STAT3 blockade ameliorates LPS-induced kidney injury through macrophage-driven inflammation.

BACKGROUND: Signal transducer and activator of transcription 3 (STAT3), a multifaceted transcription factor, modulates host immune responses by activating cellular response to signaling ligands. STAT3 has a pivotal role in the pathophysiology of kidney injury by counterbalancing resident macrophage phenotypes under inflammation conditions. However, STAT3's role in acute kidney injury (AKI), particularly in macrophage migration, and in chronic kidney disease (CKD) through fibrosis development, remains unclear. METHODS: Stattic (a JAK2/STAT3 inhibitor, 5 mg/kg or 10 mg/kg) was administered to evaluate the therapeutic effect on LPS-induced AKI (L-AKI) and LPS-induced CKD (L-CKD), with animals sacrificed 6-24 h and 14 days post-LPS induction, respectively. The immune mechanisms of STAT3 blockade were determined by comparing the macrophage phenotypes and correlated with renal function parameters. Also, the transcriptomic analysis was used to confirm the anti-inflammatory effect of L-AKI, and the anti-fibrotic role was further evaluated in the L-CKD model. RESULTS: In the L-AKI model, sequential increases in BUN and blood creatinine levels were time-dependent, with a marked elevation of 0-6 h after LPS injection. Notably, two newly identified macrophage subpopulations (CD11bhighF4/80low and CD11blowF4/80high), exhibited population changes, with an increase in the CD11bhighF4/80low population and a decrease in the CD11blowF4/80high macrophages. Corresponding to the FACS results, the tubular injury score, NGAL, F4/80, and p-STAT3 expression in the tubular regions were elevated. STAT3 inhibitor injection in L-AKI and L-CKD mice reduced renal injury and fibrosis. M2-type subpopulation with CD206 in CD11blowF4/80high population increased in the Stattic-treated group compared with that in the LPS-alone group in the L-AKI model. Additionally, STAT3 inhibitor reduced inflammation driven by LPS-stimulated macrophages and epithelial cells injury in the co-culture system. Transcriptomic profiling identified 3 common genes in the JAK-STAT, TLR, and TNF signaling pathways and 11 common genes in the LPS with macrophage response. The PI3K-AKT (IL-6, Akt3, and Pik3r1) and JAK-STAT pathways were determined as potential Stattic targets. Further confirmation through mRNA and protein expressions analyses showed that Stattic treatment reduced inflammation in the L-AKI and fibrosis in the L-CKD mice. CONCLUSIONS: STAT3 blockade effectively mitigated inflammation by retrieving the CD11blowF4/80high population, further emphasizing the role of STAT3-associated macrophage-driven inflammation in kidney injury.

This study investigated the role of STAT3 in LPS-induced acute kidney injury (AKI) and its prolonged pathophysiological effect. In a mouse model, blocking STAT3 with Stattic reduced inflammation and fibrosis, decreased the levels of inflammatory and extracellular matrix (ECM) substances, reduced the number of certain immune cells (macrophages), and influenced specific genes related to inflammation. The findings suggest that targeting STAT3 is a promising approach to treat AKI and CKD by controlling the inflammation and the immune response as well as ECM accumulation. This study provides novel insights into AKI and CKD progression and will facilitate the development of new treatments for kidney injuries at various stages.

Novel Activity of ODZ10117, a STAT3 Inhibitor, for Regulation of NLRP3 Inflammasome Activation.

The NLRP3 inflammasome serves as a host defense mechanism against various pathogens, but there is growing evidence linking its activation in sterile condition to diverse inflammatory diseases. Therefore, the identification of specific inhibitors that target NLRP3 inflammasome activation is meaningful and important for novel therapies for NLRP3 inflammasome-associated diseases. In this study, we identified a chemical compound, namely ODZ10117 (ODZ), that showed NLRP3 inflammasome-targeting anti-inflammatory effects during the screening of a chemical library for anti-inflammatory activity. Although ODZ was initially discovered as a STAT3 inhibitor, here we found it also has inhibitory activity on NLRP3 inflammasome activation. ODZ inhibited the cleavage of caspase-1 and IL-1ß-induced canonical NLRP3 inflammasome triggers, but had no effect on those induced by AIM2 or NLRC4 triggers. Mechanistically, ODZ impairs NLRP3 inflammasome activation through the inhibition of NLRP3-NEK7 interaction that is required for inflammasome formation. Moreover, the results obtained from the in silico docking experiment suggested that ODZ targets NLRP3 protein, which provides evidence for the specificity of ODZ to the NLRP3 inflammasome. Furthermore, ODZ administration significantly reduced MSU-induced IL-1ß release and the mortality rate of mice with LPS-induced sepsis. Collectively, these results demonstrate a novel effect of ODZ10117 in regulating NLRP3 inflammasome activation both in vitro and in vivo, making it a promising candidate for the treatment of NLRP3-inflammasome-associated immune disorders and cancer.

STAT3 activation in microglia increases pericyte apoptosis in diabetic retinas through TNF-ɑ/AKT/p70S6 kinase signaling.

Diabetic retinopathy (DR) is one of the vascular complications associated with diabetes mellitus. Pericyte loss is an early characteristic phenomenon in DR. However, the mechanism by which pericyte apoptosis occurs in DR is not fully understood. We have focused on the increased STAT3 activation in diabetic retinas because STAT3 activation is associated with inflammation, and persistent chronic inflammation is closely related to retinal lesions. In this study, we demonstrated that STAT3 was activated by IFN-γ and IL-6 that highly expressed in diabetic retinas. We identified TNF-α as a potent inducer of pericyte apoptosis in diabetic retinas from the gene expression analysis and found that STAT3 activation in microglia increased TNF-α expression in the diabetic retinas. We also demonstrated that increased TNF-α expression in microglia caused pericyte apoptosis through downregulating AKT/p70S6 kinase signaling. Moreover, we took advantage of mice lacking STAT3 in microglia and demonstrated that STAT3 ablation in microglia reduced the pericyte apoptosis and TNF-α expression in the diabetic retinas. These results suggest that STAT3 activation in microglia plays an important role in pericyte apoptosis in the diabetic retinas through increased TNF-α expression and provide STAT3 activation in microglia as a potential therapeutic target for preventing pericyte loss in DR.

The acidic tumor microenvironment enhances PD-L1 expression via activation of STAT3 in MDA-MB-231 breast cancer cells.

Tumor acidosis, a common phenomenon in solid cancers such as breast cancer, is caused by the abnormal metabolism of cancer cells. The low pH affects cells surrounding the cancer, and tumor acidosis has been shown to inhibit the activity of immune cells. Despite many previous studies, the immune surveillance mechanisms are not fully understood. We found that the expression of PD-L1 was significantly increased under conditions of extracellular acidosis in MDA-MB-231 cells. We also confirmed that the increased expression of PD-L1 mediated by extracellular acidosis was decreased when the pH was raised to the normal range. Gene set enrichment analysis (GSEA) of public breast cancer patient databases showed that PD-L1 expression was also highly correlated with IL-6/JAK/STAT3 signaling. Surprisingly, the expression of both phospho-tyrosine STAT3 and PD-L1 was significantly increased under conditions of extracellular acidosis, and inhibition of STAT3 did not increase the expression of PD-L1 even under acidic conditions in MDA-MB-231 cells. Based on these results, we suggest that the expression of PD-L1 is increased by tumor acidosis via activation of STAT3 in MDA-MB-231 cells.

STAT3 activation in microglia exacerbates hippocampal neuronal apoptosis in diabetic brains.

Diabetes mellitus (DM) characterized by hyperglycemia leads to a variety of complications, including cognitive impairment or memory loss. The hippocampus is a key brain area for learning and memory and is one of the regions that is most sensitive to diabetes. However, the pathogenesis of diabetic neuronal lesion is not yet completely understood. We focused on the association of microglia activation and brain lesions in diabetes. In this study, we investigated whether and how signal transducer and activator of transcription 3 (STAT3) activation in microglia affects neuronal lesions in diabetic brains. Using a streptozotocin-induced type 1 DM model, we showed enhanced hippocampal neuronal apoptosis that was associated with increased STAT3 activation. We found that hyperglycemia increased the expression of inflammatory cytokines such as interferon-γ (IFN-γ) and interleukin-6, in the diabetic hippocampus. In particular, IFN-γ induced autocrine activation of microglia, and STAT3 activation is important for this process. We also demonstrated that STAT3 activation in microglia increased tumor necrosis factor-α (TNF-α) expression; subsequently, TNF-α increased neuronal apoptosis by increasing reactive oxygen species (ROS) levels in the neuronal cells. We also took advantage of mice lacking STAT3 in microglia and demonstrated that depletion of microglial STAT3 reduced neuronal apoptosis in the diabetic hippocampus. Taken together, these results suggest that STAT3 activation in microglia plays an important role in hyperglycemia-induced neuronal apoptosis in the diabetic hippocampus and provide a potential therapeutic benefit of STAT3 inhibition in microglia for preventing diabetic neuronal lesions.

Inhibition of mTORC1 through ATF4-induced REDD1 and Sestrin2 expression by Metformin.

BACKGROUND: Although the major anticancer effect of metformin involves AMPK-dependent or AMPK-independent mTORC1 inhibition, the mechanisms of action are still not fully understood. METHODS: To investigate the molecular mechanisms underlying the effect of metformin on the mTORC1 inhibition, MTT assay, RT-PCR, and western blot analysis were performed. RESULTS: Metformin induced the expression of ATF4, REDD1, and Sestrin2 concomitant with its inhibition of mTORC1 activity. Treatment with REDD1 or Sestrin2 siRNA reversed the mTORC1 inhibition induced by metformin, indicating that REDD1 and Sestrin2 are important for the inhibition of mTORC1 triggered by metformin treatment. Moreover, REDD1- and Sestrin2-mediated mTORC1 inhibition in response to metformin was independent of AMPK activation. Additionally, lapatinib enhances cell sensitivity to metformin, and knockdown of REDD1 and Sestrin2 decreased cell sensitivity to metformin and lapatinib. CONCLUSIONS: ATF4-induced REDD1 and Sestrin2 expression in response to metformin plays an important role in mTORC1 inhibition independent of AMPK activation, and this signalling pathway could have therapeutic value.

Tubulosine selectively inhibits JAK3 signalling by binding to the ATP-binding site of the kinase of JAK3.

Gain- or loss-of-function mutations in Janus kinase 3 (JAK3) contribute to the pathogenesis of various haematopoietic malignancies and immune disorders, suggesting that aberrant JAK3 signalling is an attractive therapeutic target to treat these disorders. In this study, we performed structure-based computational database screening using the 3D structure of the JAK3 kinase domain and the National Cancer Institute diversity set and identified tubulosine as a novel JAK3 inhibitor. Tubulosine directly blocked the catalytic activity of JAK3 by selective interacting with the JAK3 kinase domain. Consistently, tubulosine potently inhibited persistently activated and interleukin-2-dependent JAK3, and JAK3-mediated downstream targets. Importantly, it did not affect the activity of other JAK family members, particularly prolactin-induced JAK2/signal transducer and activator of transcription 5 and interferon alpha-induced JAK1-TYK2/STAT1. Tubulosine specifically decreased survival and proliferation of cancer cells, in which persistently active JAK3 is expressed, by inducing apoptotic and necrotic/autophagic cell death without affecting other oncogenic signalling. Collectively, tubulosine is a potential small-molecule compound that selectively inhibits JAK3 activity, suggesting that it may serve as a promising therapeutic candidate for treating disorders caused by aberrant activation of JAK3 signalling.

TGFam-Finder: a novel solution for target-gene family annotation in plants.

Whole-genome annotation error that omits essential protein-coding genes hinders further research. We developed Target Gene Family Finder (TGFam-Finder), an alternative tool for the structural annotation of protein-coding genes containing target domain(s) of interest in plant genomes. TGFam-Finder took considerably reduced annotation run-time and improved accuracy compared to conventional annotation tools. Large-scale re-annotation of 50 plant genomes identified an average of 150, 166 and 86 additional far-red-impaired response 1, nucleotide-binding and leucine-rich-repeat, and cytochrome P450 genes, respectively, that were missed in previous annotations. We detected significantly higher number of translated genes in the new annotations using mass spectrometry data from seven plant species compared to previous annotations. TGFam-Finder along with the new gene models can provide an optimized platform for comprehensive functional, comparative, and evolutionary studies in plants.

Increased inward rectifier K+ current of coronary artery smooth muscle cells in spontaneously hypertensive rats; partial compensation of the attenuated endothelium-dependent relaxation via Ca2+ -activated K+ channels.

Endothelium-dependent vasorelaxation is partly mediated by small-conductance (SK3) and intermediate-conductance Ca2+ -activated K+ channels (SK4) in the endothelium that results in endothelium-dependent hyperpolarization (EDH). Apart from the electrical propagation through myoendothelial gap junctions, the K+ released from the endothelium facilitates EDH by increasing inward rectifier K+ channel (Kir) conductance in smooth muscle cells. The EDH-dependent relaxation of coronary artery (CA) and Kir current in smooth muscle cells (CASMCs) of hypertensive animals are poorly understood despite the critical role of coronary flow in the hypertrophic heart. In spontaneously hypertensive (SHR) and control (WKY) rats, we found attenuation of the CA relaxation by activators of SK3 and SK4 (NS309 and 1-EBIO) in SHR. In isolated CASMCs, whole-cell patch-clamp study revealed larger IKir in SHR than WKY, whereas the myocytes of skeletal and cerebral arteries showed smaller IKir in SHR than WKY. While the treatment with IKir inhibitor (0.1 mmol/L Ba2+ ) alone did not affect the WKY-CA, the SHR-CA showed significant contractile response, suggesting relaxing influence of the higher IKir in the CASMCs of SHR. Furthermore, the attenuation of NS309-induced relaxation of CA by the combined treatment with 0.1 mmol/L Ba2+ was more prominent in SHR than WKY. Our study firstly shows a distinct increase of IKir in the CASMCs of SHR, which could partly compensate for the attenuated relaxation via endothelial SK3 and SK4.

Anti-cancer effect of doxorubicin is mediated by downregulation of HMG-Co A reductase via inhibition of EGFR/Src pathway.

Doxorubicin is a widely used DNA damage-inducing anti-cancer drug. However, its use is limited by its dose-dependent side effects, such as cardiac toxicity. Cholesterol-lowering statin drugs increase the efficacy of some anti-cancer drugs. Cholesterol is important for cell growth and a critical component of lipid rafts, which are plasma membrane microdomains important for cell signaling. 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (HMG-CR) is a critical enzyme in cholesterol synthesis. Here, we show that doxorubicin downregulated HMG-CR protein levels and thus reduced levels of cholesterol and lipid rafts. Cholesterol addition attenuated doxorubicin-induced cell death, and cholesterol depletion enhanced it. Reduction of HMG-CR activity by simvastatin, a statin that acts as an HMG-CR inhibitor, or by siRNA-mediated HMG-CR knockdown enhanced doxorubicin cytotoxicity. Doxorubicin-induced HMG-CR downregulation was associated with inactivation of the EGFR-Src pathway. Furthermore, a high-cholesterol-diet attenuated the anti-cancer activity of doxorubicin in a tumor xenograft mouse model. In a multivulva model of Caenorhabditis elegans expressing an active-EGFR mutant, doxorubicin decreased hyperplasia more efficiently in the absence than in the presence of cholesterol. These data indicate that EGFR/Src/HMG-CR is a new pathway mediating doxorubicin-induced cell death and that cholesterol control could be combined with doxorubicin treatment to enhance efficacy and thus reduce side effects.

Transcriptional modulation of the T helper 17/interleukin 17 axis ameliorates renal ischemia-reperfusion injury.

BACKGROUND: Signal transducer and activator of transcription 3 (STAT3) is a latent transcription factor critical for T-cell function. Although inhibition of the Janus kinase 2 (JAK2)/STAT3 pathway has been reported to be protective against ischemia-reperfusion injury (IRI), the role of T cell-associated STAT3 in the pathogenesis of renal IRI has not been specifically defined. METHODS: We induced renal IRI in both mice with T cell-specific STAT3 knockout (Lck-Cre;STAT3flox/flox) and wild-type controls (C57BL/6) and assessed renal damage and inflammation at 48 h after IRI. Human proximal tubular epithelial cells grown under hypoxia were treated with a JAK2 inhibitor, caffeic acid 3,4-dihydroxy-phenylethyl ester, to determine the effect of JAK2/STAT3 inhibition on renal epithelia. Independently, we disrupted Cln 3-requiring 9 (Ctr9) to inhibit T helper 17 (Th17) activation via RNA interference and determined if Ctr9 inhibition aggravates renal injury through upregulated Th17 activation. RESULTS: The Lck-Cre;STAT3flox/flox mice exhibited significantly reduced kidney damage compared with controls. This protective effect was associated with reduced intrarenal Th17 infiltration and proinflammatory cytokines. Human proximal tubular epithelial cells under hypoxia exhibited significant upregulation of interleukin 17 receptors, and pharmacologic inhibition of JAK2 significantly ameliorated this change. RNA interference with Ctr9 in splenocytes enhanced differentiation into Th17 cells. In vivo knockdown of Ctr9 in mice with renal IRI further aggravated Th17-associated inflammation and kidney injury. CONCLUSIONS: STAT3 in T cells contributes to renal IRI through Th17 activation. Inhibition of Ctr9 further enhances Th17 activation and aggravates kidney injury, further supporting the role of Th17 cells in renal IRI.

Evaluation and Optimization of the Anti-Melanogenic Activity of 1-(2-Cyclohexylmethoxy-6-hydroxy-phenyl)-3-(4-hydroxymethyl-phenyl)-propenone Derivatives.

The chemical modification and optimization of biologically active compounds are essential steps in the identification of promising lead compounds for drug development. We previously reported the anti-melanogenic activity of 1-(2-cyclohexylmethoxy-6-hydroxy-phenyl)-3-(4-hydroxymethyl-phenyl)-propenone (chalcone 21). In this study, we synthesized 21 derivatives of chalcone 21 and evaluated their anti-melanogenic activity in -MSH-induced B16F10 cells. (E)-N-(4-(3-(2-(Cyclohexylmethoxy)phenyl)-3-oxoprop-1-en-1-yl)phenyl)acetamide (chalcone 21-21) exhibited the strongest inhibition of cellular melanin production, with an IC50 value of 0.54 M. It was more potent than chalcone 21 and the known anti-melanogenic agents kojic acid and arbutin, whose IC50 values were 4.9, 38.5, and 148.4 M, respectively. Chalcone 21-21 decreased the expression and activity of tyrosinase. It also decreased the expression of TRP1, TRP2 and MITF, the phosphorylation of CREB and ERK1/2, and the transcriptional activity of MITF and CRE. Our results demonstrate that chalcone-21-21 is an effective lead compound with anti-melanogenic activity.

Molecular cloning and anti-invasive activity of cathepsin L propeptide-like protein from Calotropis procera R. Br. against cancer cells.

Cathepsin L of cancer cells has been shown to play an important role in degradation of extracellular matrix for metastasis. In order to reduce cell invasion, cathepsin L propeptide-like proteins which are classified as the I29 family in the MEROPS peptidase database were characterized from Calotropis procera R. Br., rich in cysteine protease. Of 19 candidates, the cloned and expressed recombinant SnuCalCp03-propeptide (rSnuCalCp03-propeptide) showed a low nanomolar Ki value of 2.3 ± 0.2 nM against cathepsin L. A significant inhibition of tumor cell invasion was observed with H1975, HT29, MDA-BM-231, PANC1, and PC3 with a 76, 67, 67, 63, and 79% reduction, respectively, in invasion observed in the presence of 400 nM of the rSnuCalCp03-propeptide. In addition, thermal and pH study showed rSnuCalCp03-propeptide consisting of secondary structures was stable at a broad range of temperatures (30-70 °C) and pH (2-10, except for 5 which is close to the isoelectric point of 5.2).

Time-averaged simulated microgravity (taSMG) inhibits proliferation of lymphoma cells, L-540 and HDLM-2, using a 3D clinostat.

BACKGROUND: Gravity is omnipresent on Earth; however, humans in space, such as astronauts at the International Space Station, experience microgravity. Long-term exposure to microgravity is considered to elicit physiological changes, such as muscle atrophy, in the human body. In addition, certain types of cancer cells demonstrate inhibited proliferation under condition of time-averaged simulated microgravity (taSMG). However, the response of human Hodgkin's lymphoma cancer cells to reduced gravity, and the associated physiological changes in these cells, have not been elucidated. METHODS: In this study, the proliferation of human Hodgkin's lymphoma cancer cells (L-540 and HDLM-2) under taSMG condition (<10-3 G, 1 G is defined as 9.8 m/s2) was studied using a 3D clinostat. Normal human dermal fibroblast (HDF) was proliferated in the same condition as a control group. For the development of 3D clinostat, two motors were used to actuate the frames. Electrical wires for power supply and communication were connected via slip ring. For symmetrical path of gravitational vector, optimal angular velocities of the motors were found using simulation results. Under the condition of taSMG implemented by the 3D clinostat, proliferation of the cells was observed for 3 days. RESULTS: The results indicated that proliferation of these cancer cells was significantly (p < 0.0005) inhibited under taSMG, whereas proliferation of normal HDF cells was not affected. CONCLUSIONS: Findings in this study could be significantly valuable in developing novel strategies for selective killing of cancer cells such as lymphoma.

Inhibition of tyrosinase activity and melanin production by the chalcone derivative 1-(2-cyclohexylmethoxy-6-hydroxy-phenyl)-3-(4-hydroxymethyl-phenyl)-propenone.

Abnormal accumulation of melanin pigments in the skin can be lead to hyperpigmentation disorders and melanoma. Melanin biosynthesis is ultimately regulated by the rate-limiting enzyme tyrosinase. In the present study, we synthesized chalcone derivatives and identified 1-(2-cyclohexylmethoxy-6-hydroxy-phenyl)-3-(4-hydroxymethyl-phenyl)-propenone (chalcone-21) as an anti-melanogenic substance in B16F10 melanoma cells. Chalcone-21 strongly inhibited cellular melanin production and tyrosinase activity in B16F10 melanoma cells stimulated with α-melanocyte stimulating hormone (α-MSH) or protoporphyrin IX. In addition, the compound suppressed not only the expression of tyrosinase, tyrosinase-related protein-1 (TRP-1), TRP-2, and microphthalmia-associated transcription factor (MITF), but also the transcriptional activity of tyrosinase and MITF. Our results demonstrated chalcone-21 to be an effective depigmenting agent.

Targeting ODC1 inhibits tumor growth through reduction of lipid metabolism in human hepatocellular carcinoma.

Ornithine decarboxylase 1 (ODC1), a metabolic enzyme critically involved in the polyamine biosynthesis, is commonly upregulated in hepatocellular carcinoma (HCC). Despite its altered expression in human HCC tissues, the molecular mechanism by which ODC1 alters the course of HCC progression and functions in HCC cell survival is unknown. Here we identified that silencing of ODC1 expression with small interfering (si) RNA causes inhibition of HCC cell growth through blockade of cell cycle progression and induction of apoptosis. Next, to obtain insights into the molecular changes in response to ODC1 knockdown, global changes in gene expression were examined using RNA sequencing. It revealed that 119 genes show same directional regulation (76 up- and 43 down-regulated) in both Huh1 and Huh7 cells and were considered as a common ODC1 knockdown signature. Particularly, we found through a network analysis that KLF2, which is known to inhibit PPARγ expression and adipogenesis, was commonly up-regulated. Subsequent Western blotting affirmed that the downregulation of ODC1 was accompanied by a decrease in the levels of PPARγ as well as of PARP-1, cyclin E1 and pro-caspase 9 delaying cell cycle progression and accelerating apoptotic signaling. Following the down-regulation of PPARγ expression, ODC1 silencing resulted in a strong inhibition in the expression of important regulators of glucose transport and lipid biogenesis, and caused a marked decrease in lipid droplet accumulation. In addition, ODC1 silencing significantly inhibited the growth of human HCC xenografts in nude mice. These findings indicate that the function of ODC1 is correlated with HCC lipogenesis and suggest that targeting ODC1 could be an attractive option for molecular therapy of HCC.

Targeting HIF-1α is a prerequisite for cell sensitivity to dichloroacetate (DCA) and metformin.

Recently, targeting deregulated energy metabolism is an emerging strategy for cancer therapy. In the present study, combination of DCA and metformin markedly induced cell death, compared with each drug alone. Furthermore, the expression levels of glycolytic enzymes including HK2, LDHA and ENO1 were downregulated by two drugs. Interestingly, HIF-1α activation markedly suppressed DCA/metformin-induced cell death and recovered the expressions of glycolytic enzymes that were decreased by two drugs. Based on these findings, we propose that targeting HIF-1α is necessary for cancer metabolism targeted therapy.

Signal transducer and activator of transcription 3-mediated CD133 up-regulation contributes to promotion of hepatocellular carcinoma.

UNLABELLED: Enhanced expression of the cancer stem cell (CSC) marker, CD133, is closely associated with a higher rate of tumor formation and poor prognosis in hepatocellular carcinoma (HCC) patients. Despite its clinical significance, the molecular mechanism underlying the deregulation of CD133 during tumor progression remains to be clarified. Here, we report on a novel mechanism by which interleukin-6/signal transducer and activator of transcription 3 (IL-6/STAT3) signaling up-regulates expression of CD133 and promotes HCC progression. STAT3 activated by IL-6 rapidly bound to CD133 promoter and increased protein levels of CD133 in HCC cells. Reversely, in hypoxic conditions, RNA interference silencing of STAT3 resulted in decrease of CD133 levels, even in the presence of IL-6, with a concomitant decrease of hypoxia-inducible factor 1 alpha (HIF-1α) expression. Active STAT3 interacted with nuclear factor kappa B (NF-κB) p65 subunit to positively regulate the transcription of HIF-1α providing a mechanistic explanation on how those three oncogenes work together to increase the activity of CD133 in a hypoxic liver microenvironment. Activation of STAT3 and its consequent induction of HIF-1α and CD133 expression were not observed in Toll-like receptor 4/IL-6 double-knockout mice. Long-term silencing of CD133 by a lentiviral-based approach inhibited cancer cell-cycle progression and suppressed in vivo tumorigenicity by down-regulating expression of cytokinesis-related genes, such as TACC1, ACF7, and CKAP5. We also found that sorafenib and STAT3 inhibitor nifuroxazide inhibit HCC xenograft formation by blocking activation of STAT3 and expression of CD133 and HIF-1α proteins. CONCLUSION: IL-6/STAT3 signaling induces expression of CD133 through functional cooperation with NF-κB and HIF-1α during liver carcinogenesis. Targeting STAT3-mediated CD133 up-regulation may represent a novel, effective treatment by eradicating the liver tumor microenvironment.

Down-regulation of lipid raft-associated onco-proteins via cholesterol-dependent lipid raft internalization in docosahexaenoic acid-induced apoptosis.

Lipid rafts, plasma membrane microdomains, are important for cell survival signaling and cholesterol is a critical lipid component for lipid raft integrity and function. DHA is known to have poor affinity for cholesterol and it influences lipid rafts. Here, we investigated a mechanism underlying the anti-cancer effects of DHA using a human breast cancer cell line, MDA-MB-231. We found that DHA decreased cell surface levels of lipid rafts via their internalization, which was partially reversed by cholesterol addition. With DHA treatment, caveolin-1, a marker for rafts, and EGFR were colocalized with LAMP-1, a lysosomal marker, in a cholesterol-dependent manner, indicating that DHA induces raft fusion with lysosomes. DHA not only displaced several raft-associated onco-proteins, including EGFR, Hsp90, Akt, and Src, from the rafts but also decreased total levels of those proteins via multiple pathways, including the proteasomal and lysosomal pathways, thereby decreasing their activities. Hsp90 overexpression maintained its client proteins, EGFR and Akt, and attenuated DHA-induced cell death. In addition, overexpression of Akt or constitutively active Akt attenuated DHA-induced apoptosis. All these data indicate that the anti-proliferative effect of DHA is mediated by targeting of lipid rafts via decreasing cell surface lipid rafts by their internalization, thereby decreasing raft-associated onco-proteins via proteasomal and lysosomal pathways and decreasing Hsp90 chaperone function.