Oral Delivery of Photopolymerizable Nanogels Loaded with Gemcitabine for Pancreatic Cancer Therapy: Formulation Design, and in vitro and in vivo Evaluations.

Background: Gemcitabine (GEM) faces challenges of poor oral bioavailability and extensive first-pass metabolism. Currently, only injectable formulations are available for clinical use. Hence, there is an urgent demand for the development of advanced, efficacious, and user-friendly dosage forms to maintain its status as the primary treatment for pancreatic ductal adenocarcinoma (PDAC). Nanogels (NGs) offer a novel oral drug delivery system, ideal for hydrophilic compounds like GEM. This study aims to develop NGs tailored for GEM delivery, with the goal of enhancing cellular uptake and gastrointestinal permeability for improved administration in PDAC patients. Methods: We developed cross-linked NGs via photopolymerization of methacryloyl for drug delivery of GEM. We reveal characterization, cytotoxicity, and cellular uptake studies in Caco-2 and MIA PaCa-2 cells. In addition, studies of in vitro permeability and pharmacokinetics were carried out to evaluate the bioavailability of the drug. Results: Our results show NGs, formed via photopolymerization of methacryloyl, had a spherical shape with a size of 233.91±7.75 nm. Gemcitabine-loaded NGs (NGs-GEM) with 5% GelMA exhibited efficient drug loading (particle size: 244.07±19.52 nm). In vitro drug release from NGs-GEM was slower at pH 1.2 than pH 6.8. Cellular uptake studies indicated significantly enhanced uptake in both MIA PaCa-2 and Caco-2 cells. While there was no significant difference in GEM's AUC and Cmax between NGs-GEM and free-GEM groups, NGs-GEM showed markedly lower dFdU content (10.07 hr∙µg/mL) compared to oral free-GEM (19.04 hr∙µg/mL) after oral administration (p<0.01), highlighting NGs' efficacy in impeding rapid drug metabolism and enhancing retention. Conclusion: In summary, NGs enhance cellular uptake, inhibit rapid metabolic degradation of GEM, and prolong retention after oral administration. These findings suggest NGs-GEM as a promising candidate for clinical use in oral pancreatic cancer therapy.

Suppression of lipopolysaccharide-induced COX-2 expression via p38MAPK, JNK, and C/EBPß phosphorylation inhibition by furomagydarin A, a benzofuran glycoside from Magydaris pastinacea.

The phytochemical investigation of the methanol extract of the seeds of Magydaris pastinacea afforded two undescribed benzofuran glycosides, furomagydarins A-B (1, 2), together with three known coumarins. The structures of the new isolates were elucidated after extensive 1D and 2D NMR experiments as well as HR MS. Compound 1 was able to inhibit the COX-2 expression in RAW264.7 macrophages exposed to lipopolysaccharide, a pro-inflammatory stimulus. RT-qPCR and luciferase reporter assays suggested that compound 1 reduces COX-2 expression at the transcriptional level. Further studies highlighted the capability of compound 1 to suppress the LPS-induced p38MAPK, JNK, and C/EBPß phosphorylation, leading to COX-2 down-regulation in RAW264.7 macrophages.

Role of the Autism Risk Gene Shank3 in the Development of Atherosclerosis: Insights from Big Data and Mechanistic Analyses.

Increased medical attention is needed as the prevalence of autism spectrum disorder (ASD) rises. Both cardiovascular disorder (CVD) and hyperlipidemia are closely associated with adult ASD. Shank3 plays a key genetic role in ASD. We hypothesized that Shank3 contributes to CVD development in young adults with ASD. In this study, we investigated whether Shank3 facilitates the development of atherosclerosis. Using Gene Set Enrichment Analysis software (Version No.: GSEA-4.0.3), we analyzed the data obtained from Shank3 knockout mice (Gene Expression Omnibus database), a human population-based study cohort (from Taiwan's National Health Insurance Research Database), and a Shank3 knockdown cellular model. Shank3 knockout upregulated the expression of genes of cholesterol homeostasis and fatty acid metabolism but downregulated the expression of genes associated with inflammatory responses. Individuals with autism had higher risks of hyperlipidemia (adjusted hazard ratio (aHR): 1.39; p < 0.001), major adverse cardiac events (aHR: 2.67; p < 0.001), and stroke (aHR: 3.55; p < 0.001) than age- and sex-matched individuals without autism did. Shank3 downregulation suppressed tumor necrosis factor-α-induced fatty acid synthase expression; vascular cell adhesion molecule 1 expression; and downstream signaling pathways involving p38, Jun N-terminal kinase, and nuclear factor-κB. Thus, Shank3 may influence the development of early-onset atherosclerosis and CVD in ASD. Furthermore, regulating Shank3 expression may reduce inflammation-related disorders, such as atherosclerosis, by inhibiting tumor necrosis factor-alpha-mediated inflammatory cascades.

Differential cellular responses to FDA-approved nanomedicines: an exploration of albumin-based nanocarriers and liposomes in protein corona formation.

Albumin nanoparticles (NPs) and PEGylated liposomes have garnered tremendous interest as therapeutic drug carriers due to their unique physicochemical properties. These unique properties also have significant effects on the composition and structure of the protein corona formed around these NPs in a biological environment. Herein, protein corona formation on albumin NPs and liposomes was simultaneously evaluated through in vitro and simulation studies. The sizes of both types of NPs increased with more negatively charged interfaces upon being introduced into fetal bovine serum. Gel electrophoresis and label-free quantitative proteomics were performed to identify proteins recruited to the hard corona, and fewer proteins were found in albumin NPs than in liposomes, which is in accordance with isothermal titration calorimetry. The cellular uptake efficiency of the two NPs significantly differed in different serum concentrations, which was further scrutinized by loading an anticancer compound into albumin NPs. The presence of the hard protein corona increased the cellular uptake of albumin NPs in comparison with liposomes. In our simulation study, a specific receptor present in the membrane was greatly attracted to the albumin-apolipoprotein E complex. Overall, this study not only evaluated protein corona formation on albumin NPs, but also made promising advancements toward albumin- and liposome-based therapeutic systems.

Src-FAK Signaling Mediates Interleukin 6-Induced HCT116 Colorectal Cancer Epithelial-Mesenchymal Transition.

Colorectal cancer is one of the most prevalent and lethal malignancies, affecting approximately 900,000 individuals each year worldwide. Patients with colorectal cancer are found with elevated serum interleukin-6 (IL-6), which is associated with advanced tumor grades and is related to their poor survival outcomes. Although IL-6 is recognized as a potent inducer of colorectal cancer progression, the detail mechanisms underlying IL-6-induced colorectal cancer epithelial-mesenchymal transition (EMT), one of the major process of tumor metastasis, remain unclear. In the present study, we investigated the regulatory role of IL-6 signaling in colorectal cancer EMT using HCT116 human colorectal cancer cells. We noted that the expression of epithelial marker E-cadherin was reduced in HCT116 cells exposed to IL-6, along with the increase in a set of mesenchymal cell markers including vimentin and α-smooth muscle actin (α-SMA), as well as EMT transcription regulators-twist, snail and slug. The changes of EMT phenotype were related to the activation of Src, FAK, ERK1/2, p38 mitogen-activated protein kinase (p38MAPK), as well as transcription factors STAT3, κB and C/EBPß. IL-6 treatment has promoted the recruitment of STAT3, κB and C/EBPß toward the Twist promoter region. Furthermore, the Src-FAK signaling blockade resulted in the decline of IL-6 induced activation of ERK1/2, p38MAPK, κB, C/EBPß and STAT3, as well as the decreasing mesenchymal state of HCT116 cells. These results suggested that IL-6 activates the Src-FAK-ERK/p38MAPK signaling cascade to cause the EMT of colorectal cancer cells. Pharmacological approaches targeting Src-FAK signaling may provide potential therapeutic strategies for rescuing colorectal cancer progression.

Anti-Angiogenetic and Anti-Lymphangiogenic Effects of a Novel 2-Aminobenzimidazole Derivative, MFB.

Background and Purpose: Benzimidazoles have attracted much attention over the last few decades due to their broad-spectrum pharmacological properties. Increasing evidence is showing the potential use of benzimidazoles as anti-angiogenic agents, although the mechanisms that impact angiogenesis remain to be fully defined. In this study, we aim to investigate the anti-angiogenic mechanisms of MFB, a novel 2-aminobenzimidazole derivative, to develop a novel angiogenesis inhibitor. Experimental Approach: MTT, BrdU, migration and invasion assays, and immunoblotting were employed to examine MFB's effects on vascular endothelial growth factor (VEGF)-induced endothelial cell proliferation, migration, invasion, as well as signaling molecules activation. The anti-angiogenic effects of MFB were analyzed by tube formation, aorta ring sprouting, and matrigel plug assays. We also used a mouse model of lung metastasis to determine the MFB's anti-metastatic effects. Key Results: MFB suppressed cell proliferation, migration, invasion, and endothelial tube formation of VEGF-A-stimulated human umbilical vascular endothelial cells (HUVECs) or VEGF-C-stimulated lymphatic endothelial cells (LECs). MFB suppressed VEGF-A and VEGF-C signaling in HUVECs or LECs. In addition, MFB reduced VEGF-A- or tumor cells-induced neovascularization in vivo. MFB also diminished B16F10 melanoma lung metastasis. The molecular docking results further showed that MFB may bind to VEGFR-2 rather than VEGF-A with high affinity. Conclusions and Implications: These observations indicated that MFB may target VEGF/VEGFR signaling to suppress angiogenesis and lymphangiogenesis. It also supports the role of MFB as a potential lead in developing novel agents for the treatment of angiogenesis- or lymphangiogenesis-associated diseases and cancer.

Suppressing VEGF-A/VEGFR-2 Signaling Contributes to the Anti-Angiogenic Effects of PPE8, a Novel Naphthoquinone-Based Compound.

Natural naphthoquinones and their derivatives exhibit a broad spectrum of pharmacological activities and have thus attracted much attention in modern drug discovery. However, it remains unclear whether naphthoquinones are potential drug candidates for anti-angiogenic agents. The aim of this study was to evaluate the anti-angiogenic properties of a novel naphthoquinone derivative, PPE8, and explore its underlying mechanisms. Determined by various assays including BrdU, migration, invasion, and tube formation analyses, PPE8 treatment resulted in the reduction of VEGF-A-induced proliferation, migration, and invasion, as well as tube formation in human umbilical vein endothelial cells (HUVECs). We also used an aorta ring sprouting assay, Matrigel plug assay, and immunoblotting analysis to examine PPE8's ex vivo and in vivo anti-angiogenic activities and its actions on VEGF-A signaling. It has been revealed that PPE8 inhibited VEGF-A-induced micro vessel sprouting and was capable of suppressing angiogenesis in in vivo models. In addition, PPE8 inhibited VEGF receptor (VEGFR)-2, Src, FAK, ERK1/2, or AKT phosphorylation in HUVECs exposed to VEGF-A, and it also showed significant decline in xenograft tumor growth in vivo. Taken together, these observations indicated that PPE8 may target VEGF-A-VEGFR-2 signaling to reduce angiogenesis. It also supports the role of PPE8 as a potential drug candidate for the development of therapeutic agents in the treatment of angiogenesis-related diseases including cancer.

Lovastatin-mediated MCF-7 cancer cell death involves LKB1-AMPK-p38MAPK-p53-survivin signalling cascade.

There is increasing evidence that statins, which are widely used in lowering serum cholesterol and the incidence of cardiovascular diseases, also exhibits anti-tumour properties. The underlying mechanisms by which statins-induced cancer cell death, however, remain incompletely understood. In this study, we explored the anti-tumour mechanisms of a lipophilic statin, lovastatin, in MCF-7 breast cancer cells. Lovastatin inhibited cell proliferation and induced cell apoptosis. Lovastatin caused p21 elevation while reduced cyclin D1 and survivin levels. Lovastatin also increased p53 phosphorylation, acetylation and its reporter activities. Results from chromatin immunoprecipitation analysis showed that p53 binding to the survivin promoter region was increased, while Sp1 binding to the region was decreased, in MCF-7 cells after lovastatin exposure. These actions were associated with liver kinase B1 (LKB1), AMP-activated protein kinase (AMPK) and p38 mitogen-activated protein kinase (p38MAPK) activation. Lovastatin's enhancing effects on p53 activation, p21 elevation and survivin reduction were significantly reduced in the presence of p38MAPK signalling inhibitor. Furthermore, LKB1-AMPK signalling blockade abrogated lovastatin-induced p38MAPK and p53 phosphorylation. Together these results suggest that lovastatin may activate LKB1-AMPK-p38MAPK-p53-survivin cascade to cause MCF-7 cell death. The present study establishes, at least in part, the signalling cascade by which lovastatin induces breast cancer cell death.

WMJ-S-001, a Novel Aliphatic Hydroxamate-Based Compound, Suppresses Lymphangiogenesis Through p38mapk-p53-survivin Signaling Cascade.

Background and purpose: Angiogenesis and lymphangiogenesis are major routes for metastatic spread of tumor cells. It thus represent the rational targets for therapeutic intervention of cancer. Recently, we showed that a novel aliphatic hydroxamate-based compound, WMJ-S-001, exhibits anti-angiogenic, anti-inflammatory and anti-tumor properties. However, whether WMJ-S-001 is capable of suppressing lymphangiogenesis remains unclear. We are thus interested in exploring WMJ-S-001's anti-lymphangiogenic mechanisms in lymphatic endothelial cell (LECs). Experimental approach: WMJ-S-001's effects on LEC proliferation, migration and invasion, as well as signaling molecules activation were analyzed by immunoblotting, flow-cytometry, MTT, BrdU, migration and invasion assays. We performed tube formation assay to examine WMJ-S-001's ex vivo anti-lymphangiogenic effects. Key results: WMJ-S-001 inhibited serum-induced cell proliferation, migration, invasion in murine LECs (SV-LECs). WMJ-S-001 reduced the mRNA and protein levels of survivin. Survivin siRNA significantly suppressed serum-induced SV-LEC invasion. WMJ-S-001 induced p53 phosphorylation and increased its reporter activities. In addition, WMJ-S-001 increased p53 binding to the promoter region of survivin, while Sp1 binding to the region was decreased. WMJ-S-001 induced p38 mitogen-activated protein kinase (p38MAPK) activation. p38MPAK signaling blockade significantly inhibited p53 phosphorylation and restored survivin reduction in WMJ-S-001-stimulated SV-LCEs. Furthermore, WMJ-S-001 induced survivin reduction and inhibited cell proliferation, invasion and tube formation of primary human LECs. Conclusions and Implications: These observations indicate that WMJ-S-001 may suppress lymphatic endothelial remodeling and reduce lymphangiogenesis through p38MAPK-p53-survivin signaling. It also suggests that WMJ-S-001 is a potential lead compound in developing novel agents for the treatment of lymphangiogenesis-associated diseases and cancer.

A novel 2-aminobenzimidazole-based compound Jzu 17 exhibits anti-angiogenesis effects by targeting VEGFR-2 signalling.

BACKGROUND AND PURPOSE: Recent development in drug discovery have shown benzimidazole to be an important pharmacophore,. Benzimidazole derivatives exhibit broad-spectrum pharmacological properties including anti-microbial, anti-diabetic and anti-tumour activity. However, whether benzimidazole derivatives are effective in suppressing angiogenesis and its underlying mechanisms remain incompletely understood. In this study, we aim to characterize the anti-angiogenic mechanisms of a novel 2-aminobenzimidazole-based compound, Jzu 17, in an effort to develop novel angiogenesis inhibitor. EXPERIMENTAL APPROACH: Effects of Jzu 17 on endothelial cell proliferation, migration, invasion, and activation of signalling molecules induced by VEGF-A, were analysed by immunoblotting, MTT, BrdU, migration, and invasion assays. We performed tube formation assay, aorta ring sprouting assay, matrigel plug assay, and a mouse model of metastasis to evaluate ex vivo and in vivo anti-angiogenic effects of Jzu 17. KEY RESULTS: Jzu 17 inhibited VEGF-A-induced cell proliferation, migration, invasion, and endothelial tube formation of HUVECs. Jzu 17 suppressed VEGF-A-induced microvessel sprouting ex vivo and attenuated VEGF-A- or tumour cell-induced neovascularization in vivo. Jzu 17 also reduced B16F10 melanoma lung metastasis. In addition, Jzu 17 inhibited the phosphorylation of VEGFR-2 and its downstream signalling molecules in VEGF-A-stimulated HUVECs. Results from computer modelling further showed that Jzu 17 binds to VEGFR-2 with high affinity. CONCLUSIONS AND IMPLICATIONS: Jzu 17 may inhibit endothelial remodelling and suppress angiogenesis through targeting VEGF-A-VEGFR-2 signalling. These results also suggest Jzu 17 as a potential lead compound and warrant the clinical development of similar agents in the treatment of cancer and angiogenesis-related diseases.

A Novel Hydroxamate-Based Compound WMJ-J-09 Causes Head and Neck Squamous Cell Carcinoma Cell Death via LKB1-AMPK-p38MAPK-p63-Survivin Cascade.

Growing evidence shows that hydroxamate-based compounds exhibit broad-spectrum pharmacological properties including anti-tumor activity. However, the precise mechanisms underlying hydroxamate derivative-induced cancer cell death remain incomplete understood. In this study, we explored the anti-tumor mechanisms of a novel aliphatic hydroxamate-based compound, WMJ-J-09, in FaDu head and neck squamous cell carcinoma (HNSCC) cells. WMJ-J-09 induced G2/M cell cycle arrest and apoptosis in FaDu cells. These actions were associated with liver kinase B1 (LKB1), AMP-activated protein kinase (AMPK) and p38 mitogen-activated protein kinase (p38MAPK) activation, transcription factor p63 phosphorylation, as well as modulation of p21 and survivin. LKB1-AMPK-p38MAPK signaling blockade reduced WMJ-J-09's enhancing effects in p63 phosphorylation, p21 elevation and survivin reduction. Moreover, WMJ-J-09 caused an increase in α-tubulin acetylation and interfered with microtubule assembly. Furthermore, WMJ-J-09 suppressed the growth of subcutaneous FaDu xenografts in vivo. Taken together, WMJ-J-09-induced FaDu cell death may involve LKB1-AMPK-p38MAPK-p63-survivin signaling cascade. HDACs inhibition and disruption of microtubule assembly may also contribute to WMJ-J-09's actions in FaDu cells. This study suggests that WMJ-J-09 may be a potential lead compound and warrant the clinical development in the treatment of HNSCC.

CME-1, a novel polysaccharide, suppresses iNOS expression in lipopolysaccharide-stimulated macrophages through ceramide-initiated protein phosphatase 2A activation.

CME-1, a novel water-soluble polysaccharide purified from Ophiocordyceps sinensis mycelia, has anti-oxidative, antithrombotic and antitumour properties. In this study, other major attributes of CME-1, namely anti-inflammatory and immunomodulatory properties, were investigated. Treating lipopolysaccharide (LPS)-stimulated RAW 264.7 cells with CME-1 concentration-dependently suppressed nitric oxide formation and inducible nitric oxide synthase (iNOS) expression. In the CME-1-treated RAW 264.7 cells, LPS-induced IκBα degradation and the phosphorylation of p65, Akt and mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated kinase, c-Jun N-terminal kinase and p38, were reduced. Treatment with a protein phosphatase 2A (PP2A)-specific inhibitor, significantly reversed the CME-1-suppressed iNOS expression; IκBα degradation; and p65, Akt and MAPK phosphorylation. PP2A activity up-regulation and PP2A demethylation reduction were also observed in the cells. Moreover, CME-1-induced PP2A activation and its subsequent suppression of LPS-activated RAW 264.7 cells were diminished by the inhibition of ceramide signals. LPS-induced reactive oxygen species (ROS) and hydroxyl radical formation were eliminated by treating RAW 264.7 cells with CME-1. Furthermore, the role of ceramide signalling pathway and anti-oxidative property were also demonstrated in CME-1-mediated inhibition of LPS-activated primary peritoneal macrophages. In conclusion, CME-1 suppressed iNOS expression by up-regulating ceramide-induced PP2A activation and reducing ROS production in LPS-stimulated macrophages. CME-1 is a potential therapeutic agent for treating inflammatory diseases.

Ketamine, a Clinically Used Anesthetic, Inhibits Vascular Smooth Muscle Cell Proliferation via PP2A-Activated PI3K/Akt/ERK Inhibition.

Abnormal proliferation of vascular smooth muscle cells (VSMCs) gives rise to major pathological processes involved in the development of cardiovascular diseases. The use of anti-proliferative agents for VSMCs offers potential for the treatment of vascular disorders. Intravenous anesthetics are firmly established to have direct effects on VSMCs, resulting in modulation of blood pressure. Ketamine has been used for many years in the intensive care unit (ICU) for sedation, and has recently been considered for adjunctive therapy. In the present study, we investigated the effects of ketamine on platelet-derived growth factor BB (PDGF-BB)-induced VSMC proliferation and the associated mechanism. Ketamine concentration-dependently inhibited PDGF-BB-induced VSMC proliferation without cytotoxicity, and phosphatidylinositol 3-kinase (PI3K) and extracellular signal-regulated protein kinase (ERK) inhibitors, LY294002 and PD98059, respectively, have similar inhibitory effects. Ketamine was shown to attenuate PI3K, Akt, and ERK1/2 phosphorylation induced by PDGF-BB. Okadaic acid, a selective protein phosphatase 2A (PP2A) inhibitor, significantly reversed ketamine-mediated PDGF-BB-induced PI3K, Akt, and ERK1/2 phosphorylation; a transfected protein phosphatse 2a (pp2a) siRNA reversed Akt and ERK1/2 phosphorylation; and 3-O-Methyl-sphingomyeline (3-OME), an inhibitor of sphingomyelinase, also significantly reversed ERK1/2 phosphorylation. Moreover, ketamine alone significantly inhibited tyrosine phosphorylation and demethylation of PP2A in a concentration-dependent manner. In addition, the pp2a siRNA potently reversed the ketamine-activated catalytic subunit (PP2A-C) of PP2A. These results provide evidence of an anti-proliferating effect of ketamine in VSMCs, showing activation of PP2A blocks PI3K, Akt, and ERK phosphorylation that subsequently inhibits the proliferation of VSMCs. Thus, ketamine may be considered a potential effective therapeutic agent for reducing atherosclerotic process by blocking the proliferation of VSMCs.

WMJ-8-B, a novel hydroxamate derivative, induces MDA-MB-231 breast cancer cell death via the SHP-1-STAT3-survivin cascade.

BACKGROUND AND PURPOSE: Histone deacetylase (HDAC) inhibitors have been demonstrate to have broad-spectrum anti-tumour properties and have attracted lots of attention in the field of drug discovery. However, the underlying anti-tumour mechanisms of HDAC inhibitors remain incompletely understood. In this study, we aimed to characterize the underlying mechanisms through which the novel hydroxamate-based HDAC inhibitor, WMJ-8-B, induces the death of MDA-MB-231 breast cancer cells. EXPERIMENTAL APPROACH: Effects of WMJ-8-B on cell viability, cell cycle distribution, apoptosis and signalling molecules were analysed by the MTT assay, flowcytometric analysis, immunoblotting, reporter assay, chromatin immunoprecipitation analysis and use of siRNAs. A xenograft model was used to determine anti-tumour effects of WMJ-8-B in vivo. KEY RESULTS: WMJ-8-B induced survivin reduction, G2/M cell cycle arrest and apoptosis in MDA-MB-231 cells. STAT3 phosphorylation, transactivity and its binding to the survivin promoter region were reduced in WMJ-8-B-treated cells. WMJ-8-B activated the protein phosphatase SHP-1 and when SHP-1 signalling was blocked, the effects of WMJ-8-B on STAT3 phosphorylation and survivin levels were abolished. However, WMJ-8-B increased the transcription factor Sp1 binding to the p21 promoter region and enhanced p21 levels. Moreover, WMJ-8-B induced α-tubulin acetylation and disrupted microtubule assembly. Inhibition of HDACs was shown to contribute to WMJ-8-B's actions. Furthermore, WMJ-8-B suppressed the growth of MDA-MB-231 xenografts in mammary fat pads in vivo. CONCLUSIONS AND IMPLICATIONS: The SHP-1-STAT3-survivin and Sp1-p21 cascades are involved in WMJ-8-B-induced MDA-MB-231 breast cancer cell death. These results also indicate the potential of WMJ-8-B as a lead compound for treatment of breast cancer and warrant its clinical development.

Synthesis and biological evaluation of lovastatin-derived aliphatic hydroxamates that induce reactive oxygen species.

Some hydroxamate compounds induce cancer cell death by intracellular reactive oxygen species (ROS). This study introduced the hydroxamate core into lovastatin, a fungus metabolite clinically used for the treatment of hypercholesterolemia. The resulting compounds were evaluated for the activity for inducing ROS production. Most compounds exhibited higher activity than original lovastatin. Of these compounds, compound 3c had the most potent activity. Test of cytotoxicity in a panel of human cancer cell lines indicated compound 3c had activities superior to cisplatin in prostate cancer PC-3 cells and breast cancer T47D cells. In contrast, it in amounts up to 40µM had a much lower cytotoxic effect on normal human IMR-90 cells. Further profiling of cell cycle progression, cell apoptosis, and DNA damage activated checkpoint signaling pathway revealed the important role of compound 3c-mediated cytotoxicity in ROS generation.

Interleukin-6 Induces Vascular Endothelial Growth Factor-C Expression via Src-FAK-STAT3 Signaling in Lymphatic Endothelial Cells.

Elevated serum interleukin-6 (IL-6) levels correlates with tumor grade and poor prognosis in cancer patients. IL-6 has been shown to promote tumor lymphangiogenesis through vascular endothelial growth factor-C (VEGF-C) induction in tumor cells. We recently showed that IL-6 also induced VEGF-C expression in lymphatic endothelial cells (LECs). However, the signaling mechanisms involved in IL-6-induces VEGF-C induction in LECs remain incompletely understood. In this study, we explored the causal role of focal adhesion kinase (FAK) in inducing VEGF-C expression in IL-6-stimulated murine LECs (SV-LECs). FAK signaling blockade by NSC 667249 (a FAK inhibitor) attenuated IL-6-induced VEGF-C expression and VEGF-C promoter-luciferase activities. IL-6's enhancing effects of increasing FAK, ERK1/2, p38MAPK, C/EBPß, p65 and STAT3 phosphorylation as well as C/EBPß-, κB- and STAT3-luciferase activities were reduced in the presence of NSC 667249. STAT3 knockdown by STAT3 siRNA abrogated IL-6's actions in elevating VEGF-C mRNA and protein levels. Moreover, Src-FAK signaling blockade reduced IL-6's enhancing effects of increasing STAT3 binding to the VEGF-C promoter region, cell migration and endothelial tube formation of SV-LECs. Together these results suggest that IL-6 increases VEGF-C induction and lymphangiogenesis may involve, at least in part, Src-FAK-STAT3 cascade in LECs.

Lovastatin causes FaDu hypopharyngeal carcinoma cell death via AMPK-p63-survivin signaling cascade.

Statins are used widely to lower serum cholesterol and the incidence of cardiovascular diseases. Growing evidence shows that statins also exhibit beneficial effects against cancers. In this study, we investigated the molecular mechanisms involved in lovastatin-induced cell death in Fadu hypopharyngeal carcinoma cells. Lovastatin caused cell cycle arrest and apoptosis in FaDu cells. Lovastatin increased p21(cip/Waf1) level while the survivin level was decreased in the presence of lovastatin. Survivin siRNA reduced cell viability and induced cell apoptosis in FaDu cells. Lovastatin induced phosphorylation of AMP-activated protein kinase (AMPK), p38 mitogen-activated protein kinase (MAPK) and transcription factor p63. Lovastatin also caused p63 acetylation and increased p63 binding to survivin promoter region in FaDu cells. AMPK-p38MAPK signaling blockade abrogated lovastatin-induced p63 phosphorylation. Lovastatin's enhancing effect on p63 acetylation was reduced in HDAC3- or HDAC4- transfected cells. Moreover, transfection of cells with AMPK dominant negative mutant (AMPK-DN), HDAC3, HDAC4 or p63 siRNA significantly reduced lovastatin's effects on p21(cip/Waf1) and survivin. Furthermore, lovastatin inhibited subcutaneous FaDu xenografts growth in vivo. Taken together, lovastatin may activate AMPK-p38MAPK-p63-survivin cascade to cause FaDu cell death. This study establishes, at least in part, the signaling cascade by which lovastatin induces hypopharyngeal carcinoma cell death.

Andrographolide stimulates p38 mitogen-activated protein kinase-nuclear factor erythroid-2-related factor 2-heme oxygenase 1 signaling in primary cerebral endothelial cells for definite protection against ischemic stroke in rats.

Stroke pathogenesis involves complex oxidative stress-related pathways. The nuclear factor erythroid-2-related factor 2 (Nrf2) and heme oxygenase 1 (HO-1) pathways have been considered molecular targets in pharmacologic intervention for ischemic diseases. Andrographolide, a labdane diterpene, has received increasing attention in recent years because of its various pharmacologic activities. We determined that andrographolide modulates the mitogen-activated protein kinase (MAPK)-Nrf2-HO-1 signaling cascade in primary cerebral endothelial cells (CECs) to provide positive protection against middle cerebral artery occlusion (MCAO)-induced ischemic stroke in rats. In the present study, andrographolide (10 µM) increased HO-1 protein and messenger RNA expressions, Nrf2 phosphorylation, and nuclear translocation in CECs, and these activities were disrupted by a p38 MAPK inhibitor, SB203580, but not by the extracellular signal-regulated kinase inhibitor PD98059 or c-Jun amino-terminal kinase inhibitor SP600125. Similar results were observed in confocal microscopy analysis. Moreover, andrographolide-induced Nrf2 and HO-1 protein expressions were significantly inhibited by Nrf2 small interfering RNA. Moreover, HO-1 knockdown attenuated the protective effect of andrographolide against oxygen-glucose deprivation-induced CEC death. Andrographolide (0.1 mg/kg) significantly suppressed free radical formation, blood-brain barrier disruption, and brain infarction in MCAO-insulted rats, and these effects were reversed by the HO-1 inhibitor zinc protoporphyrin IX. The mechanism is attributable to HO-1 activation, as directly evidenced by andrographolide-induced pronounced HO-1 expression in brain tissues, which was highly localized in the cerebral capillary. In conclusion, andrographolide increased Nrf2-HO-1 expression through p38 MAPK regulation, confirming that it provides protection against MCAO-induced brain injury. These findings provide strong evidence that andrographolide could be a therapeutic agent for treating ischemic stroke or neurodegenerative diseases.

The effects of a novel aliphatic-chain hydroxamate derivative WMJ-S-001 in HCT116 colorectal cancer cell death.

Hydroxamate derivatives have attracted considerable attention due to their broad pharmacological properties and have been extensively investigated. We recently demonstrated that WMJ-S-001, a novel aliphatic hydroxamate derivative, exhibits anti-inflammatory and anti-angiogenic activities. In this study, we explored the underlying mechanisms by which WMJ-S-001 induces HCT116 colorectal cancer cell death. WMJ-S-001 inhibited cell proliferation and induced cell apoptosis in HCT116 cells. These actions were associated with AMP-activated protein kinase (AMPK) and p38 mitogen-activated protein kinase (MAPK) activation, p53 phosphorylation and acetylation, as well as the modulation of p21(cip/Waf1), cyclin D1, survivin and Bax. AMPK-p38MAPK signaling blockade reduced WMJ-S-001-induced p53 phosphorylation. Transfection with AMPK dominant negative mutant (DN) reduced WMJ-S-001's effects on p53 and Sp1 binding to the survivn promoter region. Transfection with HDAC3-Flag or HDAC4-Flag also abrogated WMJ-S-001's enhancing effect on p53 acetylation. WMJ-S-001's actions on p21(cip/Waf1), cyclin D1, survivin, Bax were reduced in p53-null HCT116 cells. Furthermore, WMJ-S-001 was shown to suppress the growth of subcutaneous xenografts of HCT116 cells in vivo. In summary, the death of HCT116 colorectal cancer cells exposed to WMJ-S-001 may involve AMPK-p38MAPK-p53-survivin cascade. These results support the role of WMJ-S-001 as a potential drug candidate and warrant the clinical development in the treatment of cancer.

Protein Phosphatase 2A in Lipopolysaccharide-Induced Cyclooxygenase-2 Expression in Murine Lymphatic Endothelial Cells.

The lymphatic endothelium plays an important role in the maintenance of tissue fluid homeostasis. It also participates in the pathogenesis of several inflammatory diseases. However, little is known about the underlying mechanisms by which lymphatic endothelial cell responds to inflammatory stimuli. In this study, we explored the mechanisms by which lipopolysaccharide (LPS) induces cyclooxygenase (COX)-2 expression in murine lymphatic endothelial cells (SV-LECs). LPS caused increases in cox-2 mRNA and protein levels, as well as in COX-2 promoter luciferase activity in SV-LECs. These actions were associated with protein phosphatase 2A (PP2A), apoptosis signal-regulating kinase 1 (ASK1), JNK1/2 and p38MAPK activation, and NF-κB subunit p65 and C/EBPß phosphorylation. PP2A-ASK1 signaling blockade reduced LPS-induced JNK1/2, p38MAPK, p65 and C/EBPß phosphorylation. Transfection with PP2A siRNA reduced LPS's effects on p65 and C/EBPß binding to the COX-2 promoter region. Transfected with the NF-κB or C/EBPß site deletion of COX-2 reporter construct also abrogated LPS's enhancing effect on COX-2 promoter luciferase activity in SV-LECs. Taken together, the induction of COX-2 in SV-LECs exposed to LPS may involve PP2A-ASK1-JNK and/or p38MAPK-NF-κB and/or C/EBPß cascade.