Visfatin and Resveratrol Differentially Regulate the Expression of Thymidylate Synthase to Control the Sensitivity of Human Colorectal Cancer Cells to Capecitabine Cytotoxicity.

Colorectal cancer (CRC) is a highly lethal malignant cancer. Capecitabine, a 5-fluororacil (5-FU) derivate, is its first-line drug, but the resistance of CRC to capecitabine is still the most challenging factor for curing patients. It has been suggested that thymidylate synthase (TYMS) level might affect the capecitabine efficacy in CRC patients, but the mechanism still needs more elucidation. Obesity is a risk factor for CRC. Recently, a correlation between serum visfatin, an obesity-elicited adipokine, and CRC development has been found. Thus, the aim of present study is to examine the visfatin capacity in TYMS expression and in the development of capecitabine resistance of CRC. Moreover, an attractive natural component, i.e., resveratrol, has been proposed in anticancer therapy and has hence been examined in the present study to see its potential capacity in the alleviation of CRC resistance. Our results found that visfatin significantly reduces the CRC sensitivity to capecitabine by controlling the TYMS expression via p38 signaling and Sp1 transcription factor. Moreover, resveratrol could significantly alleviate the visfatin effect on capecitabine-treated CRC cells. These results provided new insights to understand the capecitabine susceptibility of CRC under a visfatin-containing environment and a possible therapeutic application of resveratrol in CRC patients with obesity.

Truncated Pneumolysin from Streptococcus pneumoniae as a TLR4-Antagonizing New Drug for Chronic Inflammatory Conditions.

Microbial proteins have recently been found to have more benefits in clinical disease treatment because of their better-developed strategy and properties than traditional medicine. In this study, we investigated the effectiveness of a truncated peptide synthesized from the C-terminal sequence of pneumolysin, i.e., C70PLY4, in Streptococcus pneumoniae, in treating chronic inflammatory conditions. It has been shown that C70PLY4 significantly blocks the transendothelial migration of neutrophils and attenuates the formation of atherosclerotic plaque and the secretion of soluble forms of the intercellular adhesion molecule-1 (ICAM-1), the vascular cell adhesion molecule 1 (VCAM-1), and E-selectin in high-fat-diet/streptozotocin-induced inflammatory rats. The mechanism and the docking simulation analysis further indicated that C70PLY4 might serve as a Toll-like receptor 4 (TLR4) antagonist by competing for the binding site of MD2, an indispensable protein for lipopolysaccharide (LPS)-TLR4 interaction signaling, on the TLR4 structure. Moreover, compared to the full-length PLY, C70PLY4 seems to have no cytotoxicity in human vascular endothelial cells. Our study elucidated a possible therapeutic efficacy of C70PLY4 in reducing chronic inflammatory conditions and clarified the underlying mechanism. Thus, our findings identify a new drug candidate that, by blocking TLR4 activity, could be an effective treatment for patients with chronic inflammatory diseases.

Differential regulation of human aortic smooth muscle cell proliferation by monocyte-derived macrophages from diabetic patients.

Macrophage accumulation in the arterial wall and smooth muscle cell (SMC) proliferation are features of type 2 diabetes mellitus (DM) and its vascular complications. However, the effects of diabetic monocyte-derived macrophages on vascular SMC proliferation are not clearly understood. In the present study, we investigated the pro-proliferative effect of macrophages isolated from DM patients on vascular SMCs. Macrophage-conditioned media (MCM) were prepared from macrophages isolated from DM patients. DM-MCM treatment induced HASMC proliferation, decreased p21(Cip1) and p27(Kip1) expressions, and increased microRNA (miR)-17-5p and miR-221 expressions. Inhibition of either miR-17-5p or miR-221 inhibited DM-MCM-induced cell proliferation. Inhibition of miR-17-5p abolished DM-MCM-induced p21(Cip1) down-regulation; and inhibition of miR-221 attenuated the DM-MCM-induced p27(Kip1) down-regulation. Furthermore, blocking assays demonstrated that PDGF-CC in DM-MCM is the major mediators of cell proliferation in SMCs. In conclusion, our present data support the hypothesis that SMC proliferation stimulated by macrophages may play critical roles in vascular complications in DM patients and suggest a new mechanism by which arterial disease is accelerated in diabetes.

High glucose-treated macrophages augment E-selectin expression in endothelial cells.

E-selectin expression by endothelial cells (ECs) is crucial for leukocyte recruitment during the inflammatory response. Macrophage accumulation and serum E-selectin elevation are features of type 2 diabetes mellitus. However, the interactions between macrophages and ECs in regulating vascular endothelial function are not clearly understood. We investigated the mechanisms underlying the modulation of EC E-selectin expression by high glucose (HG)-treated macrophages. Macrophage-conditioned media (MCM) were prepared from HG-treated macrophages. EC stimulation with HG-MCM induced increases the expression and secretion of E-selectin. By using specific inhibitors and small interfering RNAs, we demonstrate that the activation of the JNK and p38 MAPK pathways are critical for HG-MCM-induced E-selectin expression. Transcription factor ELISA and chromatin immunoprecipitation assays further showed that HG-MCM increases the NF-κB- and AP-1 DNA-binding activities in ECs. The inhibition of NF-κB and AP-1 activation by specific siRNAs blocks the HG-MCM-induced E-selectin promoter activity and expression. Protein arrays and blocking assays using neutralizing antibodies demonstrated that macrophage inflammatory protein 1α and 1ß in HG-MCM are major mediators for the induction of EC E-selectin expression. These data support the hypothesis that E-selectin up-regulation stimulated by macrophages may play an active role in atherogenesis in the HG condition and suggest a new mechanism by which arterial disease is accelerated in diabetes.

Homocysteine induces smooth muscle cell proliferation through differential regulation of cyclins A and D1 expression.

The mechanism of homocysteine-induced cell proliferation in human vascular smooth muscle cells (SMCs) remains unclear. We investigated the molecular mechanisms by which homocysteine affects the expression of cyclins A and D1 in human umbilical artery SMCs (HUASMCs). Homocysteine treatment induced proliferation of HUASMCs and increased the expression levels of cyclins A and D1. Knocking down either cyclin A or cyclin D1 by small interfering RNA (siRNA) inhibited homocysteine-induced cell proliferation. Furthermore, treatment with extracellular signal-related kinase (ERK) inhibitor (PD98059) and dominant negative Ras (RasN17) abolished homocysteine-induced cyclin A expression; and treatment with phosphatidylinositol 3-kinase (PI3K) inhibitor (LY294002) and mammalian target of rapamycin (mTOR) inhibitor (rapamycin) attenuated the homocysteine-induced cyclin D1 expression. Homocysteine also induced transient phosphorylation of ERK, Akt, and p70 ribosomal S6 kinase (p70S6K). Neutralizing antibody and siRNA for ß1 integrin blocked cell proliferation, expression of cyclins A and D1, and phosphorylation of ERK and Akt. In conclusion, homocysteine-induced differential activation of Ras/ERK and PI3K/Akt/p70S6K signaling pathways and consequent expression of cyclins A and D1 are dependent on ß1 integrin. Homocysteine may accelerate progression of atherosclerotic lesions by promoting SMC proliferation.

Butyrate reduced lipopolysaccharide-mediated macrophage migration by suppression of Src enhancement and focal adhesion kinase activity.

Macrophage motility is vital in innate immunity. Lipopolysaccharide (LPS)-mediated macrophage migration requires the enhancement of Src expression and enzymatic activity, which can be regulated by inducible nitric oxide synthase (iNOS). As a major short-chain fatty acid with histone deacetylase (HDAC) inhibitor activity, butyrate exerts anti-inflammatory effect by regulating the expression of cytokines. However, the influence of butyrate on macrophage movement was vague. In this study, we observed that butyrate inhibited migration of both RAW264.7 and rat peritoneal macrophages elicited by LPS. Unlike its myeloid relatives (i.e. Lyn, Fgr and Hck) whose expression was almost unaltered in the presence or absence of butyrate in LPS-treated macrophages, LPS-mediated Src induction was greatly suppressed by butyrate and that could be attributable to reduced level of the src transcript. Similar phenomenon was also detected in LPS-treated macrophages exposed to another HDAC inhibitor, trichostatin A (TSA). Consistent with the indispensability of iNOS in promoting macrophage mobilization via Src up-regulation and the activation of both Src and FAK, we did observe concomitant decrement of iNOS, Src and the suppressed activity of Src and FAK in butyrate- or TSA-pretreated macrophages following LPS exposure. These results imply that by virtue of reduction of Src, butyrate could effectively hamper LPS-triggered macrophage locomotion.

Shear stress inhibits homocysteine-induced stromal cell-derived factor-1 expression in endothelial cells.

RATIONALE: Hyperhomocysteinemia contributes to vascular dysfunction and risks of cardiovascular diseases. Stromal cell-derived factor (SDF)-1, a chemokine expressed by endothelial cells (ECs), is highly expressed in advanced atherosclerotic lesions. The interplays among homocysteine, chemokines, and shear stress in regulating vascular endothelial function are not clearly understood. OBJECTIVE: To investigate the mechanisms for modulations of EC SDF-1 expression by homocysteine and shear stress. METHODS AND RESULTS: Homocysteine stimulation induced dose- and time-dependent SDF-1 expression and phosphorylation of mitogen-activated protein kinases extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38. By using specific inhibitors, small interfering (si)RNA, and dominant negative mutants, we demonstrated that activation of JNK pathway is critical for the homocysteine-induced SDF-1 expression. Transcription factor ELISA and chromatin immunoprecipitation assays showed that homocysteine increased Sp1- and AP-1-DNA binding activities in ECs. Inhibition of Sp1 and AP-1 activations by specific siRNA blocked the homocysteine-induced SDF-1 promoter activity and expression. Preshearing of ECs for 1 to 4 hours at 20 dyn/cm2 inhibited the homocysteine-induced JNK phosphorylation, Sp1 and AP-1 activation, and SDF-1 expression. The homocysteine-induced SDF-1 expression was suppressed by NO donor. Inhibitor or siRNA for endothelial NO synthase abolished the shear inhibition of SDF-1 expression. CONCLUSIONS: Our findings serve to elucidate the molecular mechanisms underlying the homocysteine induction of SDF-1 expression in ECs and the shear stress protection against this induction.

Lipopolysaccharide-induced c-Src expression plays a role in nitric oxide and TNFalpha secretion in macrophages.

As tyrosine kinases are indispensable in lipopolysaccharide (LPS)-induced macrophage activation, the myeloid-specific Src members (i.e. Lyn, Fgr and Hck) are speculated to play important roles in this process. However, the normal LPS responsiveness in lyn(-/-)fgr(-/-)hck(-/-) macrophages implicates the presence of an elusive, compensating tyrosine kinase(s). In this study, we demonstrate the upregulation of c-Src in Raw264.7 and peritoneal macrophages (PEMs) by LPS, which is inhibited by PP2 (an inhibitor for Src family kinases), pyrrolidinedithiocarbamate (PDTC; NF-kappaB inhibitor) and LY294002 (PI3K inhibitor). And this LPS-mediated c-Src induction is also observed in macrophages recovered from LPS-challenged rats. Intriguingly, PP2 attenuates the ability of PEMs to elicit COX-2 expression and nitric oxide production in response to LPS. Similar results are also observed when macrophages recovered from rats receiving either LPS alone or LPS and PP2 both are compared. Furthermore, administration of PP2 in Raw264.7 and animal models of sepsis greatly suppresses TNFalpha secretion and serum TNFalpha level, respectively. Therefore, we conclude that c-Src, with its LPS induction, has an unperceived role in transmitting LPS signaling in macrophages.

Butyrate regulates the expression of c-Src and focal adhesion kinase and inhibits cell invasion of human colon cancer cells.

Epidemiological studies indicate that dietary fiber-derived fermentation products such as butyrate can prevent colon cancer development. To further dissect the role of butyrate in anticarcinogenesis, its effect on cellular growth and invasion as well as the expression of c-Src and FAK, two mutually interactive nonreceptor tyrosine kinases, in three different human colon cancer cell lines (Caco-2, SW480, and SW620) were investigated. In addition to growth inhibition, butyrate treatment results in a significant downregulation of c-Src and FAK in human colon cancer cells, which can be attributable to their reduced transcripts and implicates the participation of a butyrate-sensitive pathway in modulating their expression. Concurrent to butyrate-reduced c-Src and FAK expression is the decrease of FAK Tyr-decrease 397 phosphorylation. Besides, butyrate also abolished the secretion of MMP-2 and MMP-9. And these butyrate-mediated effects severely impaired invasion of SW620 cells through Matrigel in vitro. Interestingly, in situ parallel enhancement of c-Src and FAK was also observed in human colorectal tumor specimens. These results imply that by virtue of suppression of c-Src and FAK along with other butyrate targets in colonocytes, butyrate could effectively inhibit tumor growth and invasion.