Proteasome activation is critical for cell death induced by inhibitors of polo-like kinase 1 (PLK1) in multiple cancers.

Inhibitors of polo-like kinase (PLK) are currently being evaluated as anticancer drugs. However, the molecular mechanism of PLK inhibitor-induced cell death is not fully understood. In this study, we found that GW843682X and BI2536, two inhibitors of PLK1, significantly induced cell death in multiple type cells. The induction of cell death was related to the preferring expression of PLK1. However, in human umbilical vascular endothelial cells (HUVEC) and human colorectal carcinoma cells, which expressed higher levels of both PLK1 and PLK2, PLK1 inhibitors induced very low levels of cell death. Clinical analysis reveals PLK1 presence in 26 of 30 NPC tumor tissues. In in vivo NPC lung metastasis nude mouse models, PLK1 inhibitors decreased NPC progress. Mechanistically, the PLK1 inhibitor did not activate p53, and the cell death was not reversed by p53 inhibition. Moreover, PLK1 inhibitor-induced cell death was PARP- and caspase-independent. Although PLK1 inhibitors induced down-regulation of calpain inhibitor calpastatin and calpain was activated by PLK1 inhibition, calpain blocking did not reverse cell death induced by PLK1 inhibitors, suggesting the non-involvement of calpain. Surprisingly, we found that PLK1 inhibitors induced the activation of proteasome, and the treatment of cells with PLK1 inhibitors reduced the levels of ubiquitinated proteins. And proteasome inhibitors reversed cell death induced by PLK1 inhibitors in various cell types in which PLK1 was preferentially expressed. Moreover, PLK1 inhibition reversed the degradation of proteins including p53, caspase 8, PARP and calpastatin. These results suggest that the activation of proteasome is critical for cell death induced by PLK1 inhibition.

STAT3 promotes cytoplasmic-nuclear translocation of RNA-binding protein HuR to inhibit IL-1ß-induced IL-8 production.

Signal transducer and activator of transcription 3 (STAT3) functions to regulate inflammation and immune response, but its mechanism is not fully understood. We report here that STAT3 inhibitors Stattic and Niclosamide up-regulated IL-1ß-induced IL-8 production in C33A, CaSki, and Siha cervical cancer cells. As expected, IL-1ß-induced IL-8 production was also up-regulated through the molecular inhibition of STAT3 by use of CRISPR/Cas9 technology. Unexpectedly, IL-1ß induced IL-8 production via activating ERK and P38 signal pathways, but neither STAT3 inhibitors nor STAT3 knockout affected IL-1ß-induced signal transduction, suggesting that STAT3 decreases IL-8 production not via inhibition of signal transduction. To our surprise, STAT3 inhibition increased the stabilization, and decreased the degradation of IL-8 mRNA, suggesting a post-transcriptional regulation of IL-1ß-induced IL-8. Moreover, Dihydrotanshinone I, an inhibitor of RNA-binding protein HuR, down-regulated IL-1ß-induced IL-8 dose-dependently. HuR inhibition by CRISPR/Cas9 also decreased IL-8 production induced by IL-1ß. Mechanistically, co-immunoprecipitation results showed that STAT3 did not react with HuR directly, but STAT3 inhibition increased the protein levels of HuR in cytoplasm. And IL-6 activation of STAT3 induced HuR cytoplasmic-nuclear transport. Taken together, these results suggest that STAT3 contributes to HuR nuclear localization and inhibits Il-1ß-induced IL-8 production through this non-transcriptional mechanism.

A modified U-Net convolutional neural network for segmenting periprostatic adipose tissue based on contour feature learning.

Objective: This study trains a U-shaped fully convolutional neural network (U-Net) model based on peripheral contour measures to achieve rapid, accurate, automated identification and segmentation of periprostatic adipose tissue (PPAT). Methods: Currently, no studies are using deep learning methods to discriminate and segment periprostatic adipose tissue. This paper proposes a novel and modified, U-shaped convolutional neural network contour control points on a small number of datasets of MRI T2W images of PPAT combined with its gradient images as a feature learning method to reduce feature ambiguity caused by the differences in PPAT contours of different patients. This paper adopts a supervised learning method on the labeled dataset, combining the probability and spatial distribution of control points, and proposes a weighted loss function to optimize the neural network's convergence speed and detection performance. Based on high-precision detection of control points, this paper uses a convex curve fitting to obtain the final PPAT contour. The imaging segmentation results were compared with those of a fully convolutional network (FCN), U-Net, and semantic segmentation convolutional network (SegNet) on three evaluation metrics: Dice similarity coefficient (DSC), Hausdorff distance (HD), and intersection over union ratio (IoU). Results: Cropped images with a 270 × 270-pixel matrix had DSC, HD, and IoU values of 70.1%, 27 mm, and 56.1%, respectively; downscaled images with a 256 × 256-pixel matrix had 68.7%, 26.7 mm, and 54.1%. A U-Net network based on peripheral contour characteristics predicted the complete periprostatic adipose tissue contours on T2W images at different levels. FCN, U-Net, and SegNet could not completely predict them. Conclusion: This U-Net convolutional neural network based on peripheral contour features can identify and segment periprostatic adipose tissue quite well. Cropped images with a 270 × 270-pixel matrix are more appropriate for use with the U-Net convolutional neural network based on contour features; reducing the resolution of the original image will lower the accuracy of the U-Net convolutional neural network. FCN and SegNet are not appropriate for identifying PPAT on T2 sequence MR images. Our method can automatically segment PPAT rapidly and accurately, laying a foundation for PPAT image analysis.

Magnified x ray ghost imaging with tapered polycapillary optics free of the penumbra effect.

X ray ghost imaging (XGI) offers both radiation dose-reduction potential and cost-effective benefits owing to the utilization of a single-pixel detector. Most XGI schemes with laboratory x ray sources require a mechanically moving mask for either structured illumination or structured detection. In either configuration, however, its resolution remains limited by the source size and the unit size of the mask. Upon propagation, the details of the object can actually be magnified by the divergence of x rays, but at the same time, the penumbra effect produced by the finite source size is dramatically intensified, which ultimately leads to a degradation of image quality in XGI. To address these limitations, this work proposes a magnified XGI scheme using structured detection equipped with tapered polycapillary optics, which can efficiently suppress the object's penumbra as well as resolve the magnified details of the object. In general, the resolution of this scheme is no longer affected by the source size but by the microcapillary size of polycapillary. Our work fundamentally achieves cancellation of penumbra effect-induced aberration, thus paving the way for high-resolution magnified XGI.

Endoplasmic reticulum stress is upregulated in inflammatory bowel disease and contributed TLR2 pathway-mediated inflammatory response.

OBJECTIVE: Endoplasmic reticulum stress (ERS) and Toll-like receptor 2 (TLR2) signaling play an important role in inflammatory bowel disease (IBD); however, the link between TLR2 and ERS in IBD is unclear. This study investigated whether Thapsigargin (TG) -induced ER protein expression levels contributed to TLR2-mediated inflammatory response. METHODS: The THP-1 cells were treated with TLR2 agonist (Pam3CSK4), ERS inducer Thapsigargin (TG) or inhibitor (TUDCA). The mRNA expressions of TLR1-TLR10 were detected by qPCR. The production and secretion of inflammatory factors were detected by PCR and ELISA. Immunohistochemistry was used to detect the expressions of GRP78 and TLR2 in the intestinal mucosa of patients with Crohn's disease (CD). The IBD mouse model was established by TNBS in the modeling group. ERS inhibitor (TUDCA) was used in the treatment group. RESULTS: The expression of TLRs was detected via polymerase chain reaction (PCR) in THP-1 cells treated by ERS agonist Thapsigargin (TG). According to the findings, TG could promote TLR2 and TLR5 expression. Subsequently, in TLR2 agonist Pam3CSK4 induced THP-1 cells, TG could lead to increased expression of the inflammatory factors such as TNF-α, IL-1ß and IL-8, and ERS inhibitor (TUDCA) could block this effect. However, Pam3CSK4 did not significantly impact the GRP78 and CHOP expression. Based upon the immunohistochemical results, TLR2 and GRP78 expression were significantly increased in the intestinal mucosa of patients with Crohn's disease (CD). For in vivo experiments, TUDCA displayed the ability to inhibit intestinal mucosal inflammation and reduce GRP78 and TLR2 proteins. CONCLUSIONS: ERS and TLR2 is upregulated in inflammatory bowel disease, ERS may promote TLR2 pathway-mediated inflammatory response. Moreover, ERS and TLR2 signaling could be novel therapeutic targets for IBD.

Natural and synthetic compounds for glioma treatment based on ROS-mediated strategy.

Glioma is the most frequent and most malignant tumor of the central nervous system (CNS)，accounting for about 50% of all CNS tumor and approximately 80% of the malignant primary tumors in the CNS. Patients with glioma benefit from surgical resection, chemo- and radio-therapy. However these therapeutical strategies do not significantly improve the prognosis, nor increase survival rates owing to restricted drug contribution in the CNS and to the malignant characteristics of glioma. Reactive oxygen species (ROS) are important oxygen-containing molecules that regulate tumorigenesis and tumor progression. When ROS accumulates to cytotoxic levels, this can lead to anti-tumor effects. Multiple chemicals used as therapeutic strategies are based on this mechanism. They regulate intracellular ROS levels directly or indirectly, resulting in the inability of glioma cells to adapt to the damage induced by these substances. In the current review, we summarize the natural products, synthetic compounds and interdisciplinary techniques used for the treatment of glioma. Their possible molecular mechanisms are also presented. Some of them are also used as sensitizers: they modulate ROS levels to improve the outcomes of chemo- and radio-therapy. In addition, we summarize some new targets upstream or downstream of ROS to provide ideas for developing new anti-glioma therapies.

Visualization and quantification of coconut using advanced computed tomography postprocessing technology.

INTRODUCTION: Computed tomography (CT) is a non-invasive examination tool that is widely used in medicine. In this study, we explored its value in visualizing and quantifying coconut. MATERIALS AND METHODS: Twelve coconuts were scanned using CT for three months. Axial CT images of the coconuts were obtained using a dual-source CT scanner. In postprocessing process, various three-dimensional models were created by volume rendering (VR), and the plane sections of different angles were obtained through multiplanar reformation (MPR). The morphological parameters and the CT values of the exocarp, mesocarp, endocarp, embryo, bud, solid endosperm, liquid endosperm, and coconut apple were measured. The analysis of variances was used for temporal repeated measures and linear and non-linear regressions were used to analyze the relationship between the data. RESULTS: The MPR images and VR models provide excellent visualization of the different structures of the coconut. The statistical results showed that the weight of coconut and liquid endosperm volume decreased significantly during the three months, while the CT value of coconut apple decreased slightly. We observed a complete germination of a coconut, its data showed a significant negative correlation between the CT value of the bud and the liquid endosperm volume (y = -2.6955x + 244.91; R2 = 0.9859), and a strong positive correlation between the height and CT value of the bud (y = 1.9576 ln(x) -2.1655; R2 = 0.9691). CONCLUSION: CT technology can be used for visualization and quantitative analysis of the internal structure of the coconut, and some morphological changes and composition changes of the coconut during the germination process were observed during the three-month experiment. Therefore, CT is a potential tool for analyzing coconuts.

LCS-1 inhibition of superoxide dismutase 1 induces ROS-dependent death of glioma cells and degradates PARP and BRCA1.

Gliomas are characterized by high morbidity and mortality, and have only slightly increased survival with recent considerable improvements for treatment. An innovative therapeutic strategy had been developed via inducing ROS-dependent cell death by targeting antioxidant proteins. In this study, we found that glioma tissues expressed high levels of superoxide dismutase 1 (SOD1). The expression of SOD1 was upregulated in glioma grade III and V tissues compared with that in normal brain tissues or glioma grade I tissues. U251 and U87 glioma cells expressed high levels of SOD1, low levels of SOD2 and very low levels of SOD3. LCS-1, an inhibitor of SOD1, increased the expression SOD1 at both mRNA and protein levels slightly but significantly. As expected, LCS-1 caused ROS production in a dose- and time-dependent manner. SOD1 inhibition also induced the gene expression of HO-1, GCLC, GCLM and NQO1 which are targeting genes of nuclear factor erythroid 2-related factor 2, suggesting the activation of ROS signal pathway. Importantly, LCS-1 induced death of U251 and U87 cells dose- and time-dependently. The cell death was reversed by the pretreatment of cells with ROS scavenges NAC or GSH. Furthermore, LCS-1 decreased the growth of xenograft tumors formed by U87 glioma cells in nude mice. Mechanistically, the inhibition of P53, caspases did not reverse LCS-1-induced cell death, indicating the failure of these molecules involving in cell death. Moreover, we found that LCS-1 treatment induced the degradation of both PARP and BRCA1 simultaneously, suggesting that LCS-1-induced cell death may be associated with the failure of DNA damage repair. Taking together, these results suggest that the degradation of both PARP and BRCA1 may contribute to cell death induced by SOD1 inhibition, and SOD1 may be a target for glioma therapy.

Endoplasmic reticulum stress contributed to inflammatory bowel disease by activating p38 MAPK pathway.

Recent evidence suggests that endoplasmic reticulum (ER) stress plays a vital role in inflammatory bowel disease (IBD). Therefore, the aim of this study was to investigate the mechanism by which ER stress promotes inflammatory response in IBD. The expression of Gro-α, IL-8 and ER stress indicator Grp78 in colon tissues from patients with Crohn's disease (CD) and colonic carcinoma was analyzed by immunohistochemistry staining. Colitis mouse model was established by the induction of trinitrobenzene sulphonic acid (TNBS), and the mice were treated with ER stress inhibitor tauroursodeoxycholic acid (TUDCA). Then the body weight, colon length and colon inflammation were evaluated, and Grp78 and Gro-α in colon tissues were detected by immunohistochemistry. Epithelial cells of colon cancer HCT116 cells were treated with tunicamycin to induce ER stress. Grp78 was detected by Western blot, and chemokines were measured by PCR and ELISA. The expression levels of Grp78, Gro-α and IL-8 were significantly upregulated in intestinal tissues of CD patients. Mice with TNBS induced colitis had increased expression of Grp78 and Gro-α in colonic epithelia. TUDCA reduced the severity of TNBS-induced colitis. In HCT116 cells, tunicamycin increased the expression of Grp78, Gro-α and IL-8 in a concentration-dependent manner. Furthermore, p38 MAPK inhibitor significantly inhibited the upregulation of Gro-α and IL-8 induced by tunicamycin. In conclusion, ER stress promotes inflammatory response in IBD, and the effects may be mediated by the activation of p38 MAPK signaling pathway.

TLR4 promoted endoplasmic reticulum stress induced inflammatory bowel disease via the activation of p38 MAPK pathway.

Endoplasmic reticulum (ER) stress contribute to inflammatory bowel disease (IBD). However, the mechanistic link between toll-like receptor 4 (TLR4) and ER stress in IBD remains elusive. This study aimed to investigate the mechanism by which ER stress and TLR4 promote inflammation in IBD. IBD mouse model was established by the induction of TNBS, and Grp78 and TLR4 in intestine tissues were detected by immunohistochemistry. THP-1 cells were treated with lipopolysaccharides (LPS), ER stress inducer or inhibitor tauroursodeoxycholic acid (TUDCA), or p38 MAPK inhibitor. The activation of MAPK signaling was detected by Western blot, and the production and secretion of inflammatory factors were detected by PCR and ELISA. We found that the expression levels of TLR4 and GRP78 were significantly higher in the intestine of IBD model mice compared with control mice but were significantly lower in the intestine of IBD model mice treated with ER stress inhibitor TUDCA. ER stress inducer significantly increased while ER stress inhibitor TUDCA significantly decreased the expression and secretion of TNF-α, IL-1ß and IL-8 in THP-1 cells treated by LPS. Only p38 MAPK signaling was activated in THP-1 cells treated by ER stress inducer. Furthermore, p38 inhibitor SB203580 inhibited the production and secretion of TNF-α, IL-1ß and IL-8 in THP-1 cells treated with LPS. In conclusion, TLR4 promotes ER stress induced inflammation in IBD, and the effects may be mediated by p38 MAPK signaling. TLR4 and p38 MAPK signaling are novel therapeutic targets for IBD.

Proteasome inhibitors decrease paclitaxel­induced cell death in nasopharyngeal carcinoma with the accumulation of CDK1/cyclin B1.

Southeast Asia is a region with high incidence of nasopharyngeal carcinoma (NPC). Paclitaxel is the mainstay for the treatment of advanced nasopharyngeal cancer. The present study investigated the effect of proteasome inhibitors on the therapeutic effect of paclitaxel and its related mechanism. The present data from Cell Counting Kit­8 and flow cytometry assays demonstrated that appropriate concentrations of proteasome inhibitors (30 nM PS341 or 700 nM MG132) reduced the lethal effect of paclitaxel on the nasopharyngeal cancer cells. While 400 nM paclitaxel effectively inhibited cell division and induced cell death, proteasome inhibitors (PS341 30 nM or MG132 700 nM) could reverse these effects. Additionally, the western blotting results demonstrated accumulation of cell cycle regulation protein CDK1 and cyclin B1 in proteasome inhibitor­treated cells. In addition, proteasome inhibitors combined with paclitaxel led to decreased MCL1 apoptosis regulator, BCL2 family member/Caspase­9/poly (ADP­ribose) polymerase apoptosis signaling triggered by CDK1/cyclin B1. Therefore, dysfunction of CDK1/cyclin B1 could be defining the loss of paclitaxel lethality against cancer cells, a phenomenon affirmed by the CDK1 inhibitor Ro3306. Overall, the present results demonstrated that a combination of paclitaxel with proteasome inhibitors or CDK1 inhibitors is antagonistic to effective clinical management of NPC.

A Novel Biological Activity of the STAT3 Inhibitor Stattic in Inhibiting Glutathione Reductase and Suppressing the Tumorigenicity of Human Cervical Cancer Cells via a ROS-Dependent Pathway.

INTRODUCTION: Glutathione reductase (GSR) provides reduced glutathione (GSH) to maintain redox homeostasis. Inhibition of GSR disrupts this balance, resulting in cell damage, which benefits cancer therapy. However, the effect of GSR inhibition on the tumorigenicity of human cervical cancer is not fully understood. MATERIALS AND METHODS: Tissue microarray analysis was employed to determine GSR expression in cervical cancer tissues by immunohistochemical staining. Cell death was measured with PI/FITC-annexin V staining. mRNA levels were measured via quantitative RT-PCR. Protein expression was measured by Western blotting and flow cytometry. STAT3 deletion was performed with CRISPR/Cas9 technology. GSR knockdown was achieved by RNA interference. Reactive oxygen species (ROS) levels were measured by DCF staining. GSR enzymatic activity was measured with a GSR assay kit. The effect of GSR inhibition on the growth of tumors formed by cervical cancer cells was investigated using a xenograft model. RESULTS: The expression of GSR was increased in human cervical cancer tissues, as shown by immunohistochemical staining. GSR knockdown by RNA interference in human cervical cancer cell lines resulted in cell death, suggesting the ability of GSR to maintain cancer cell survival. The STAT3 inhibitor 6-nitrobenzo[b]thiophene 1,1-dioxide (Stattic) also inhibited the enzymatic activity of GSR and induced the death of cervical cancer cells. More importantly, Stattic decreased the growth of xenograft tumors formed by cervical cancer cells in nude mice. Mechanistically, tumor cell death induced by Stattic-mediated GSR inhibition was ROS-dependent, since the ROS scavengers GSH and N-acetyl cysteine (NAC) reversed the effect of Stattic. In contrast, pharmacological and molecular inhibition of STAT3 did not induce the death of cervical cancer cells, suggesting a STAT3-independent activity of Stattic. CONCLUSION: Stattic inhibits the enzymatic activity of GSR and induces STAT3-independent but ROS-dependent death of cervical cancer cells, suggesting its potential application as a therapeutic agent for human cervical cancers.

DNA damage/cGAS-triggered up-regulation of MALAT1 promotes undesirable inflammatory responses in radiotherapy of cancer.

Radiotherapy is the most common strategy for treating cancer. However, the radiation-induced inflammatory responses, acute or chronic, in the normal tissues of the irradiated region may result in undesirable side effects, such as lung injury and atherosclerosis. MALAT1 is believed to function in the onset, development, progression and metastasis of various cancers. Silencing MALAT1 may be a promising treatment for rescuing cancer. Nevertheless, whether MALAT1 promotes the radiation-induced undesirable inflammatory response is still uncovered. The present study reveals that radiation-induced DNA damage triggers cGAS signaling and subsequently increases the expression of MALAT1. Overexpression of MALAT1 inhibits the function of miR146a in the suppression of STAT1, which results in the boost of adhesion molecules and eventually induces acute lung injury and atherosclerosis. Thus, silencing MALAT1 may facilitate the reduction of radiation-induced acute and chronic complications in the radiotherapy of cancer.

Pharmacological inhibition of GSK3 promotes TNFα-induced GM-CSF via up-regulation of ERK signaling in nasopharyngeal carcinoma (NPC).

Granulocyte-macrophage colony stimulating factor (GM-CSF) functions to drive nasopharyngeal cancer (NPC) metastasis via recruitment and activation of macrophages. However, the source and the regulation of GM-CSF in tumor microenvironment of NPC are not fully understood. In this study, we found that TNFα induced GM-CSF production in NPC CNE1, CNE2, and 5-8F cells in time- and dose-dependent manners. GM-CSF production was tolerant, because the pre-treatment of NPC cells with TNFα down-regulated the GM-CSF production induced by TNFα re-treatment. TNFα activated glycogen synthase kinase-3 (GSK-3), which is an enzyme to regulate glycogen synthesis, and also is a critical downstream element of the PI3K/Akt to regulate cell survival. GSK3 inhibitors up-regulated TNFα-induced GM-CSF, and reversed GM-CSF tolerance induced by TNFα pre-treatment, suggesting that GSK3 activation down-regulated GM-CSF production. GM-CSF down-regulation was not related to ubiquitin-editing enzyme A20. The over-expression of A20 did not regulate GM-CSF production induced by TNFα. However, GSK3 inhibitors up-regulated ERK activation, which contributed to the production of GM-CSF induced by TNFα, suggesting that GSK3 negatively regulated TNFα-induced GM-CSF via down-regulation of ERK signaling. Taking together, these results suggested that GSK3 pathway may be a target for the regulation of TNFα-induced GM-CSF in the tumor microenvironment.

H19 regulates angiogenic capacity of extravillous trophoblasts by H19/miR-106a-5p/VEGFA axis.

PURPOSE: To investigate the role and underlying mechanism of H19 in regulating angiogenic capacity of extravillous trophoblasts. METHODS: Gain and loss of function experiments were performed using a human first-trimester extravillous trophoblast (EVT) cell line, HTR-8/SVneo cells. H19 was overexpressed or knocked down in HTR-8 cells by transfecting plasmid harboring whole-length H19 sequence (pH19) or siRNA specially targeting H19, respectively (siH19). Cell migration and tube-formation assay were assessed in the indicated groups. Gene expression was detected by RT-qPCR, Western blot, and ELISA assay. RESULTS: Overexpression of H19 in EVT cells increased cell migration and tube formation, while downregulation of H19 in EVT cells decreased cell migration and tube formation. Furthermore, we found that H19 played its role by VEGFA. In addition, we demonstrated the H19/miR-106a-5p/VEGFA regulatory axis in EVT. Experiments of the clinical specimen showed that H19 was very abundantly expressed in human first-trimester trophoblasts, and we found that the expression of H19 and VEGFA were significantly downregulated in the villous tissues from idiopathic recurrent miscarriage (RM) patients; moreover, the expression of H19 and VEGFA was positively correlated. CONCLUSION: H19/miR-106a-5p/VEGFA axis plays a role in regulating the angiogenic capacity of EVT, which might contribute to idiopathic RM.

X-rays induced IL-8 production in lung cancer cells via p38/MAPK and NF-κB pathway.

PURPOSE: It is reported inflammatory cytokine interleukin-8 (IL-8) could predict radiation-induced lung toxicity (RILT). RILT is believed to be a consequence of a cascade of cytokine production. It is considered that vascular endothelial cell and macrophages are the mainly source of cytokines. This study was investigated the production of IL-8 from cancer cells induced by X-rays may involve in the radiation-induced inflammation. MATERIALS AND METHODS: We analyzed IL-8 in human lung cancer cell lines after expose to X-rays, and we also detect IL-8 in HUVEC cells and THP1 cells as endothelial cell and macrophage model to identify the change in normal cells after expose. Furthermore, we added the inhibitors to the culture with or without radiation to identify the role of MAPK and NF-κB pathways on the radiation-induced secretion of IL-8. RESULTS: Radiation could induce IL-8 production both in non-lung cancer cells (HUVECs and THP1 cells) and in lung cancer cells (A549 cells, H446 cells, PC-9 cells). Simultaneously, radiation activated p38/MAPK and NF-κB signal pathways in lung cancer cells. Moreover, p38/MAPK inhibitor SB203580 and NF-κB inhibitor BAY11-7082 could block the IL-8 up-regulated by X-rays but JNK inhibitor SP600125, ERK inhibitor U0126, ROS Scavenger NAC could not inhibit this phenomenon. CONCLUSIONS: X-rays could induce IL-8 production in lung cancer cells, which may be related to the activation of p38/MAPK and NF-κB signaling pathway, providing a new point for elucidating the mechanism of radiation pneumonitis.

VE-822 mediated inhibition of ATR signaling sensitizes chondrosarcoma to cisplatin via reversion of the DNA damage response.

INTRODUCTION: Cisplatin has been reported to elicit the DNA damage response (DDR) via activation of the ATR-Chk1 pathway, which in turn contributes to the induction of cisplatin resistance. Inhibition of ATR-Chk1 signaling reverses cisplatin resistance in some cancers. However, the influence of inhibiting ATR-Chk1 signaling on cisplatin resistance in chondrosarcoma cancer has not been reported. MATERIALS AND METHODS: We compared the expression levels of ATR kinases in human nasopharyngeal carcinoma, choriocarcinoma and chondrosarcoma cell lines. We inhibited ATR kinase function with VE-822, a selective ATR inhibitor, and suppressed ATR kinase expression with shRNA. Western blotting, the CCK-8 assay, cell cycle distribution assay and apoptosis analysis were used to study the influence of inhibiting ATR-Chk1 signaling on reversing cisplatin resistance in chondrosarcoma cell lines. RESULTS: We found that chondrosarcoma cells expressed very low basal levels of phosphorylated ATR, but cisplatin treatment induced the activation of ATR-Chk1 signaling in a dose- and time-dependent manner, suggesting the induction of DDR. As expected, ATR inhibition with VE-822 reversed cisplatin-induced DDR and enhanced cisplatin-induced activation of H2AX, which is an important marker of DNA damage. Meanwhile, ATR inhibition by RNA interference also reversed DDR and promoted DNA damage. Furthermore, both pharmacological and molecular inhibition of ATR accelerated cisplatin-induced inhibition of cell proliferation and cell death. CONCLUSION: Our results suggested that inhibiting ATR activation promoted cisplatin-induced cell death via reversion of DDR, and VE-822 may be a valuable strategy for the prevention of cisplatin resistance in chondrosarcoma.

IL-1α and IL-1ß promote NOD2-induced immune responses by enhancing MAPK signaling.

Both toll-like receptors (TLRs) and nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) induce a tightly regulated inflammatory response at risk of causing tissue damage, depending on the effectiveness of ensuing negative feedback regulatory mechanisms. Cross-regulation between TLRs, NLRs, and cytokine receptors has been observed. However, the cross-regulation between interleukin-1 (IL-1) receptors and NOD2 is not completely understood. In this study, we found that IL-1α/ß increased NOD2-induced inflammatory response in human monocytic THP1 cells, peripheral blood mononuclear cells (PBMCs), mouse macrophage RWA264.7 cells and spleen cells, and in an in vivo experiment. IL-1α/ß pre-treatment induced the production of CXC chemokines, including growth-regulated oncogene (GRO)-α, GRO-ß, and IL-8, and proinflammatory cytokines, including IL-1ß, IL-6, and TNFα, which are induced by the activation of NOD2, in a dose- and time-dependent manner. However, pre-treatment with the NOD2 ligand muramyl dipeptide (MDP) did not up-regulate the expression of cytokines induced by IL-1α/ß re-treatment. IL-1ß treatment increased the expression of A20, which is an important inhibitor of the innate immune response. However, the overexpression of A20 failed to inhibit MDP-induced cytokine production, suggesting that A20 had no effects on the NOD2-induced immune response. In addition, IL-1α/ß increased the expression of NOD2 and its downstream adaptor RIP2, and IL-1α/ß pre-treatment increased MDP-induced activation of mitogen-activated protein kinases (MAPKs), including ERK, JNK, and P38, which contributed to MDP-induced cytokine production. Based on these results, IL-1α/ß promote the NOD2-induced immune responses by enhancing MDP-induced activation of MAPK signaling pathways.

AKF-PD alleviates diabetic nephropathy via blocking the RAGE/AGEs/NOX and PKC/NOX Pathways.

Diabetic nephropathy (DN) is a major complication of diabetes. Currently, drugs are not available to effectively control the disease. Fluorofenidone (AKF-PD) is a recently developed drug; it possesses activities in reducing DN progression in preclinical research. Nonetheless, its renal protection and the underlying mechanisms have not been thoroughly investigated. We report here that AKF-PD significantly alleviatesrenal oxidative stress (OS) in db/dbmice through downregulation of Nicotinamide Adenine Dinucleotide Phosphate (NADPH) oxidase and upregulation of glutathione peroxidase and superoxide dismutase, thereby protecting kidney from DN pathogenesis. AKF-PD likely reduces OS through the advanced glycation end products (AGE) and protein kinase C (PKC) pathways. While renal AGEs, PKCα, PKCß, and NADPH oxidase 4 (NOX4) were all substantially upregulated in db/db mice compared to db/m animals, AKF-PD robustly downregulated all these events to the basal levelsdetected in db/m mice. In primary human renal mesangial cells (HMCs), high glucose (HG) elevated receptor for advanced glycation endproducts (RAGE), PKCα, PKCß and NOX4 activity, and induced the production of reactive oxygen species (ROS); these events were all inhibited by AKF-PD. Furthermore, HG led to mitochondrial damagein HMCs;AKF-PD conferred protection on the damage. Knockdown of either PKCα or PKCß reduced HG-induced ROS production and mitochondrial damage in HMCs. The knockdown significantly enhanced AKF-PD-mediated inhibition of ROS production and mitochondrial damage in HG-treated HMCs. Collectively, our study demonstrates that AKF-PD protects renal function under diabetes conditions in part through inhibition of OS during DN pathogenesis. AKF-PD can be explored for clinical applications in DN therapy.

The activation of Toll-like receptor 4 reverses tumor differentiation in human glioma U251 cells via Notch pathway.

Toll-like receptors (TLRs) are closely related to cancer. However, the mechanism for TLR regulation of cancer is not fully understood. Our previous studies demonstrated that toll-like receptor (TLR) 4 functions to maintain the un-differential stem cell phenotypes of human endothelial progenitor cells. In this study, we found that human glioma cells expressed several TLRs. The activation of TLR4 by LPS in glioma U251 cells induced the expression of cytokines, including IL-1ß, IL-6, IL-8, and TNFα, suggesting the functional expression of TLR4. Nude mouse in vivo studies showed that LPS treatment promoted tumor growth, and decreased mouse survival. But LPS treatment did not promote tumor cell proliferation in vitro. Meanwhile, we found that LPS treatment down-regulated the expression of glial fibrillary acidic protein (GFAP), an important differentiation maker of glioma, at both mRNA and protein levels. TLR4 activation also down-regulated GFAP in glioma Hs683 cells. LPS did not induce the activation of MAPKs, but induced the activation of NF-κB. However, pharmacological inhibition of NF-κB signaling did not reverse the down-regulation of GFAP. Furthermore, we found that LPS induced the activation of Notch pathway, which was MyD88-dependent, and Notch inhibition reversed the down-regulation of GFAP. In addition, LPS treatment up-regulated stem cell makers, including CD34 and CD133. Taken together, these results suggested that in human glioma U251 cells, TLR4 functions to reverse tumor differentiation, and it may be a target for glioma prevention and therapy.