM. The inhibition in cell proliferation after 5-ALA-PDT treatment was correlated to more cells being arrested in the G0/G1 phase of the cell cycle (p<0.01). Immunocytochemical observations using anti-active caspase-3 antibodies showed active caspase-3 was translocated from cytoplasm to nuclear during apoptosis. STAT3 and its downstream gene Bax and BCL-2 were changed after 5-ALA-PDT treatment for the mRNA and protein expression. Our studies confirmed that 5-ALA-PDT might be an effective treatment for human squamous carcinoma by inhibiting the tumor cell A431growth and for the first time demonstrated that the expression of STAT3 was significantly reduced at 24h after 5-ALA-PDT treatment.

Protein tyrosine phosphatases, TC-PTP, SHP1, and SHP2, cooperate in rapid dephosphorylation of Stat3 in keratinocytes following UVB irradiation.

Stat3 is initially dephosphorylated in murine keratinocytes in response to UVB irradiation. Treatment with Na(3)VO(4) desensitized keratinocytes to UVB-induced apoptosis with the recovery of phosphorylated Stat3 protein levels, implying that a protein tyrosine phosphatase (PTP) is involved in this mechanism. In the current work, we report that three PTPs including TC45 (the nuclear form of TC-PTP), SHP1, and SHP2 are involved in this rapid dephosphorylation of Stat3 in keratinocytes induced by UVB irradiation. Dephosphorylation of Stat3 was increased rapidly after UVB irradiation of cultured keratinocytes. Knockdown of TC-PTP, SHP1, or SHP2 using RNAi showed that these PTPs are likely responsible for most of the rapid Stat3 dephosphorylation observed following UVB irradiation. The level of phosphorylated Stat3 was significantly higher in keratinocytes transfected with TC-PTP, SHP1, or SHP2 siRNA in the presence or absence of UVB compared with keratinocytes transfected with control siRNA. TC45 was mainly localized in the cytoplasm of keratinocytes and translocated from cytoplasm to nucleus upon UVB irradiation. Stat3 dephosphorylation was associated with nuclear translocation of TC45. Further studies revealed that knockdown of all three phosphatases, using RNAi, prevented the rapid dephosphorylation of Stat3 following UVB irradiation. In mouse epidermis, the level of phosphorylated Stat3 was initially decreased, followed by a significant increase at later time points after UVB exposure. The levels of Stat3 target genes, such as cyclin D1 and c-Myc, followed the changes in activated Stat3 in response to UVB irradiation. Collectively, these results suggest that three phosphatases, TC45, SHP1, and SHP2, are primarily responsible for UVB-mediated Stat3 dephosphorylation and may serve as part of an initial protective mechanism against UV skin carcinogenesis.

Heavy ion irradiation increases apoptosis and STAT-3 expression, led to the cells arrested at G2/M phase in human hepatoma SMMC-7721 cells.

BACKGROUND: The impact of STAT-3 expression on the apoptosis of human hepatomas cell SMMC-7721 line induced by X-ray and carbon ion irradiations was investigated. METHODS: Human hepatoma SMMC-7721 cells were irradiated with a carbon ion beam and X-ray. Cell survival was determined by a standard colony-forming assay. STAT-3 protein expression was analysed by Western Immunoblots. Cell cycle and apoptosis were performed by flow cytometry. RESULTS: The viability of SMMC-7721 cells decreased with increasing dose of the carbon ion beam, and the high-LET carbon ion beam led to the cells getting arrested at G(2)/M phase. Western Blot analyses show that STAT-3 expression increased with increasing radiation dose. The carbon ion irradiation induced cell apoptosis and significantly promoted the expression of STAT-3 gene compared with the X-ray irradiation. The apoptosis rate is correlated with the expression of STAT-3 in human hepatoma SMMC-7721 cells after exposure to different doses of X-ray and heavy ion beam. CONCLUSIONS: Heavy ion irradiation increases the expression of STAT-3 gene, makes SMMC-7721 cells arrested at G(2)/M phase and increases cell apoptosis in comparison with that induced by low-LET X-ray. The STAT-3 expression may be regarded as a protected reaction when the cancerous cells suffer a strong stimulus such as high-LET irradiation. The interaction of STAT-3 expression and other cytokines in human hepatoma and the relationship between STAT-3 and radiation-induced apoptosis remain to be clarified in the future.

Radiation-induced HIF-1alpha cell survival pathway is inhibited by soy isoflavones in prostate cancer cells.

We previously showed that treatment of prostate cancer cells with soy isoflavones and radiation resulted in greater cell killing in vitro, and caused downregulation of NF-kappaB and APE1/Ref-1. APE1/Ref-1 functions as a redox activator of transcription factors, including NF-kappaB and HIF-1alpha. These molecules are upregulated by radiation and implicated in radioresistance of cancer cells. We extended our studies to investigate the role of HIF-1alpha survival pathway and its upstream Src and STAT3 molecules in isoflavones and radiation interaction. Radiation induced phosphorylation of Src and STAT3 leading to induction of HIF-1alpha. Genistein, daidzein or a mixture of soy isoflavones did not activate this pathway. These data were observed both in PC-3 (AR-) and C4-2B (AR+) androgen-independent cell lines. Pretreatment with isoflavones inhibited Src/STAT3/HIF-1alpha activation by radiation and nuclear translocation of HIF-1alpha. These findings correlated with decreased expression of APE1/Ref-1 and DNA binding activity of HIF-1alpha and NF-kappaB. In APE1/Ref-1 cDNA transfected cells, radiation caused a greater increase in HIF-1alpha and NF-kappaB activities but this effect was inhibited by pretreatment with soy prior to radiation. Transfection experiments indicate that APE1/Ref-1 inhibition by isoflavones impairs the radiation-induced transcription activity of NF-kappaB and HIF-1alpha. This mechanism could result in the inhibition of genes essential for tumor growth and angiogenesis, as demonstrated by inhibition of VEGF production and HUVECs tube formation. Our novel findings suggest that the increased responsiveness to radiation mediated by soy isoflavones could be due to pleiotropic effects of isoflavones blocking cell survival pathways induced by radiation including Src/STAT3/HIF-1alpha, APE1/Ref-1 and NF-kappaB.