Chaperonin counteracts diet-induced non-alcoholic fatty liver disease by aiding sirtuin 3 in the control of fatty acid oxidation.

AIMS/HYPOTHESIS: The mitochondrial chaperonin heat shock protein (HSP) 60 is indispensable in protein folding and the mitochondrial stress response; however, its role in nutrient metabolism remains uncertain. This study investigated the role of HSP60 in diet-induced non-alcoholic fatty liver disease (NAFLD). METHODS: We studied human biopsies from individuals with NAFLD, murine high-fat-diet (HFD; a diet with 60% energy from fat)-induced obesity (DIO), transgenic (Tg) mice overexpressing Hsp60 (Hsp60-Tg), and human HepG2 cells transfected with HSP60 cDNA or with HSP60 siRNA. Histomorphometry was used to assess hepatic steatosis, biochemistry kits were used to measure insulin resistance and glucose tolerance, and an automated home cage phenotyping system was used to assess energy expenditure. Body fat was assessed using MRI. Macrophage infiltration, the lipid oxidation marker 4-hydroxy-2-nonenal (4-HNE) and the oxidative damage marker 8-hydroxy-2'-deoxyguanosine (8-OHdG) were detected using immunohistochemistry. Intracellular lipid droplets were evaluated by Nile red staining. Expression of HSP60, and markers of lipogenesis and fatty acid oxidation were quantified using RT-PCR and immunoblotting. Investigations were analysed using the two-way ANOVA test. RESULTS: Decreased HSP60 expression correlated with severe steatosis in human NAFLD biopsies and murine DIO. Hsp60-Tg mice developed less body fat, had reduced serum triglyceride levels, lower levels of insulin resistance and higher serum adiponectin levels than wild-type mice upon HFD feeding. Respiratory quotient profile indicated that fat in Hsp60-Tg mice may be metabolised to meet energy demands. Hsp60-Tg mice showed amelioration of HFD-mediated hepatic steatosis, M1/M2 macrophage dysregulation, and 4-HNE and 8-OHdG overproduction. Forced HSP60 expression reduced the mitochondrial unfolded protein response, while preserving mitochondrial respiratory complex activity and enhancing fatty acid oxidation. Furthermore, HSP60 knockdown enhanced intracellular lipid formation and loss of sirtuin 3 (SIRT3) signalling in HepG2 cells upon incubation with palmitic acid (PA). Forced HSP60 expression improved SIRT3 signalling and repressed PA-mediated intracellular lipid formation. SIRT3 inhibition compromised HSP60-induced promotion of AMP-activated protein kinase (AMPK) phosphorylation and peroxisome proliferator-activated receptor α (PPARα levels), while also decreasing levels of fatty acid oxidation markers. CONCLUSION/INTERPRETATION: Mitochondrial HSP60 promotes fatty acid oxidation while repressing mitochondrial stress and inflammation to ameliorate the development of NAFLD by preserving SIRT3 signalling. This study reveals the hepatoprotective effects of HSP60 and indicates that HSP60 could play a fundamental role in the development of therapeutics for NAFLD or type 2 diabetes.

ONC201 Suppresses Neuroblastoma Growth by Interrupting Mitochondrial Function and Reactivating Nuclear ATRX Expression While Decreasing MYCN.

Neuroblastoma (NB) is characterized by several malignant phenotypes that are difficult to treat effectively without combination therapy. The therapeutic implication of mitochondrial ClpXP protease ClpP and ClpX has been verified in several malignancies, but is unknown in NB. Firstly, we observed a significant increase in ClpP and ClpX expression in immature and mature ganglion cells as compared to more malignant neuroblasts and less malignant Schwannian-stroma-dominant cell types in human neuroblastoma tissues. We used ONC201 targeting ClpXP to treat NB cells, and found a significant suppression of mitochondrial protease, i.e., ClpP and ClpX, expression and downregulation of mitochondrial respiratory chain subunits SDHB and NDUFS1. The latter was associated with a state of energy depletion, increased reactive oxygen species, and decreased mitochondrial membrane potential, consequently promoting apoptosis and suppressing cell growth of NB. Treatment of NB cells with ONC201 as well as the genetic attenuation of ClpP and ClpX through specific short interfering RNA (siRNA) resulted in the significant upregulation of the tumor suppressor alpha thalassemia/mental retardation X-linked (ATRX) and promotion of neurite outgrowth, implicating mitochondrial ClpXP proteases in MYCN-amplified NB cell differentiation. Furthermore, ONC201 treatment significantly decreased MYCN protein expression and suppressed tumor formation with the reactivation of ATRX expression in MYCN-amplified NB-cell-derived xenograft tumors. Taken together, ONC201 could be the potential agent to provide diversified therapeutic application in NB, particularly in NB with MYCN amplification.

Inappropriate manipulation and drainage exacerbate post-operative pain and prolong the hospital stay after laparoscopic appendectomy for pediatric complicated appendicitis.

BACKGROUND: This study examined whether drain placement or not is associated with the postoperative outcomes of pediatric patients following trans-umbilical single-port laparoscopic appendectomy (TUSPLA) for complicated appendicitis. METHODS: The medical records of pediatric patients undergoing TUSPLA for acute complicated appendicitis from January 2012 to September 2018 in Kaohsiung Chang Gung Memorial Hospital were reviewed retrospectively. They were classified according to whether they received passive drainage with a Penrose drain (Penrose group) (19), active drainage with a Jackson-Pratt drain with a vacuum bulb (JP group) (16), or no drain (non-drain group) (86). The postoperative outcomes of the three groups were compared. RESULTS: Postoperative visual analog scale pain score was significantly higher in the non-drain group than in either the JP group or Penrose group. Patients in the Penrose group had a significantly longer postoperative hospital stay than those in the non-drain group and a higher rate of intra-abdominal abscess, while patients in the JP group had a significantly shorter postoperative hospital stay; moreover, no patient in JP group developed a postoperative intra-abdominal abscess. CONCLUSIONS: Compared to passive drainage with a Penrose drain or no drain, active drainage with a JP drain shorter the postoperative hospital stay and decreased the risk of postoperative intra-abdominal abscess.

Immunomodulator polyinosinic-polycytidylic acid enhances the inhibitory effect of 13-cis-retinoic acid on neuroblastoma through a TLR3-related immunogenic-apoptotic response.

High-risk neuroblastoma is associated with low long-term survival rates due to recurrence or metastasis. Retinoids, including 13-cis-retinoic acid (13cRA), are commonly used for the treatment of high-risk neuroblastoma after myeloablative therapy; however, there are significant side effects and resistance rates. In this study, we demonstrated that 13cRA has a better antiproliferative effect in MYCN-amplified neuroblastoma cells than in MYCN-nonamplified neuroblastoma cells. In MYCN-amplified SK-N-DZ cells, 13cRA induced significant upregulation of toll-like receptor 3 (TLR3) and mitochondrial antiviral-signaling protein (MAVS) expression in a time-dependent manner. Furthermore, poly (I:C), a synthetic agonist of TLR3, effectively synergized with 13cRA to enhance antiproliferative effects through upregulation of the innate immune signaling and the mitochondrial stress response, leading to augmentation of the apoptotic response in 13cRA-responsive cancer cells. In addition, the 13cRA/poly (I:C) combination induced neural differentiation through activation of retinoic acid receptors beta (RAR-ß), restoring expression of α-thalassemia/mental retardation syndrome X-linked (ATRX) protein, and inhibiting vessel formation, leading to retarded tumor growth in a mouse xenograft model. These results suggest that the combination of poly (I:C) and RA may provide synergistic therapeutic benefits for treatment of patients with high-risk neuroblastoma.

Constant Light Dysregulates Cochlear Circadian Clock and Exacerbates Noise-Induced Hearing Loss.

Noise-induced hearing loss is one of the major causes of acquired sensorineural hearing loss in modern society. While people with excessive exposure to noise are frequently the population with a lifestyle of irregular circadian rhythms, the effects of circadian dysregulation on the auditory system are still little known. Here, we disturbed the circadian clock in the cochlea of male CBA/CaJ mice by constant light (LL) or constant dark. LL significantly repressed circadian rhythmicity of circadian clock genes Per1, Per2, Rev-erbα, Bmal1, and Clock in the cochlea, whereas the auditory brainstem response thresholds were unaffected. After exposure to low-intensity (92 dB) noise, mice under LL condition initially showed similar temporary threshold shifts to mice under normal light-dark cycle, and mice under both conditions returned to normal thresholds after 3 weeks. However, LL augmented high-intensity (106 dB) noise-induced permanent threshold shifts, particularly at 32 kHz. The loss of outer hair cells (OHCs) and the reduction of synaptic ribbons were also higher in mice under LL after noise exposure. Additionally, LL enhanced high-intensity noise-induced 4-hydroxynonenal in the OHCs. Our findings convey new insight into the deleterious effect of an irregular biological clock on the auditory system.

Innate immune sensor laboratory of genetics and physiology 2 suppresses tumor cell growth and functions as a prognostic marker in neuroblastoma.

The innate immune receptors, such as toll-like receptor 3 (TLR3), melanoma differentiation-associated 5 (MDA5) and retinoic acid-inducible gene-I (RIG-I), have been shown to be differentially expressed in neuroblastoma (NB) and promote dsRNA poly (I:C)-induced NB suppression in vitro and in vivo. However, the role of another important innate immune cytosolic sensor, laboratory of genetics and physiology 2 (LGP2), in the cancer behavior of NB remains unclear. Here, we demonstrated that the expression levels of LGP2 were either low or undetectable in all NB cell lines tested with or without MYCN amplification. LGP2 expression levels were significantly increased only in NB cells without MYCN amplification, including SK-N-AS and SK-N-FI after poly (I:C) treatment in vitro and in mouse xenograft models. Ectopic expression of LGP2 in NB cells significantly enhanced poly (I:C)-induced NB cell death associated with downregulation of MDA5, RIG-I, MAVS and Bcl-2, as well as upregulation of Noxa and tBid. By immunofluorescence analyses, LGP2 localized mainly in the cytoplasm of NB cells after poly (I:C) treatment. In human NB tissue samples, cytoplasmic LGP2 expression was positively correlated with histological differentiation and inversely correlated with MYCN amplification. Positive cytoplasmic LGP2 expression in tumor tissues could predict a favorable outcome in NB patients independent of other prognostic factors. In short, LGP2 was effective in promoting poly (I:C)-induced NB suppression and cytoplasmic LGP2 can serve as an independent favorable prognostic factor in NB patients.

Soluble delta-like 1 homolog (DLK1) stimulates angiogenesis through Notch1/Akt/eNOS signaling in endothelial cells.

AIM: Delta-like 1 homolog (DLK1) is a non-canonical ligand of Notch signaling, which plays a pivotal role in vascular development and tumor angiogenesis. This study aimed to elucidate the function and mechanism of DLK1 in angiogenesis. METHODS AND RESULTS: By using in situ hybridization and immunohistochemical studies, expression analysis revealed a unique vascular tropism of DLK1 in vasculature of neuroblastoma and vascular tumors. Thus, it was hypothesized that DLK1 may be cleaved and then bound to endothelial cells, thereby regulating the endothelial function. To test such hypothesis, soluble DLK1 encompassing DLK1 extracellular domain (DLK1-EC) was generated and validated by its inhibitory function in adipogenesis assay. Recombinant DLK1-EC exhibited the preferential binding capability toward endothelial cells and stimulated the microvessels sprouting in aorta rings. Above all, implantation of DLK1-EC dose-dependently elicited the cornea neovascularization in rats. By using various angiogenesis assays, it was delineated that DLK1-EC stimulated the angiogenesis by promoting the proliferation, motility and tube formation of endothelial cells. By immunoblot and luciferase analysis, it was elucidated that DLK1-EC enhanced the expression and activities of Notch1/Akt/eNOS/Hes-1 signaling in dose- and time-dependent manners. Pharmaceutical blockage of Notch signaling using γ-secretase inhibitor DAPT abrogated the DLK1-EC-induced endothelial migration and Hes-1-driven luciferase activities. Furthermore, Notch1 inactivation by neutralizing antibodies or RNA interference reversed the DLK1-EC-induced angiogenesis. CONCLUSIONS: The present study unveils the pro-angiogenic function and mechanism of soluble DLK1 through activation of Notch1 signaling in endothelial cells.

Correction to: Soluble delta-like 1 homolog (DLK1) stimulates angiogenesis through Notch1/Akt/eNOS signaling in endothelial cells.

In the original publication of the article, there is an error in one of the citations in the Discussion section.

Increased expression of senescence-associated cell cycle regulators in the progression of biliary atresia: an immunohistochemical study.

AIMS: Cellular senescence plays a role in tumour suppression and in the pathogenesis of various non-neoplastic diseases, including primary biliary cholangitis and other adult cholangiopathies. Less is known about the role of cellular senescence in cholangiopathies in children. With that in mind, we examined the expression of senescence-associated cell cycle regulators in biliary atresia, the most common form of paediatric obliterative cholangiopathy. METHODS AND RESULTS: The expression of senescence-associated cell cycle regulators (p16Ink4a and p21WAF1/Cip1 ) and a ductular reaction related marker (neural cell adhesion molecule: NCAM) was examined in bile ducts and bile ductules in liver samples taken from the patients with biliary atresia [n = 80; including 23 samples at the time of the Kasai procedure (KP) and 63 obtained from the explanted liver (LT) (six cases with samples at both surgical stages of disease)] and from appropriate controls (n = 17). The degree of ductular reaction and cholestasis was significantly more extensive in LT than KP (P < 0.01). The expression of p16INK4a and NCAM was significantly more extensive in bile ducts and bile ductules in ductular reaction in both KP and LT compared to controls and in LT compared to KP (P < 0.05). The expression of p21WAF1/Cip1 was significantly more extensive in bile ducts and bile ductules in KP compared to both LT and controls (P < 0.01). CONCLUSIONS: Cellular senescence may play a role in the progression of bile duct loss in biliary atresia in a manner similar to that of adult cholangiopathies.

Increased expression of interleukin-23 associated with progression of colorectal cancer.

BACKGROUND AND OBJECTIVES: The prognostic significance of interleukin-23 in colorectal cancer remains unclear. We designed this study to investigate the association between colorectal cancer and interleukin-23 (IL-23) or interleukin-23 receptor (IL-23R) expression and the resulting clinical features and survival. METHODS: Immunohistochemical staining was performed for IL-23 and IL-23R in colorectal cancer samples. H-score was calculated to compare the expression of IL-23 and IL-23R. The median of H-score was used as the cut-off value to separate patients into high or low expression groups. The differences in clinicopathological features were evaluated. Cox regression hazard ratios were used for survival analysis. RESULTS: A total of 129 colorectal cancer patients were enrolled. H-score for the late TNM stage patients was higher than that for the early TNM stage patients (P = 0.002). Patients with high IL-23 expression were associated with advanced pathological T category (P < 0.001) and late TNM stage (P = 0.003). High IL-23 expression was associated with poor 5-year disease-free survival and overall survival in patients (P = 0.048 and P = 0.028, respectively). Multivariate adjustment demonstrated a significant association between high IL-23 expression and overall survival (hazard ratio = 1.865, P = 0.041). CONCLUSIONS: Elevated IL-23 expression was associated with poor outcome and can be used as a prognostic biomarker for colorectal cancer. J. Surg. Oncol. 2017;115:208-212. © 2016 Wiley Periodicals, Inc.

mtDNA as a Mediator for Expression of Hypoxia-Inducible Factor 1α and ROS in Hypoxic Neuroblastoma Cells.

Mitochondria consume O2 to produce ATP and are critical for adaption of hypoxia, but the role of mitochondria in HIF-1α pathway is as yet unclear. In this study, mitochondrial DNA (mtDNA) enriched (SK-N-AS) and depleted (ρ°) cells of neuroblastoma were cultured in a hypoxic chamber to simulate a hypoxic condition and then the major components involved in mitochondrial related pathways, hypoxia-inducible factor 1α (HIF-1α) and reactive oxygen species (ROS) were measured. The results showed that hypoxia-stimulated exposure elevated expression of HIF-1α, which was additionally influenced by level of generated ROS within the cytosol. Moreover, elevation of HIF-1α also resulted in increases of lactate dehydrogenase A (LDH-A) and pyruvate dehydrogenase kinase 1 (PDK1) in both hypoxic cells. The expression of mitochondrial biogenesis related proteins and metabolic components were noted to increase significantly in hypoxic SK-N-AS cells, indicating that mtDNA was involved in mitochondrial retrograde signaling and metabolic pathways. An analysis of dynamic proteins found elevated levels of HIF-1α causing an increased expression of dynamin-related protein 1 (DRP1) during hypoxia; further, the existence of mtDNA also resulted in higher expression of DRP1 during hypoxia. By using siRNA of HIF-1α or DRP1, expression of DRP1 decreased after suppression of HIF-1α; moreover, the expression of HIF-1α was also affected by the suppression of DRP1. In this study, we demonstrated that mtDNA is a mediator of HIF-1α in eliciting metabolic reprogramming, and mitochondrial biogenesis. Identification of a mutual relationship between HIF-1α and DRP1 may be a critical tool in the future development of clinical applications.

Downregulation of c-Myc is involved in TLR3-mediated tumor death of neuroblastoma xenografts.

Neuroblastoma (NB) is the deadliest pediatric solid tumor due to its pleomorphic molecular characteristics. In the innate immune system, toll-like receptor 3 (TLR3) recognizes viral double-stranded RNAs to initiate immune signaling. Positive TLR3 expression indicates a favorable prognosis in NB patients, and is associated with MYCN-non-amplified. However, TLR3-mediated innate immune responses remain elusive in NB. In this study, we attempted to dissect the molecular mechanism underlying TLR3-agonist polyinosinic-polycytidylic acid [poly(I:C)] treatment in NB in vivo. We established NB xenograft models in non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice with MYCN-amplified SK-N-DZ (DZ) cells or MYCN-non-amplified SK-N-AS (AS) cells. Poly(I:C) treatment led to significant tumor regression in AS xenografts, but not in DZ xenografts. Through immunohistochemical analysis, significant suppression of tumor proliferation, downregulation of c-Myc expression, and upregulation of TLR3 expression were found in the treatment group. Poly(I:C) inducing activation of TLR3/IRF3-mediated innate immunity associated with downregulation of c-Myc can be found in MYCN-non-amplified SK-N-AS cells, but not in MYCN-amplified BE(2)-M17 cells. Knockdown of TLR3 disturbed poly(I:C)-induced suppression of c-Myc and upregulation of p-IRF3 in AS cells. Furthermore, poly(I:C) treatment upregulated active NF-κB, mitochondrial antioxidant manganese superoxide dismutase and 8-hydroxydeoxyguanosine, which works with reactive oxygen species (ROS) generation and DNA damage. Upregulation of active caspase 3 and cleaved poly [ADP-ribose] polymerase 1 were found in poly(I:C)-treated AS xenografts, which indicates the induction of apoptosis. Thus, our results suggest that c-Myc overexpression may increase sensitivity to poly(I:C)-induced tumor growth arrest and ROS-mediated apoptosis in NB. This study demonstrates that c-Myc protein expression has an important role in TLR3-induced innate immune responses, providing future treatment recommendations.

Associations of mitochondrial haplogroups b4 and e with biliary atresia and differential susceptibility to hydrophobic bile Acid.

Mitochondrial dysfunction has been implicated in the pathogenesis of biliary atresia (BA). This study aimed to determine whether a specific mitochondrial DNA haplogroup is implicated in the pathogenesis and prognosis of BA. We determined 40 mitochondrial single nucleotide polymorphisms in 15 major mitochondrial haplogroups by the use of 24-plex PCR and fluorescent beads combined with sequence-specific oligonucleotide probes in 71 patients with BA and in 200 controls in the Taiwanese population of ethnic Chinese background. The haplogroup B4 and E prevalence were significantly lower and higher respectively, in the patients with BA than in the controls (odds ratios, 0.82 [p = 0.007] and 7.36 [p = 0.032] respectively) in multivariate logistic-regression analysis. The 3-year survival rate with native liver was significantly lower in haplogroup E than the other haplogroups (P = 0.037). A cytoplasmic hybrid (cybrid) was obtained from human 143B osteosarcoma cells devoid of mtDNA (ρ(0) cell) and was fused with specific mtDNA bearing E and B4 haplogroups donated by healthy Taiwanese subjects. Chenodeoxycholic acid treatment resulted in significantly lower free radical production, higher mitochondrial membrane potential, more viable cells, and fewer apoptotic cybrid B4 cells than parental 143B and cybrid E cells. Bile acid treatment resulted in a significantly greater protective mitochondrial reaction with significantly higher mitochondrial DNA copy number and mitofusin 1 and 2 concentrations in cybrid B4 and parental cells than in cybrid E cells. The results of the study suggested that the specific mitochondrial DNA haplogroups B4 and E were not only associated with lower and higher prevalence of BA respectively, in the study population, but also with differential susceptibility to hydrophobic bile acid in the cybrid harboring different haplogroups.

Microarray Study of Pathway Analysis Expression Profile Associated with MicroRNA-29a with Regard to Murine Cholestatic Liver Injuries.

UNLABELLED: Accumulating evidence demonstrates that microRNA-29 (miR-29) expression is prominently decreased in patients with hepatic fibrosis, which consequently stimulates hepatic stellate cells' (HSCs) activation. We used a cDNA microarray study to gain a more comprehensive understanding of genome-wide gene expressions by adjusting miR-29a expression in a bile duct-ligation (BDL) animal model. METHODS: Using miR-29a transgenic mice and wild-type littermates and applying the BDL mouse model, we characterized the function of miR-29a with regard to cholestatic liver fibrosis. Pathway enrichment analysis and/or specific validation were performed for differentially expressed genes found within the comparisons. RESULTS: Analysis of the microarray data identified a number of differentially expressed genes due to the miR-29a transgene, BDL, or both. Additional pathway enrichment analysis revealed that TGF-ß signaling had a significantly differential activated pathway depending on the occurrence of miR-29a overexpression or the lack thereof. Furthermore, overexpression was found to elicit changes in Wnt/ß-catenin after BDL. CONCLUSION: This study verified that an elevated miR-29a level could alleviate liver fibrosis caused by cholestasis. Furthermore, the protective effects of miR-29a correlate with the downregulation of TGF-ß and associated with Wnt/ß-catenin signal pathway following BDL.

MicroRNA-29a protects against acute liver injury in a mouse model of obstructive jaundice via inhibition of the extrinsic apoptosis pathway.

Recent studies have shown that microRNA-29 (miR-29) is significantly decreased in liver fibrosis, as demonstrated in human liver cirrhosis, and that its downregulation influences the activation of hepatic stellate cells. In addition, both cleaved caspase-3 production and apoptosis play a role in cholestatic liver injury. However, it is unknown if miR-29 is effective in modulating the extent of injury. We employed miR-29a transgenic mice (miR-29aTg mice) and wild-type (WT) littermates to clarify the role of miR-29 in hepatic injury and fibrogenesis, using the bile duct-ligation (BDL) mouse model. After BDL, all three members of the miR-29 family were significantly downregulated in the livers of WT mice, and miR-29b and miR-29c were significantly downregulated in the livers of the miR-29aTg mice. Liver function, as measured by alanine transaminase and aspartate transaminase activity, was significantly improved in the miR-29aTg mice than in the WT littermates, following 1 week of obstructive jaundice. In addition, overexpression of miR-29a was associated with a significant downregulation of the expression of collagen-1α1, collagen-4α1, phospho-FADD, cleaved caspase-8, cleaved caspase-3, Bax, Bcl-2, PARP, and nuclear factor-κB, as well as an upregulation of phospho-AKT expression. In addition, there were significantly fewer TUNEL-positive liver cells in the miR-29aTg group than in the WT littermates after BDL. Our results indicate that miR-29a decreases cholestatic liver injury and fibrosis after BDL, at least partially, by modulating the extrinsic rather than intrinsic pathway of apoptosis.

Upregulation of TLRs and IL-6 as a marker in human colorectal cancer.

Toll-like receptors (TLRs) not only form an important part of the innate immune system but also serve to activate the adaptive immune system in response to cancer. Real-time PCR; immunohistochemical stain and Western blotting analyses were performed to clarify molecular alterations in colorectal cancer (CRC) patients. We identified Toll-like receptor 1 (TLR1), TLR2, TLR4 and TLR8 gene expression levels and downstream gene, i.e., interleukin-6 (IL-6), IL-8, interferon-α (IFN-α) and myeloid differentiation primary-response protein-88 (MyD88), expression levels in CRC patients and in cancer cell lines. CRC tissues have higher TLR1, TLR2, TLR4, TLR8, IL-6 and IL-8 gene expression levels than do the normal colon mucosa (p < 0.05). TLR2 expression varied in different cell types (mucosa and lymphocytes). There was no difference in the MyD88 and IFN-α gene expression levels between cancerous and normal colon mucosa. CRC patients had higher levels of IL-6 (p = 0.002) and IL-8 (p = 0.038) expression than healthy volunteers did; and higher IL-6 and IL-8 expression was also found to signify a higher risk of recurrence. CL075 (3M002) treatments can reduce the production of IL-8 in different cancer cell lines. The signaling pathway of TLRs in cancer tissue is different from that in normal cells; and is MyD88-independent. Higher expression levels of TLR1, TLR2, TLR 4 and TLR 8 mRNA were related to upregulation inflammatory cytokines IL-6 and IL-8 gene expression in tissue and to the upregulation of IL-6 in blood. The concentration of IL-6 in serum can be used as an indicator of the possibility of CRC recurrence. Treatment with 3M002 can reduce IL-6 production in vitro and may prevent CRC recurrence. Our findings provide evidence that TLR1, TLR2, TLR4 and TLR8 gene expression induce downstream IL-6 and IL-8 gene expression; detection of these expression levels can serve as a CRC marker.

Resveratrol partially prevents rotenone-induced neurotoxicity in dopaminergic SH-SY5Y cells through induction of heme oxygenase-1 dependent autophagy.

Parkinson disease (PD) is a complex neurodegenerative disorder characterized by a progressive loss of dopaminergic neurons. Mitochondrial dysfunction, oxidative stress or protein misfolding and aggregation may underlie this process. Autophagy is an intracellular catabolic mechanism responsible for protein degradation and recycling of damaged proteins and cytoplasmic organelles. Autophagic dysfunction may hasten the progression of neuronal degeneration. In this study, resveratrol promoted autophagic flux and protected dopaminergic neurons against rotenone-induced apoptosis. In an in vivo PD model, rotenone induced loss of dopaminergic neurons, increased oxidation of mitochondrial proteins and promoted autophagic vesicle development in brain tissue. The natural phytoalexin resveratrol prevented rotenone-induced neuronal apoptosis in vitro, and this pro-survival effect was abolished by an autophagic inhibitor. Although both rotenone and resveratrol promoted LC3-II accumulation, autophagic flux was inhibited by rotenone and augmented by resveratrol. Further, rotenone reduced heme oxygenase-1 (HO-1) expression, whereas resveratrol increased HO-1 expression. Pharmacological inhibition of HO-1 abolished resveratrol-mediated autophagy and neuroprotection. Notably, the effects of a pharmacological inducer of HO-1 were similar to those of resveratrol, and protected against rotenone-induced cell death in an autophagy-dependent manner, validating the hypothesis of HO-1 dependent autophagy in preventing neuronal death in the in vitro PD model. Collectively, our findings suggest that resveratrol induces HO-1 expression and prevents dopaminergic cell death by regulating autophagic flux; thus protecting against rotenone-induced neuronal apoptosis.

Toll-like receptor-4 agonist inhibits motility and invasion of hepatoblastoma HepG2 cells in vitro.

BACKGROUND: Expression of toll-like receptor-4 (TLR4) on tumor cells is known to mediate innate immune responses that influence tumor cell growth and migration. This study aimed to characterize TLR4 expression and elucidate its functional significance in human hepatoblastoma (HB) cells. PROCEDURE: Immunohistochemistry (IHC) was used to determine TLR4 expression level and its distribution pattern in HB liver tissues. Transcripts of tumor necrosis factor (TNF)-α, interleukin (IL)-8, matrix metalloproteinase (MMP)-2, MMP-13, tissue inhibitor of metalloproteinases (TIMP)-1, and TIMP-2 in HB HepG2 cells with lipopolysacharide (LPS) treatment were measured by quantitative PCR. Soluble cytokines and peptides in conditioned media were measured by ELISA. MMP-2 activity was determined by using gelatin zymography. Cell motility and invasiveness was determined using wound healing migration and Matrigel invasion assays, respectively. RESULTS: TLR4 IHC staining demonstrated that TLR4 overexpression in HB liver tissues dramatically vanished after chemotherapy. In vitro study using an HB cell line, HepG2, showed that TLR4 agonist, LPS, significantly decreased transcripts of IL-8 and TNF-α, but did not affect MMP-13 mRNA level. By contrast, LPS only down-regulated IL-8 production and MMP-2 gelatinolytic activity. The latter might be in part due to the increased levels of MMP-2/TIMP-2 complex in conditioned media, thus leading to the decreased motility and invasiveness of HepG2 cells. CONCLUSIONS: HB cells overexpress TLR4, whereas TLR4 agonistic treatment inhibits migration and invasion of HB HepG2 cells. These findings suggest that TLR4 signaling pathway is a potential therapeutic target for control of HB tumor progression.

Selective activation of Toll-like receptor 7 in activated hepatic stellate cells may modulate their profibrogenic phenotype.

Cholestatic liver injury may activate HSCs (hepatic stellate cells) to a profibrogenic phenotype, contributing to liver fibrogenesis. We have previously demonstrated the involvement of TLR (Toll-like receptor) 7 in the pathogenesis of biliary atresia. In the present study we investigated the ability of TLR7 to modulate the profibrogenic phenotype in HSCs. Obstructive jaundice was associated with significant down-regulation of TLR7. Primary HSCs isolated from BDL (bile duct ligation) rats with obstructive jaundice exhibited reduced expression of TLR7 and increased expression of α-SMA (α-smooth muscle actin) and collagen-α1 compared with sham rats, reflecting HSC-mediated changes. Treatment of primary activated rat HSCs and rat T6 cells with CL075, a TLR7 and TLR8 ligand, significantly decreased expression of MCP-1 (monocyte chemotactic protein-1), TGF-ß1 (transforming growth factor-ß1), collagen-α1 and MMP-2 (matrix metalloproteinase-2), and inhibited cell proliferation and migration. In contrast, silencing TLR7 expression with shRNA (short hairpin RNA) in T6 cells effectively blocked the effects of CL075 stimulation, reversing the changes in MCP-1, TGF-ß1 and collagen-α1 expression and accelerating cell migration. Our results indicate that obstructive jaundice is associated with down-regulation of TLR7 and up-regulation of profibrogenic gene expression in HSCs. Selective activation of TLR7 may modulate the profibrogenic phenotype in activated HSCs associated with cholestatic liver injury.