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Background/aim Colorectal cancer is one of the most common malignancies worldwide. Small molecule-based chemotherapy is an attractive approach for the chemoprevention and treatment of colorectal cancer. Methylsulfonylmethane (MSM) is a natural organosulfur compound with anticancer properties, as revealed by studies on in vitro models of gingival, prostate, lung, hepatic, and breast cancer. However, the molecular mechanisms underlying the effects of MSM in colon cancer cells remain unclear. Materials and methods Here, we investigated the effects of MSM, especially on the cell cycle arrest and apoptosis, in HT-29 cells. Results MSM suppressed the viability of HT-29 cells by inducing apoptosis and cell cycle arrest at the G0/G1 phase. MSM suppressed the sphere-forming ability and expression of stemness markers in HT-29 cells. Conclusion MSM has anti-cancer effects on HT-29 cells, and induces cell cycle arrest and apoptosis, while suppressing the stemness potential.

Background/aim Non-small cell lung cancer (NSCLC) is one among the most common cancers worldwide. Recently, dietary phytochemicals have been reported as an attractive approach to improve the symptoms of NSCLC patients. Tannic acid is a natural polyphenol, which is known to have anticancer effects on in vitro models of breast, gingival and colon cancer. However, the molecular mechanisms associated with the actions of tannic acid on A549 human lung cancer cells have not been elucidated. Materials and methods In this study, we analyzed the effect of tannic acid on A549 cells and their underlying mechanisms using western blotting, flow cytometry, invasion assay and tumorsphere formation assay. Results Tannic acid treatment suppressed the viability of A549 cells through cell cycle arrest and induction of the intrinsic pathways of apoptosis. In addition, the various malignant phenotypes of A549 cells including invasion, migration, and stemness were inhibited by tannic acid treatment. Conclusion Tannic acid could be used as an effective inhibitor of lung cancer progression.

Ketosis is a condition where ketone bodies are produced as an alternative energy source, due to insufficient glucose for energy production so that the body switches from carbohydrate metabolism to mostly fat metabolism. In this study, we examined the anti-ketosis effects of silibinin, a major active component of silymarin. We induced ketosis in FL83B mouse hepatocytes in vitro by culturing in low glucose media and compared results to hepatocytes maintained in high-glucose conditions. We quantified β-hydroxybutyrate (BHB) levels with a colorimetric assay. In low-glucose conditions, silibinin reduced the amount of BHB produced, compared to high-glucose conditions; thus, silibinin exhibited an anti-ketotic effect. Ketone body formation during beta oxidation is mediated by 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2). The nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) regulates the transcription of HMGCS2, and plays a vital role in BHB levels. We showed that silibinin inhibited the expression of HMGCS2 and NF-kB at transcriptional and translational levels. Silibinin also inhibited the nuclear translocation of NF-kB and its DNA binding activity. To elucidate the relationship between HMGCS2 and NF-kB, we tested inhibited and over-expressed NF-kB. We found that NF-kB acted as a positive regulator for HMGCS2 under ketosis treatment conditions.

High glucose-induced inflammation leads to atherosclerosis, which is considered a major cause of death in type 1 and type 2 diabetic patients. Nuclear factor-kappa B (NF-&kappa, B) plays a central role in high glucose-induced inflammation and is activated through toll-like receptors (TLRs) as well as canonical and protein kinase C-dependent (PKC) pathways. Non-toxic sulfur (NTS) and methylsulfonylmethane (MSM) are two sulfur-containing natural compounds that can induce anti-inflammation. Using Western blotting, real-time polymerase chain reaction, and flow cytometry, we found that high glucose-induced inflammation occurs through activation of TLRs. An effect of NTS and MSM on canonical and PKC-dependent NF-&kappa, B pathways was also demonstrated by western blotting. The effects of proinflammatory cytokines were investigated using a chromatin immunoprecipitation assay and enzyme-linked immunosorbent assay. Our results showed inhibition of the glucose-induced expression of TLR2 and TLR4 by NTS and MSM. These sulfur compounds also inhibited NF-&kappa, B activity through reactive oxygen species (ROS)-mediated canonical and PKC-dependent pathways. Finally, NTS and MSM inhibited the high glucose-induced expression of interleukin (IL)-1&beta, IL-6, and tumor necrosis factor-&alpha, and binding of NF-&kappa, B protein to the DNA of proinflammatory cytokines. Together, these results suggest that NTS and MSM may be potential drug candidates for anti-inflammation therapy.

Severe acute respiratory syndrome coronavirus nonstructural protein 13 (SCV nsP13), a superfamily 1 helicase, plays a central role in viral RNA replication through the unwinding of duplex RNA and DNA with a 5′ single-stranded tail in a 5′ to 3′ direction. Despite its putative role in viral RNA replication, nsP13 readily unwinds duplex DNA by cooperative translocation. Herein, nsP13 exhibited different characteristics in duplex RNA unwinding than that in duplex DNA. nsP13 showed very poor processivity on duplex RNA compared with that on duplex DNA. More importantly, nsP13 inefficiently unwinds duplex RNA by increasing the 5′-ss tail length. As the concentration of nsP13 increased, the amount of unwound duplex DNA increased and that of unwound duplex RNA decreased. The accumulation of duplex RNA/nsP13 complexes increased as the concentration of nsP13 increased. An increased ATP concentration in the unwinding of duplex RNA relieved the decrease in duplex RNA unwinding. Thus, nsP13 has a strong affinity for duplex RNA as a substrate for the unwinding reaction, which requires increased ATPs to processively unwind duplex RNA. Our results suggest that duplex RNA is a preferred substrate for the helicase activity of nsP13 than duplex DNA at high ATP concentrations.

Cortisol is a hormone involved in stress during exercise. The application of natural compounds is a new potential approach for controlling cortisol-induced stress. Tumour suppressor protein p53 is activated during cellular stress. Succinate dehydrogenase complex subunit A (SDHA) and hypoxanthine phosphoribosyl transferase 1 (HPRT1) are considered to be two of the most stable reference genes when measuring stress during exercise in horses. In the present study cells were considered to be in a ‘stressed state’ if the levels of these stable genes and the highly stress responsive gene p53 were altered. It was hypothesized that a natural organic sulphur-containing compound, methylsulfonylmethane (MSM), could inhibit cortisol-induced stress in racing horse skeletal muscle cells by regulating SDHA, HPRT1 and p53 expression. After assessing cell viability using MTT assays, 20 µg/ml cortisol and 50 mM MSM were applied to horse skeletal muscle cell cultures. Reverse transcription-quantitative PCR and western blot analysis demonstrated increases in SDHA, HPRT1 and p53 expression in cells in response to cortisol treatment, which was inhibited or normalized by MSM treatment. To determine the relationship between p53 and SDHA/HPRT1 expression at a transcriptional level, horse gene sequences of SDHA and HPRT1 were probed to identify novel binding sites for p53 in the gene promoters, which were confirmed using a chromatin immunoprecipitation assay. The relationship between p53 and SDHA/HPRT1 expression was confirmed using western blot analysis following the application of pifithrin-α, a p53 inhibitor. These results suggested that MSM is a potential candidate drug for the inhibition of cortisol-induced stress in racehorse skeletal muscle cells.

Ketogenesis is the production of ketone bodies, which provide energy when the body lacks glucose. Under ketogenic conditions, the body switches from primarily carbohydrate to fat metabolism to maintain energy balance. However, accumulation of high levels of ketone bodies in the blood results in ketosis. Treating ketosis with natural substances is preferable, because they are unlikely to cause side-effects. Momilactone B is an active compound isolated from Korean rice. Based on previous studies, we hypothesized that momilactone B could inhibit ketosis. We constructed an in vitro ketosis model by glucose starvation. We used this model to test the anti-ketosis effects of momilactone B. A primary target for treating ketosis is angiopoietin-like-3 (ANGPTL3), which modulates lipoprotein metabolism by inhibiting lipoprotein lipase (LPL), a multifunctional enzyme that breaks down stored fat to produce triglycerides. We showed that momilactone B could regulate the ANGPTL3-LPL pathway. However, a strong anti-ketosis candidate drug should also inhibit ketogenesis. Ketogenesis can be suppressed by inhibiting the expression of 3-hydroxy-3-methylglutaryl-CoA synthase-2 (HMGCS2), a mitochondrial enzyme that converts acetyl-CoA to ketone bodies. We found that momilactone B suppressed the expression of HMGCS2 through the increased expression of STAT5b. We also elucidated the relationship of STAT5b to ANGPTL3 and LPL expression.

The major hallmarks of tumor progression are angiogenesis, migration and metastasis. Among the components of Rhodiola rosea, salidroside (p‑hydroxyphenethyl-β‑d-glucoside) is one of the most potent, and is present in all Rhodiola species. Recent data have revealed the anticancer effects of salidroside; however, the mechanism underlying its ability to inhibit tumor angiogenesis remains unknown. The present study aimed to analyze how salidroside affects major factors involved in breast cancer, and to elucidate its ability to inhibit angiogenesis and invasion. Signal transducer and activator of transcription 3 (STAT3) is a marker for tumor angiogenesis and migration, which interacts with matrix metalloproteinases (MMPs). Specifically, MMPs act as a downstream target for STAT3. Using western blotting and reverse transcription-quantitative polymerase chain reaction analysis, the present study demonstrated that treatment of MDA‑MB 231 triple-negative breast cancer (TNBC) cells with salidroside led to inhibition of invasion and migration markers, and of STAT3 signaling. Furthermore, in vitro angiogenesis analyses in human umbilical vein endothelial cells confirmed the anti-angiogenic activity of salidroside. An electrophoretic mobility shift assay also demonstrated that salidroside may inhibit the DNA-binding activity of STAT3, preventing STAT3 from binding to a novel binding site of the MMP2 gene promoter. In conclusion, the present results demonstrated that salidroside may downregulate the STAT3 signaling pathway, and inhibit cell viability, migration and invasion through MMPs in breast cancer cells.

Targeted cancer therapy with natural compounds is more effective than nontargeted therapy. Nobiletin is a flavonoid derived from citrus peel that has anticancer activity. Cluster of differentiation 36 (CD36) is a member of the class B scavenger receptor family that is involved in importing fatty acids into cells. CD36 plays a role in tumor angiogenesis by binding to its ligand, thrombospondin-1 (TSP-1), and then interacting with transforming growth factor beta 1 (TGFβ1). CD36 is implicated in tumor metastasis through its roles in fatty acid metabolism. This study investigated the molecular mechanisms underlying nobiletin’s anticancer activity by characterizing its interactions with CD36 as the target molecule. We hypothesize that the anti-angiogenic activity of nobiletin involving its regulation of CD36 via signal transducer and activator of transcription 3 (STAT3) rather than through TSP-1. Gene analysis identified a Gamma interferon activation site (GAS) element in the CD36 gene promoter that acts as a STAT3 binding site, an interaction that was confirmed by ChIP assay. STAT3 interacts with nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), suggesting that nobiletin also acts through the CD36/ (STAT3)/NF-κB signaling axis. Nobiletin inhibited CD36-dependent breast cancer cell migration and invasion as well as CD36-mediated tumor sphere formation. Taken together, these results suggest that nobiletin inhibits cancer stem cells in multiple ways.

Breast cancer is the most common cancer in women globally. The factors that increase risk include: late age at first birth, alcohol, radiation exposure, family history of breast cancer, and postmenopausal hormone therapy. Numerous drugs are being developed to treat breast cancer. Among them, Herceptin is used for the treatment of human epidermal growth factor receptor 2 (HER2)-positive cases and targets HER2 effectively and efficiently, but it is very expensive. Methylsulfonylmethane (MSM) is an organic sulfur-containing natural compound having no reported toxicity. We examined MSM in breast cancer cell lines and found it inhibited the proliferation of estrogen receptor-positive and HER2-positive breast cancer cells in a dose-dependent manner. It also suppressed the activation of STAT5b and expression of HER2 in breast cancer cells. We determined the STAT5b binding site (GAS element) in the HER2 gene. Detailed analysis showed that MSM decreased the ability of STAT5b to bind the promoter of the HER2 gene and a luciferase assay demonstrated reduced activity. We confirmed that MSM can effectively regulate STAT5b, and thereby decrease HER2 expression. Therefore, we recommend the use of MSM as an inhibitor for the management of HER2-positive breast cancers.

Ketosis is a condition where ketone bodies are used as the energy source instead of glucose. The state of ketosis becomes dangerous if the fat metabolism in the body is high. When the glucose availability gets limited, liver activates fat metabolism to make glucose for energy. As the results of these, ketone bodies will form resulting the state of ketosis. In the present study, we analyzed the in vitro ketosis condition and the effect of glucose level in ketosis induction. The natural organic sulfur containing compound methylsulfonylmethane (MSM) was analyzed for anti-ketosis activity in normal mouse hepatocyte, FL83B cell model system. Ketosis condition was induced by modulating glucose concentration. Growth hormone receptor (GHR) has a great role in the ketosis condition. We hypothesize that MSM has the ability to regulate GHR signaling through signal transducer and activator of transcription 5b (STAT5b). The whole cell lysate analysis and DNA binding activity analysis also suggested the critical role o...

Tumor angiogenesis is one of the major hallmarks of tumor progression. Nobiletin is a natural flavonoid isolated from citrus peel that has anti-angiogenic activity. Steroid receptor coactivator (Src) is an intracellular tyrosine kinase so that focal adhesion kinase (FAK) binds to Src to play a role in tumor angiogenesis. Signal transducer and activator of transcription 3 (STAT3) is a marker for tumor angiogenesis which interacts with Src. Paxillin (PXN) acts as a downstream target for both FAK and STAT3. The main goal of this study was to assess inhibition of tumor angiogenesis by nobiletin in estrogen receptor positive (ER+) breast cancer cells via Src, FAK, and STAT3-mediated signaling through PXN. Treatment with nobiletin in MCF-7 and T47D breast cancer cells inhibited angiogenesis markers, based on western blotting and RT-PCR. Validation of in vitro angiogenesis in the human umbilical vein endothelial cells (HUVEC) endothelial cell line proved the anti-angiogenic activity of nobiletin. Electrophoretic mobility shift assay and the ChIP assay showed that nobiletin inhibits STAT3/DNA binding activity and STAT3 binding to a novel binding site of the PXN gene promoter. We also investigated the migration and invasive ability of nobiletin in ER+ cells. Nobiletin inhibited tumor angiogenesis by regulating Src, FAK, and STAT3 signaling through PXN in ER+ breast cancer cells.

Physical exercise induces gene expression changes that trigger glucose metabolism pathways in organisms. In the present study, we monitored the expression levels of LDHA (lactate dehydrogenase) and GYS1 (glycogen synthase 1) in the blood, to confirm the roles of these genes in exercise physiology. LDHA and GYS1 are related to glucose metabolism and fatigue recovery, and these processes could elicit economically important traits in racehorses. We collected blood samples from three retired thoroughbred racehorses, pre-exercise and immediately after 30 min of exercise. We extracted total RNA and small RNA (≤ 200 nucleotide-long) from the blood, and assessed the expression levels of LDHA, GYS1, and microRNAs (miRNAs), by using qRT-PCR. We showed that LDHA and GYS1 were down-regulated, whereas eca-miR-33a and miR-17 were up-regulated, after exercise. We used sequences from the 3′ UTR of LDHA and GYS1, containing eca-miR-33a and miR-17 binding sites, to observe the down-regulation activity of each gene expression. We observed that the two miRNAs, namely, eca-miR-33a and miR-17, inhibited LDHA and GYS1 expression via binding to the 3′ UTR sequences of each gene. Our results indicate that eca-miR-33a and miR-17 play important roles in the glucose metabolism pathway. In addition, our findings provide a basis for further investigation of the exercise metabolism of racehorses.

Tannic acid (TA), a naturally occurring polyphenol, is a potent anti‐oxidant with anti‐proliferative effects on multiple cancers. However, its ability to modulate gene‐specific expression of tumour suppressor genes and oncogenes has not been assessed. This work investigates the mechanism of TA to regulate canonical and non‐canonical STAT pathways to impose the gene‐specific induction of G1‐arrest and apoptosis. Regardless of the p53 status and membrane receptors, TA induced G1‐arrest and apoptosis in breast cancer cells. Tannic acid distinctly modulated both canonical and non‐canonical STAT pathways, each with a specific role in TA‐induced anti‐cancer effects. Tannic acid enhanced STAT1 ser727 phosphorylation via upstream serine kinase p38. This STAT1 ser727 phosphorylation enhanced the DNA‐binding activity of STAT1 and in turn enhanced expression of p21Waf1/Cip1. However, TA binds to EGF‐R and inhibits the tyrosine phosphorylation of both STAT1 and STAT3. This inhibition leads to the inhibition of STAT3/BCL‐2 DNA‐binding activity. As a result, the expression and mitochondrial localization of BCl‐2 are declined. This altered expression and localization of mitochondrial anti‐pore factors resulted in the release of cytochrome c and the activation of intrinsic apoptosis cascade involving caspases. Taken together, our results suggest that TA modulates EGF‐R/Jak2/STAT1/3 and P38/STAT1/p21Waf1/Cip1 pathways and induce G1‐arrest and intrinsic apoptosis in breast carcinomas.

Worldwide, breast cancer (BCa) is the most common cancer in women. Among its subtypes, triple-negative breast cancer (TNBC) is an aggressive form associated with diminished survival. TNBCs are characterized by their absence, or minimal expression, of the estrogen and progesterone receptors, as well as the human epidermal growth factor receptor 2 (i.e. ER-/-, PR-/-, Her2-/Low). Consequently, treatment for this subtype of BCa remains problematic. Silibinin, a derivative of the flavonoid silymarin, is reported to have anticancer activities against hepatic and non-small cell lung cancers. We hypothesized that silibinin might inhibit cell-extracellular matrix interactions via the regulation, expression, and activation of STAT3 in TNBCs, which could directly inhibit metastasis in silibinin-treated BCa cells. Using proliferation assays, we found that exposure to silibinin at a concentration of 200 µM inhibited the proliferation of breast cancer (BCa) cells; this concentration also inhibited phosphorylation of STAT3 and its principal upstream kinase, Jak2. Furthermore, we found that silibinin inhibited the nuclear translocation of STAT3, as well as its binding to the MMP2 gene promoter. The ability of silibinin to inhibit metastasis was further studied using an in vitro invasion assay. The results confirm the role of STAT3 as a critical mediator in the invasive potential of BCa cells, and STAT3 knock-down resulted in inhibition of invasion. The invasion ability of silibinin-treated BCa cells was studied in detail with the expression of MMP2. Prevention of STAT3 activation also resulted in the inhibition of MMP2 expression. Use of a small interfering RNA to knock down STAT3 (siSTAT3) allowed us to confirm the role of STAT3 in regulating MMP2 expression, as well as the mechanism of action of silibinin in inhibiting MMP2. Taken together, we found that silibinin inhibits the Jak2/STAT3/MMP2 signaling pathway, and inhibits the proliferation, migration, and invasion of triple-negative BCa cells.

Osteoclast differentiation is dependent on the activities of receptor activator NF-kB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). Given that RANKL plays a critical role in osteoclast formation and bone resorption, any new compounds found to alter its activity would be predicted to have therapeutic potential for disorders associated with bone loss. Methylsulfonylmethane (MSM) is a naturally occurring sulfur compound with well-documented anti-oxidant and anti-inflammatory properties; currently its effects on osteoclast differentiation are unknown. We sought to investigate whether MSM could regulate osteoclastogenesis, and if so, its mechanism of action. In this study, we investigated the effects of MSM on RANKL-induced osteoclast differentiation, together with STAT3's involvement in the expression of osteoclastic gene markers. These experiments were conducted using bone marrow derived macrophages (BMMs) and cell line material, together with analyses that interrogated both protein and mRNA levels, as well as signaling pathway activity. Although MSM was not toxic to osteoclast precursors, MSM markedly inhibited RANKL-induced TRAP activity, multinucleated osteoclast formation, and bone resorptive activity. Additionally, the expression of several osteoclastogenesis-related marker genes, including TRAF6, c-Fos, NFATc1, cathepsin K, and OSCAR were suppressed by MSM. MSM mediated suppression of RANKL-induced osteoclastogenesis involved inhibition of ITAM signaling effectors such as PLCγ and Syk, with a blockade of NF-kB rather than MAPK activity. Furthermore, MSM inhibited RANKL-induced phosphorylation of STAT3 Ser727. Knockdown of STAT3 using shRNAs resulted in reduced RANKL-mediated phosphorylation of Ser727 STAT3, and TRAF6 in cells for which depletion of STAT3 was confirmed. Additionally, the expression of RANKL-induced osteoclastogenic marker genes were significantly decreased by MSM and STAT3 knockdown. Taken together, these results indicate that STAT3 plays a pivotal role in RANKL-induced osteoclast formation, and that MSM can attenuate RANKL-induced osteoclastogenesis by blocking both NF-kB and STAT3 activity.

and has been extensively studied due toits production of two valuable alkaloids, vincristine andvinblastine which are used in the treatment of humanneoplasm, and an alkaloid from the root, ajmalicine which isused in the treatment of circulatory disorders and hyper-tension. Biologically indole alkaloids produced by plants arebelieved to play a role as antimicrobial and antifeedingcompounds.

Hypoxia, a common consequence of solid tumor growth in breast cancer or other cancers, serves to propagate a cascade of molecular pathways which include angiogenesis, glycolysis, and various cell-cycle control proteins. As we have shown previously, hypoxia activates STAT5 (signal transducer and activator of transcription 5) and increases its binding activity to the GAS element in mammary epithelial cells. In this study we attempted to elucidate the mechanism by which cyclin D1 is regulated by the STAT5 protein under hypoxic conditions. Our data demonstrate that hypoxia (2% O(2)) or desferrioxamine (DFO) induces tyrosine and serine phosphorylation of STAT5 in human breast cancer cells (MCF-7) and mammary epithelial cells (HC11). Imunoprecipitation and subsequent Western analysis showed that Jak2 leads to the tyrosine phosphorylation and activation of STAT5a or STAT5b under hypoxic conditions. Using a transfected COS-7 cell model system, we demonstrate that the activity of a cyclin D1 promoter-luciferase construct increased under hypoxic conditions or DFO treatment. The activity of the STAT5b/cyclin D1 promoter increased significantly by 12 h of hypoxia, whereas the activity of the STAT5a/cyclin D1 promoter was unaffected under hypoxic conditions. These increases in promoter activity are predominantly mediated by the Jak2/STAT5b signaling pathway. We have shown by EMSA that hypoxia induces STAT5 to bind to the cyclin D1 promoter (GAS-1) in MCF-7 and HC11 cells. These data suggest that STAT5b may mediate the transcriptional activation of cyclin D1 after hypoxic stimulation.

STATs (signal transducers and activators of transcription) are proteins with dual functions: signal transducers in the cytoplasm and transcriptional activators in the nucleus. STAT proteins act as transcription factors activated by phosphorylation on its tyrosine residues upon stimulation by various cytokines. The phosphorylated STAT molecules then form homo- or heterodimers through SH2-mediated interaction and translocate into the nucleus to activate the transcription of various target genes. STAT5 recognizes the interferon-gamma activated site TTCNNNGAA (GAS sequence) in the promoter region of the beta-casein gene. Except for prolactin-dependent beta-casein production in mammary gland cells, the biological consequences of STAT5a activation in various systems are not clear. Here we showed that STAT5a was phosphorylated 10 min after desferrioxamine (DFO) treatment, and reached a maximum induction at 4 h in mammary epithelial cells (HC11) and transfected COS-7 cells. Under hypoxic conditions (2% O2), a maximal phosphorylation of STAT5a was observed within 6 h. EMSA (electrophoretic mobility shift assay) showed that DFO or hypoxia enhanced the binding activities of STAT5a DNA to beta-casein gene promoter in mammary epithelial cells (HC11) and transfected COS-7 cells. These results showed that DFO or hypoxia induces tyrosine phosphorylation of STAT5a and also increases the binding activity of STAT5a DNA in mammary epithelial cells. Our data suggest that the STAT5 may act as a mediator in hypoxia-mediated gene expression.

Aberrantly enhanced vascular endothelial growth factor (VEGF) gene expression is associated with increased tumor growth and metastatic spread of solid malignancies, including human renal carcinomas. Persistent activation of STAT3 is linked to tumor-associated angiogenesis, but underlying mechanisms remain unclear. Therefore, we examined whether STAT3 modulates the stability and activity of hypoxia-inducible factor-1alpha (HIF-1alpha), and in turn enhances VEGF expression. We found that STAT3 was activated in ischemic rat kidneys and hypoxic human renal carcinoma cells. We also found that hypoxia-induced activation of STAT3 transactivated the VEGF promoter and increased the expression of VEGF transcripts. Consistent with these findings, STAT3 inhibition attenuated the hypoxic induction of VEGF. Interestingly, activated STAT3 increased HIF-1alpha protein levels due to the HIF-1alpha stability by blocking HIF-1alpha degradation and accelerated its de novo synthesis. The novel interaction of STAT3 with HIF-1alpha was identified in hypoxic renal carcinoma cells. Furthermore, hypoxia recruited STAT3, HIF-1alpha, and p300 to the VEGF promoter and induced histone H3 acetylation. Therefore, these findings provide compelling evidence that a causal relationship exists between STAT3 activation and HIF-1-dependent angiogenesis and suggest that therapeutic modalities designed to disrupt STAT3 signaling hold considerable promise for the blocking tumor growth and enhancing apoptosis of cancer cells and tissues.

Recently, statins have been shown to have anti-inflammatory effects on lung inflammatory diseases. However, the mechanisms of action of simvastatin in viral pneumonia have yet to be elucidated, although viral infection remains a considerable health threat. In this study, we hypothesised that simvastatin inhibits polyinosinic-polycytidylic acid (poly I:C)-induced airway inflammation, such as RANTES (regulated on activation, normal T-cell expressed and secreted) expression and inflammatory cell recruitment. In bronchial cells, the effect of simvastatin on poly I:C-induced RANTES expression and signal transducer and activator of transcription (STAT)3-mediated signal transduction was determined using an ELISA and short hairpin (sh)RNA system. In a poly I:C-induced pneumonia mouse model, immunological changes in the lungs after simvastatin inhalation, such as inflammatory cell recruitment and cytokine/chemokine release, were examined. In poly I:C-stimulated bronchial cells, RANTES secretion was increased by STAT3 activation, and simvastatin suppressed poly I:C-induced STAT3 activation, resulting in inhibition of RANTES expression. In BALB/c mice stimulated with inhaled poly I:C, RANTES expression and neutrophil infiltration into the airway were elevated. However, simvastatin treatment attenuated STAT3 activation, RANTES release and subsequent neutrophilia in the lungs. These findings suggest that simvastatin inhibits airway inflammation, but there are other mechanisms that need to be fully elucidated.

Objectives We aimed to determine the anti-arthritis effect and its mechanism of a combination of herbal extracts from Trachelospermi caulis (TC) and Moutan cortex radicis (MC) (TCMC). Methods The anti-arthritis activity of TCMC was assessed using a mouse model of type II collagen-induced arthritis (CIA). Reverse transcriptase-polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA), electrophoretic mobility shift assay (EMSA) and other biological assays were performed. Key findings TCMC significantly ameliorated various inflammatory parameters, such as clinical arthritis index, histological deformation of joints, serum levels of rheumatoid arthritis biomarkers (cartilage oligomeric matrix protein, serum amyloid P and anti-collagen type II IgG antibody), and Th1-related responses (T cell proliferation, and production of Interferon-γ and interleukin (IL)-2 in splenocytes isolated from CIA mice). The production of matrix metalloproteinases (MMPs), pro-inflammatory cytokines (tumour necrosis factor-α, IL-1β and IL-6) and chemokines (macrophage inflammatory protein-1, monocyte chemoattractant protein-1, and Regulated upon Activation, Normal T-cell Expressed, and Secreted) was suppressed by TCMC in CIA mice. In addition, the number of osteoclasts in the hind tibia was significantly decreased. With regard to the mechanism, TCMC suppressed the activation of the transcription factors nuclear factor (NF)-κB and activator protein (AP)-1. Conclusions TCMC exerts an anti-arthritis effect in CIA mice by suppression of the production of various inflammatory factors and the formation of osteoclasts through the inhibition of NF-κB and AP-1 activation.

In the mitochondrial matrix, the OXCT1 gene catalyzes the reversible transfer of coenzyme A from succinyl-CoA to acetoacetate in a reaction related to energy production from ketone bodies. Here, horse OXCT1 gene containing coenzyme A transferase domain was identified in the transcriptome analysis of cDNAs derived from skeletal muscles. Horse OXCT1 gene consisted of 1761 [corrected] nucleotide sequences with an open reading frame of 1560 nucleotides encoding a protein of 520 putative amino acid residues.The number of non-synonymous substitutions was lower than the number of synonymous substitutions in the OXCT1 genes of other species, indicating that purifying selection occurred in the OXCT1 genes during evolutionary radiation. Quantitative real-time RT-RCR analysis showed a dominant expression pattern of horse OXCT1 gene in the cerebrum, heart, and skeletal muscle. Different expression levels of horse OXCT1 transcripts between before- and after-exercise samples were also measured in the skeletal muscles of six horses. These data could be of great use for further investigation of the relationship between energy products and horse OXCT1 gene.

Cyclin D1 and insulin-like growth factor 1 receptor (IGF-1R) are key regulators of cell proliferation that are overexpressed in most breast cancers. The purpose of the present study was to investigate the molecular mechanism by which hemin exerts its inhibitory effects on aggressive breast cancer cells. We found that hemin regulates cyclin D1 and IGF-1R proteins and insulin-like growth factor-1 gene expression through STAT5b in breast cancer cells. We confirmed that STAT5b, cyclin D1, and IGF-1R is up-regulated by hypoxia, and the increased STAT5b binds strongly to the STAT5-binding sites contained within the distal 5'-flanking region of IGF-1 gene in breast cancer cells. EMSA studies showed that STAT5 binding activity to the IGF-1 and cyclin D1 promoter was distinctly decreased by hemin in STAT5b-transfected COS-7 or MDA-MB 231 cells. IGF-1 gene expression was also decreased by hemin in mammary epithelial cells. STAT5b expression was inhibited in siRNA experiments and by hemin, leading to decreased levels of IGF-1. These results provide a basis for molecular targets in cancer treatment via the STAT5b/IGF-1 or /cyclin D1 pathway in solid tumor cells. These data indicate that hemin inhibits the cyclin D1 and IGF-1 expression via STAT5b under hypoxia in ERalpha-negative breast cancer cells. These findings are valuable toward understanding the role of hemin-induced inhibition of cyclin D1 and IGF-1 expression under hypoxia in invasive and metastatic breast cancer.

and has been extensively studied due toits production of two valuable alkaloids, vincristine andvinblastine which are used in the treatment of humanneoplasm, and an alkaloid from the root, ajmalicine which isused in the treatment of circulatory disorders and hyper-tension. Biologically indole alkaloids produced by plants arebelieved to play a role as antimicrobial and antifeedingcompounds.

Ethyl pyruvate (EP) has been demonstrated to have an anti-inflammatory function. However, the molecular mechanisms underlying the anti-inflammatory action of EP are largely unknown. We here show that EP exerts its anti-inflammatory effect by inhibiting ROS-dependent STAT signaling through its antioxidant activity, like vitamin C or N-acetyl-L-cysteine. The inhibition of STAT1 and STAT3 by EP prevented their translocation to the nucleus and consequently inhibited expression of iNOS and COX-2 by inhibiting STAT1- and STAT3-mediated transcriptional activity, followed by changes in chromatin conformation via deacetylation of histones H3 and H4 in both gene promoters. EP also suppressed transcripts of other STAT-responsive inflammatory genes such as IL-1beta, IL-6, TNF-alpha, and MCP-1. We further found that the mechanism of inhibition of STAT1 and STAT3 by EP is due to inhibition of JAK2 through Rac1 inactivation and SOCS1 induction. These findings offer new therapeutic possibilities for EP based on a better understanding of the mechanism underlying the action of EP.

Several constitutively activated signal transducers and activators of transcription (STAT) proteins have been observed in a wide number of human cancer cell lines and primary tumors. Normal cells maintain normoxic conditions but tumor cells are characteristically hypoxic. We studied the altered activation and tyrosine phosphorylation of STATs under hypoxic conditions (2% O2) or desferrioxamine (DFO) treatment in mouse mammary epithelial cells (HC11) and a human breast cancer cell line (MCF-7). STAT1, -3 and -5 proteins are especially important and are observed at elevated levels in tumorigenesis. We also investigated the serine phosphorylation of STAT1, -3, and -5 under hypoxic conditions or DFO treatment in HC11 and MCF-7 cells. Here we show that DFO or hypoxia stimulates the tyrosine and/or serine phosphorylation and the expression of STAT proteins in breast cancer cells. Our data suggest that DFO or hypoxic condition is a critical stimulator for the activation of STAT proteins in breast cancer cells. These results may provide the basis for identifying another mechanism of breast tumorigenesis via the JAK/STAT pathway in hypoxia. Also, activation of STAT proteins by hypoxia may play an important role in the physiological phenomenon of embryonic stem cells and old cells with hypoxic conditions.

Breast cancer is the most aggressive form of all cancers, with high incidence and mortality rates. The purpose of the present study was to investigate the molecular mechanism by which methylsulfonylmethane (MSM) inhibits breast cancer growth in mice xenografts. MSM is an organic sulfur-containing natural compound without any toxicity. In this study, we demonstrated that MSM substantially decreased the viability of human breast cancer cells in a dose-dependent manner. MSM also suppressed the phosphorylation of STAT3, STAT5b, expression of IGF-1R, HIF-1 alpha, VEGF, BrK and p-IGF-1R and inhibited triple-negative receptor expression in receptor-positive cell lines. Moreover, expression in receptor-positive cell-lines. Moreover, MSM decreased the DNA-binding activities of STAT5b and STAT3, to the target gene promoters in MDA-MB 231 or co-transfected COS-7 cells. We confirmed that MSM significantly decreased the relative luciferase activities indicating crosstalk between STAT5b/IGF-1R, STAT5b/HSP90 alpha, and STAT3/VEGF. To confirm these findings in vivo, xenografts were established in Balb/c athymic nude mice with MDA-MB 231 cells and MSM was administered for 30 days. Concurring to our in vitro analysis, these xenografts showed decreased expression of STAT3, STAT5b, IGF-1R and VEGF. Through in vitro and in vivo analysis, we confirmed that MSM can effectively regulate multiple targets including STAT3/VEGF and STAT5b/IGF-1R. These are the major molecules involved in tumor development, progression, and metastasis: Thus, we strongly recommend the use of MSM as a trial drug for treating all types of breast cancers including triple-negative cancers.

We have shown previously that hypoxia activates the cyclin D1 promoter via the Jak2/STAT5b pathway in breast cancer cells. Most solid tumors contain hypoxic components and overexpression of cyclin D1. The purpose of the present study was to investigate the molecular mechanism by which momilactone B exerts its inhibitory effects on breast cancer cells. Momilactone B, extracted from Korean rice hulls, suppressed hypoxia-induced increases in phospho-STAT5, STAT5b, cyclin D1, and cdk4 protein levels in human breast cancer cells. STAT5b expression was inhibited by siRNA experiments leading to decreased cyclin D1. The effects of momilactone B on cell growth and apoptosis-related gene expression were investigated in breast cancer cells under hypoxic conditions (2% O2). Bax and p21 expression was found to be up-regulated, whereas ppRb and bcl-2 were down-regulated in momilactone B-treated cells under hypoxic conditions. However, the p53 protein level did not change. Flow cytometry with Annexin-FITC staining showed that the number of apoptotic cells increased in hypoxic cells treated with momilactone B compared with untreated hypoxic cells. Furthermore, caspase activity increased upon treatment with momilactone B under hypoxic conditions. These results indicate that momilactone B inhibits the growth of breast cancer cells, regulates the expression of apoptosis-related genes, and induces apoptosis through STAT5b and a caspase-3 dependent pathway. We suggest that momilactone B accelerates hypoxia-induced apoptosis of human breast cancer cells through STAT5b, and may represent an effective chemopreventive or therapeutic agent against breast cancer.