Salvianolic acid B (Sal B) is one of the most active hydrophilic compounds extracted from Salvia miltiorrhiza root. Previous in vitro and in vivo studies demonstrate the ability of Sal B to modulate adipocyte differentiation. However, the lipid-modulating effect and mechanism of Sal B in adipocytes remain controversial. Here we investigated the regulatory effect and mode of action of Sal B on lipid accumulation in 3T3-L1 preadipocyte differentiation. Lipid droplet (LD) accumulation and triglyceride (TG) content during 3T3-L1 preadipocyte differentiation were measured by Oil Red O staining and AdipoRed assay. The growth inhibition during 3T3-L1 preadipocyte differentiation was measured by cell count analysis. Western blotting and real-time qPCR analysis were utilized to determine the protein and mRNA expression in the preadipocyte differentiation. Notably, in 3T3-L1 preadipocyte differentiation, treatment with Sal B at 100 M led to a marked decrease in LD accumulation and TG content without influencing cell growth. Sal B treatment (100 M) further reduced the expression and phosphorylation levels of adipogenic transcription factors, including CCAAT/enhancer-binding protein- (C/EBP-), peroxisome proliferator-activated receptor-gamma (PPAR)-, and signal transducer and activator of transcription (STAT)-3/5. Treatment with Sal B (100 M) also reduced the expression and phosphorylation levels of fatty acid synthase (FAS) and acetyl-CoA carboxylase (ACC), two lipogenic enzymes and perilipin A, an LD-binding and stabilizing protein. These results collectively demonstrate that Sal B at 100 M strongly inhibits lipid accumulation in 3T3-L1 preadipocyte differentiation, mediated through regulation of the expression and phosphorylation levels of C/EBP-, PPAR-, STAT-3/5, FAS, ACC, and perilipin.

Cucurbitacin E is a pivotal member of the cucurbitacin family and has been shown to have anti-cancer effects. However, until now, the anti-cancer effect and mode of action of cucurbitacin E in human colon cancer cells remain unclear. In this study, we investigated whether cucurbitacin E inhibits the growth of HCT116 human colorectal cancer cells. Treatment of cucurbitacin E at 1 mM markedly reduced the survival of HCT116 cells. Moreover, treatment of cucurbitacin E at 1 mM caused nuclear DNA fragmentation in HCT116 cells, pointing out its apoptosis-inducing effect. Treatment of cucurbitacin E at 1 mM also led to the activation of caspase-9 and poly(ADP‑ribose) polymerase (PARP) cleavage without affecting expression of death receptor (DR)-4/5 in HCT116 cells. Furthermore, while treatment of cucurbitacin E at 1 mM had no effect on expression of Mcl-1, it largely increased expression and phosphorylation of eukaryotic translation initiation factor-2α (eIF-2α) and activating transcription factor-4 (ATF-4) in HCT116 cells. Treatment of cucurbitacin E at 1 mM further up-regulated phosphorylation of extracellular signal-regulated kinase-1/2 (ERK-1/2), but not c-Jun N-terminal kinase1/2 (JNK-1/2), in HCT116 cells. However, treatment with PD98059, an inhibitor of ERK-1/2, that strongly blocked activation of ERK-1/2 had no effect on reduction of survival of HCT116 cells treated with cucurbitacin E at 1 mM. Taken together, these findings demonstrate that cucurbitacin E at 1 mM has strong anti-survival and pro-apoptotic effects on HCT116 cells, which are mediated through control of the expression and phosphorylation levels of caspase-9, PARP, eIF-2α, and ATF-4.

Differentiation of preadipocyte, also named adipogenesis, leads to the phenotype of mature adipocyte that is filled with many lipid droplets. Excessive lipid accumulation in adipocytes leads to the development of obesity. In this study, we investigated the effect of 11 different natural compounds on lipid accumulation during the differentiation of 3T3-L1 preadipocytes into 3T3-L1 adipocytes. Strikingly, among the natural compounds, cryptotanshinone at 10 µM most strongly reduced triglyceride (TG) contents in 3T3-L1 cells after 8 days of the differentiation. Furthermore, cryptotanshinone at 10 µM significantly suppressed lipid accumulation in 3T3-L1 cells after 8 days of the differentiation. Cryptotanshinone at 1 to 10 µM tested did not affect the survival of 3T3-L1 cells after 8 days of the differentiation. On mechanistic levels, cryptotanshinone time-differentially decreased the expression levels of CCAAT/enhancer-binding protein-α (C/EBP-α), peroxisome proliferator-activated receptor-γ (PPAR-γ), fatty acid synthase (FAS), and perilipin A but also the phosphorylation levels of signal transducer and activator of transcription-3 (STAT-3) during the 3T3-L1 cell differentiation. Taken together, these findings demonstrate that cryptotanshinone inhibits lipid accumulation in differentiating 3T3-L1 cells, which appears to be mediated through the reduced expression and/or phosphorylation levels of C/EBP-α, PPAR-γ, FAS, Perilipin A, and STAT-3.

Differentiation of preadipocyte, also named adipogenesis, leads to the phenotype of mature adipocyte that is filled with many lipid droplets. Excessive lipid accumulation in adipocytes leads to the development of obesity. In this study, we investigated the effect of 11 different natural compounds on lipid accumulation during the differentiation of 3T3-L1 preadipocytes into 3T3-L1 adipocytes. Strikingly, among the natural compounds, cryptotanshinone at 10 µM most strongly reduced triglyceride (TG) contents in 3T3-L1 cells after 8 days of the differentiation. Furthermore, cryptotanshinone at 10 µM significantly suppressed lipid accumulation in 3T3-L1 cells after 8 days of the differentiation. Cryptotanshinone at 1 to 10 µM tested did not affect the survival of 3T3-L1 cells after 8 days of the differentiation. On mechanistic levels, cryptotanshinone time-differentially decreased the expression levels of CCAAT/enhancer-binding protein-α (C/EBP-α), peroxisome proliferator-activated receptor-γ (PPAR-γ), fatty acid synthase (FAS), and perilipin A but also the phosphorylation levels of signal transducer and activator of transcription-3 (STAT-3) during the 3T3-L1 cell differentiation. Taken together, these findings demonstrate that cryptotanshinone inhibits lipid accumulation in differentiating 3T3-L1 cells, which appears to be mediated through the reduced expression and/or phosphorylation levels of C/EBP-α, PPAR-γ, FAS, Perilipin A, and STAT-3.

Caffeic acid (CA) is a phenolic compound found naturally in plants and foods. CA and its natural derivatives are reported to have anti-cancer effects on many cancers, including melanoma. (E)-N-(4-Butylphenyl)-3-(3,4-dihydroxyphenyl)acrylamide (4-BPCA) is an amide derivative of CA. Thus far, the anti-cancer effect and mechanism of 4-BPCA in melanoma cells remain unknown. Here we investigated whether 4-BPCA inhibits the growth of B16F10 cells, a mouse melanoma cell line. Of note, treatment of 4-BPCA at 5 M for 24 or 48 h significantly reduced the growth (survival) of B16F10 cells. On mechanistic levels, treatment with 4-BPCA for 24 h led to the activation of caspase-9/3, but not caspase-8, in B16F10 cells. 4-BPCA treatment for 2 or 4 h also decreased the expression levels of myeloid B-cell lymphoma 1 (Mcl-1) in B16F10 cells. However, 4-BPCA treatment for the times tested did not influence the expression levels of X-linked inhibitor of apoptosis protein (XIAP) in B16F10 cells. Of interest, treatment of 4-BPCA for 2 or 4 h greatly reduced the phosphorylation levels of JAK-2 and STAT-5 without altering their total protein expression levels. 4-BPCA also had abilities to increase the expression and phosphorylation levels of glucose-regulated protein-78 (GRP-78) and eukaryotic translation initiation factor-2α (eIF-2α) in B16F10 cells. In summary, these results demonstrate firstly that 4-BPCA has a strong growth-inhibitory effect on B16F10 melanoma cells, mediated via activation of the intrinsic caspase pathway, inhibition of JAK-2 and STAT-5, and triggering endoplasmic reticulum (ER) stress.

Differentiation of preadipocyte, also named adipogenesis, leads to the phenotype of mature adipocyte that is filled with many lipid droplets. Excessive lipid accumulation in adipocytes leads to the development of obesity. In this study, we investigated the effect of 11 different natural compounds on lipid accumulation during the differentiation of 3T3-L1 preadipocytes into 3T3-L1 adipocytes. Strikingly, among the natural compounds, cryptotanshinone at 10 µM most strongly reduced triglyceride (TG) contents in 3T3-L1 cells after 8 days of the differentiation. Furthermore, cryptotanshinone at 10 µM significantly suppressed lipid accumulation in 3T3-L1 cells after 8 days of the differentiation. Cryptotanshinone at 1 to 10 µM tested did not affect the survival of 3T3-L1 cells after 8 days of the differentiation. On mechanistic levels, cryptotanshinone time-differentially decreased the expression levels of CCAAT/enhancer-binding protein-α (C/EBP-α), peroxisome proliferator-activated receptor-γ (PPAR-γ), fatty acid synthase (FAS), and perilipin A but also the phosphorylation levels of signal transducer and activator of transcription-3 (STAT-3) during the 3T3-L1 cell differentiation. Taken together, these findings demonstrate that cryptotanshinone inhibits lipid accumulation in differentiating 3T3-L1 cells, which appears to be mediated through the reduced expression and/or phosphorylation levels of C/EBP-α, PPAR-γ, FAS, Perilipin A, and STAT-3.
3T3-L1 Cells ; Adipocytes ; Adipogenesis ; Lipid Droplets ; Obesity ; Peroxisomes ; Phenotype ; Phosphorylation ; Transducers ; Triglycerides

PURPOSE: Overexpression of COX-2, an enzyme responsible fro the synthesis of prostaglandins, is well linked to human chronic lung diseases. The mechanism by which COX-2 expression is increased or enhanced in cancer cells remains largely unknown. Any compound which can reduce COX-2 expression may be considered as an anti-cancer agent. MATERIALS AND METHODS: Leptomycin B (LMB) is a metabolite of Streptomyces and a specific inhibitor of CRM1 nuclear export receptor. A549 is a human lung cancer cell line. To evaluate the effect of LMB on COX-2 expression induced by IL-1beta, a pro-inflammatory cytokine, in A549 cells, Western blot and RT-PCR assays were applied to measure COX-2 protein and mRNA expressions in response to IL-1beta, respectively. Luciferase experiments were done to measure promoter activity of COX-2, NF-kappaB or AP-1. CRM1 siRNA trasfection experiment was performed to knock-down endogenous CRM1. Biochemical protein fractionation method was also carried out to see intracellular localization of proteins. RESULTS: LMB at 9 nM strongly suppressed IL-1beta-induced expression of COX-2 protein that was attributable to decreased COX-2 transcript and promoter activity, but not mRNA stability. Distinctly, knock-down of CRM1 had no effect on COX-2 expression by IL-1beta. Moreover, LMB did not affect IL-1beta-induced phosphorylation of ERK-1/2, JNK- 1/2, and p38 MAPK or AP-1 promoter activity. In contrast, LMB blocked IL-1beta- mediated cytosolic IkappaB-alpha degradation, p65 NF-kappaB nuclear translocation, and NF-kappaB promoter activity. CONCLUSION: LMB potently down-regulates IL-1beta- induced COX-2 at transcriptional level in A549 cells, in part, through modulation of the IkappaB-alpha/NF-kappaB pathway but independent of CRM1, MAPKs and AP-1.
Active Transport, Cell Nucleus ; Blotting, Western ; Cell Line ; Cytosol ; Down-Regulation* ; Humans ; Luciferases ; Lung Diseases ; Lung Neoplasms* ; Lung* ; NF-kappa B ; p38 Mitogen-Activated Protein Kinases ; Phosphorylation ; Prostaglandins ; RNA Stability ; RNA, Messenger ; RNA, Small Interfering ; Streptomyces ; Transcription Factor AP-1

Inflammation is a common link in the pathophysiology of many neurological illnesses, including Alzheimer’s disease. Activated glial cells contribute to neuroinflammation by producing pro-inflammatory mediators. Naproxen and ibuprofen are nonsteroidal anti-inflammatory drugs with 2-aryl(s) propionic acid as a common pharmacophore. Here we designed a small series of naproxen and ibuprofen amide dimers and tested their effects on the expression of inducible nitric oxide synthase (iNOS), a neuroinflammatory enzyme in lipopolysaccharide (LPS)-stimulated BV2 mouse microglial cells. Of note, treatment with CNU 019, 020, 021, 023, 024, and 027 at 10 M markedly inhibited the LPS-induced iNOS expression in BV2 cells. CNU 024 was tested further at different concentrations to regulate the LPS-induced iNOS expression in BV2 cells. Treatment with CNU 024 at 5, 10, or 20 M dose-dependently suppressed the LPS-induced iNOS protein and mRNA expression levels in BV2 cells, in which maximal inhibition was seen at 20 M. CNU 024 treatment at doses tested further led to a concentration-dependent inhibition of the LPS-induced phosphorylation (activation) of p38 mitogen-activated protein kinase (MAPK) without influencing its total protein expression in BV2 cells, but it did not affect the LPS-induced activation of c-jun N-terminal kinase-1/2 and extracellular signal-regulated kinases-1/2 in these cells. In summary, our results demonstrate that CNU 024 inhibits the LPS-induced iNOS expression in BV2 cells, partly mediated by the inhibition of p38 MAPK. This work shows that CNU 024 could be a valuable ligand for further development as a potential drug candidate for treating neuroinflammatory pathologies.

Recent evidence suggests obesity as a low or systemic chronic inflammation. (Pre)adipocytes in the adipose tissue (AT) express and secrete a variety of cytokines and adipokines. Inducible nitric oxide synthase (iNOS) is an inflammatory enzyme involved in the production of NO. Until now, the inducer(s) of iNOS expression in (pre)adipocytes remains unclear. In this study, we investigated the effects of proinflammatory cytokines (interleukin-1β (IL-1β), IL-10, IL-12, interferon-γ (IFN-γ), adipokines (retinol-binding protein 4 (RBP4), adiponectin, leptin, and resistin), and lipopolysaccharide (LPS), a bacterial cell wall component, on the expression of iNOS in 3T3-L1 preadipocytes. Notably, treatment with IL-1β at 20 ng/mL for 4 h markedly increased iNOS mRNA expression in 3T3-L1 preadipocytes, but that with IL-10 (10 ng/mL), IL-12 (5 ng/mL), IFN-γ (10 ng/mL), RBP4 (5 g/mL), adiponectin (100 ng/mL), leptin (100 ng/mL), and resistin (100 ng/mL), and LPS (1 g/mL) for 4 h had little or no effect on it. Results of dose-response and time-course experiments confirmed the ability of IL-1β at 20 ng/mL for 4 h to maximally induce iNOS mRNA expression in 3T3-L1 preadipocytes. Importantly, pharmacological inhibition studies demonstrated that treatment with AG490 (an inhibitor of Janus-activated kinases (JAKs) and signal transducer and activator of transcription proteins (STATs)), GO6976 (an inhibitor of PKCs), or PP1 (an Src kinase inhibitor) suppressed IL-1β-induced iNOS mRNA expression in 3T3-L1 preadipocytes, pointing out the involvement of JAKs/STATs, PKCs, and Src in the process. This work advocates that IL-1β is a major pro-inflammatory cytokine contributing to inflammation in the AT by up-regulating iNOS.

Recent evidence suggests obesity as a low or systemic chronic inflammation. (Pre)adipocytes in the adipose tissue (AT) express and secrete a variety of cytokines and adipokines. Inducible nitric oxide synthase (iNOS) is an inflammatory enzyme involved in the production of NO. Until now, the inducer(s) of iNOS expression in (pre)adipocytes remains unclear. In this study, we investigated the effects of proinflammatory cytokines (interleukin-1β (IL-1β), IL-10, IL-12, interferon-γ (IFN-γ), adipokines (retinol-binding protein 4 (RBP4), adiponectin, leptin, and resistin), and lipopolysaccharide (LPS), a bacterial cell wall component, on the expression of iNOS in 3T3-L1 preadipocytes. Notably, treatment with IL-1β at 20 ng/mL for 4 h markedly increased iNOS mRNA expression in 3T3-L1 preadipocytes, but that with IL-10 (10 ng/mL), IL-12 (5 ng/mL), IFN-γ (10 ng/mL), RBP4 (5 g/mL), adiponectin (100 ng/mL), leptin (100 ng/mL), and resistin (100 ng/mL), and LPS (1 g/mL) for 4 h had little or no effect on it. Results of dose-response and time-course experiments confirmed the ability of IL-1β at 20 ng/mL for 4 h to maximally induce iNOS mRNA expression in 3T3-L1 preadipocytes. Importantly, pharmacological inhibition studies demonstrated that treatment with AG490 (an inhibitor of Janus-activated kinases (JAKs) and signal transducer and activator of transcription proteins (STATs)), GO6976 (an inhibitor of PKCs), or PP1 (an Src kinase inhibitor) suppressed IL-1β-induced iNOS mRNA expression in 3T3-L1 preadipocytes, pointing out the involvement of JAKs/STATs, PKCs, and Src in the process. This work advocates that IL-1β is a major pro-inflammatory cytokine contributing to inflammation in the AT by up-regulating iNOS.