Yoosik Yoon

Tanshinone IIA isolated from Salvia miltiorrhiza BUNGE induced apoptosis in HL60 human premyelocytic leukemia cell line.

Apoptosis is a new therapeutic target of cancer research. Tanshinone IIA isolated from Salvia miltiorrhiza BUNGE, a traditional oriental medical herb, was observed to induce apoptosis in HL60 human premyelocytic leukemia cell line. Tanshinone IIA induced DNA fragmentation into the multiples of 180 bp and increased the percentage of hypodiploid cells in flow cytometry after propidium iodide (PI) staining. Tanshinone IIA-induced apoptosis is accompanied by the specific proteolytic cleavage of poly(ADP-ribose) polymerase (PARP) and the activation of caspase-3, a major component in apoptotic cell death mechanism.

AICAR, an activator of AMPK, inhibits adipogenesis via the WNT/β-catenin pathway in 3T3-L1 adipocytes

AMP-activated protein kinase (AMPK) is known to sense the cellular energy state and regulates various cellular energy metabolism pathways through its activation by AMP, an indicator of a low-energy state. 5-Aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR), an activator of AMPK, efficiently inhibited the adipogenesis of 3T3-L1 cells. To elucidate its possible mechanism of action, the expression levels of β-catenin and other members of the WNT/β-catenin pathway were analyzed during the adipogenesis of 3T3-L1 cells in the presence or absence of AICAR. It was found that AICAR significantly enhanced β-catenin expression and its nuclear accumulation. Transfection of β-catenin small interfering RNA (siRNA) significantly prevented the effects of AICAR on the expression of various genes. The expression of the major genes of adipogenesis including the peroxisome proliferator-activated receptor (PPAR)γ, the CCAAT/enhancer binding protein (C/EPB)α, the fatty acid binding protein (FABP)4 and lipoprotein lipase (LPL), which were all reduced by AICAR treatment, were significantly recovered in β-catenin siRNA-transfected cells. Among the members of the WNT/β-catenin pathway, the expression of low density lipoprotein receptor-related protein (LRP)6, dishevelled (DVL)2 and DVL3 were significantly up-regulated by AICAR treatment, whereas the expression of AXIN was down-regulated. The present study provides compelling evidence that AICAR inhibits adipogenesis through the modulation of the WNT/β-catenin pathway.

The anti-adipogenic effects of (-)epigallocatechin gallate are dependent on the WNT/β-catenin pathway

(-)Epigallocatechin gallate (EGCG) is the most abundant catechin in green tea and reportedly has anti-obesity and anti-adipogenic effects. In this study, we determined that the up-regulation of the WNT/β-catenin pathway is the anti-adipogenic mechanisms of EGCG in 3T3-L1 cells. EGCG treatment down-regulates the expression of major genes involved in the adipogenesis pathway including peroxisome proliferator-activated receptor (PPAR)γ, CCAAT/enhancer binding protein (C/EBP)α, fatty acid binding protein (FABP)4 and fatty acid synthase (FASN), while up-regulating the nuclear level of β-catenin. Knockdown of β-catenin using small interfering (si) RNA attenuated the inhibitory effects of EGCG on intracellular lipid accumulation. β-catenin siRNA transfection also recovered terminal adipocyte markers such as FABP4, FASN, lipoprotein lipase and adiponectin, which were down-regulated by EGCG. The DNA binding activities as well as the expression levels of PPARγ and C/EBPα, which were down-regulated by EGCG, were significantly restored by β-catenin siRNA transfection. In addition, we found that EGCG efficiently up-regulates the WNT/β-catenin pathway. Among the members of the WNT/β-catenin pathway, the expressions of low density lipoprotein receptor-related protein (LRP)5, LRP6, disheveled (DVL)2 and DVL3 were significantly up-regulated, while AXIN expression was down-regulated by EGCG, and the phosphorylation of glycogen synthase kinase 3β was increased. These results suggest that EGCG activates the WNT/β-catenin pathway, resulting in the up-regulation of β-catenin, which down-regulates the major genes of the adipogenesis pathway. Taken together, our findings clearly show that the anti-adipogenic effects of EGCG are, at least partially, dependent on the WNT/β-catenin pathway.

Platycodin D inhibits adipogenesis of 3T3‐L1 cells by modulating kruppel‐like factor 2 and peroxisome proliferator‐activated receptor γ

In this study, platycodin D was found to inhibit intracellular triglyceride accumulation in 3T3‐L1 cells with an IC50 of 7.1 μM. The expression levels of genes involved in lipid metabolism such as fatty‐acid‐binding protein 4 and lipoprotein lipase were significantly downregulated following treatment with platycodin D. Treatment with platycodin D also resulted in a reduction of Peroxisome proliferator‐activated receptor(PPAR)γ expression and its binding to target DNA sequence. Among the various upstream regulators of PPARγ, the expression of Kruppel‐like factor(KLF)2, an anti‐adipogenic factor, was significantly upregulated following platycodin D treatment. When the upregulation of KLF2 was inhibited by KLF2 siRNA, the expression and binding of PPARγ to its target sequence were significantly recovered under these conditions. The results of this study suggested that anti‐adipogenic effect of platycodin D involves the upregulation of KLF2 and subsequent downregulation of PPARγ. Copyright

Antiobesity effect of baicalin involves the modulations of proadipogenic and antiadipogenic regulators of the adipogenesis pathway.

In this study, the antiobesity effects of baicalin, 5,6‐dihydroxyflavone‐7‐glucuronic acid, were characterized using an in vitro system of adipogenesis, i.e. fat cell formation. Baicalin‐treatment of 3T3‐L1 preadipocytes was shown to inhibit triglyceride accumulation and lipid droplet formation during induced adipogenesis. Microarray analyses showed that baicalin modulated the expression of genes located in pathways such as adipogenesis, cholesterol biosynthesis, focal adhesion and others. In the adipogenesis pathway, treatment with baicalin significantly down‐regulated terminal differentiation markers of adipocytes including fatty acid binding protein 4. The effects of baicalin on the core part of the adipogenesis pathway, however, were paradoxical; the expression levels of CCAAT/enhancer binding protein (C/EBP)β and C/EBPδ were up‐regulated, while the expression levels of the peroxisome proliferator‐activated receptor (PPAR)γ and C/EBPα were down‐regulated. The antiadipogenic mechanisms of baicalin can be explained by its effects on the upstream part of adipogenesis pathway; baicalin not only up‐regulates the antiadipogenic regulators, C/EBPγ, C/EBP homologous protein and Kruppel‐like factor (KLF)2, but also down‐regulates the proadipogenic regulator, KLF15. The overall effects of baicalin on these upstream regulators of adipogenesis were antiadipogenic, resulting in the down‐regulation of downstream genes and the inhibition of cellular fat accumulation. Copyright

WNT/β-catenin pathway is modulated in asthma patients and LPS-stimulated RAW264.7 macrophage cell line

In the present study, we investigated the possibility that the WNT/β-catenin pathway plays a role in inflammatory responses both in an human inflammatory condition and in an in vitro inflammation model. First, we analyzed gene expression patterns of the peripheral blood cells from asthma patients compared with those from normal subjects using microarray analyses. We found that intracellular signaling molecules of the WNT/β-catenin pathway were significantly changed in asthma patients compared with the levels in the controls. Next, we determined whether major components of the WNT/β-catenin pathway were involved in the lipopolysaccharide (LPS)-induced inflammatory response of the RAW264.7 macrophage cell line. Among the members of WNT/β-catenin pathway, the protein levels of low-density lipoprotein receptor-related protein (LRP) 6, dishevelled (DVL) 2, and AXIN1, which were measured using western blotting, did not significantly change in the presence of LPS. In contrast, the LPS induced a rapid phosphorylation of glycogen synthase kinase (GSK) 3β and accumulation of β-catenin protein. It was found that β-catenin plays a significant role in the LPS-induced inflammatory response through the performance of small interfering RNA (siRNA) transfection experiments. The mRNA level of IL-6 was significantly elevated in β-catenin siRNA-transfected cells compared with that in control siRNA-transfected cells after LPS treatment. Furthermore, nuclear factor-κB (NF-κB) activity was also significantly increased in β-catenin siRNA-transfected cells compared with the level seen in control siRNA-transfected cells. Taken together, these results suggest that β-catenin plays a role as a negative regulator, preventing the overproduction of inflammatory cytokines such as IL-6 in LPS-induced inflammatory responses.

Anti-adipogenic effects of 1,25-dihydroxyvitamin D3 are mediated by the maintenance of the wingless-type MMTV integration site/β-catenin pathway

1,25-dihydroxyvitamin D3 (1,25(OH)2D3), the active metabolite of vitamin D, was found to have anti-adipogenic activity, however, its mechanism of action has not been fully elucidated. In this study, 3T3-L1 preadipocytes were differentiated in the presence and absence of 1,25(OH)2D3, and the expression of the genes and proteins of the wingless-type MMTV integration site (WNT)/β-catenin pathway were analyzed. While the expression of the members of the WNT/β-catenin pathway were significantly downregulated during the adipogenesis of untreated 3T3-L1 cells, 1,25(OH)2D3 treatment was found to maintain the WNT/β-catenin pathway. Among the members of the WNT/β-catenin pathway, the levels of WNT10B and disheveled (DVL)2 as well as the phosphorylation of glycogen synthase kinase (GSK)3β were maintained by 1,25(OH)2D3 treatment. The levels of nuclear β-catenin, which were downregulated during adipogenesis, were also maintained by 1,25(OH)2D3 treatment. The results of this study suggested that the anti-adipogenic effect of 1,25(OH)2D3 was mediated by the maintenance of the WNT/β-catenin pathway, which was normally downregulated during adipogenesis.

WNT/β-catenin pathway mediates the anti-adipogenic effect of platycodin D, a natural compound found in Platycodon grandiflorum

AIMS This study was conducted to suggest the role of WNT/β-catenin pathway in the anti-adipogenic effect of platycodin D, a natural compound found in Platycodon grandiflorum. MAIN METHODS Gene knockdown experiments using small interfering RNA (siRNA) transfection were conducted to elucidate crucial role of β-catenin in the anti-adipogenic effects of platycodin D. Real-Time PCR and Western blot were used to analyze the expression levels of mRNAs and proteins in the WNT/β-catenin pathway. KEY FINDINGS During the adipocyte differentiation of 3 T3-L1 cells, members of the WNT/β-catenin pathway were normally down-regulated, whereas platycodin D significantly reinstated the WNT/β-catenin pathway. The mRNA and protein expressions of disheveled (DVL) 2, which stabilize β-catenin, were increased by platycodin D treatment, but the protein level of AXIN, which induces the degradation of β-catenin, was decreased in platycodin D-treated cells. The nuclear level of β-catenin was normally down-regulated during adipogenesis, but platycodin D treatment led to the accumulation of β-catenin in the nucleus which resulted in the up-regulation of its target genes, cyclin D (CCND) 1 and peroxisome proliferator-activated receptor gamma (PPAR)γ. The anti-adipogenic effects of platycodin D were significantly attenuated in β-catenin siRNA-transfected cells compared with those of control siRNA-transfected cells. β-catenin siRNA transfection significantly recovered the levels of PPARγ, CCAAT/enhancer binding protein (C/EBP)α and fatty acid binding protein (FABP)4 as well as intracellular lipid droplet formation, all of which were reduced by platycodin D treatment. SIGNIFICANCE WNT/β-catenin pathway can be used as a therapeutic target of natural compounds for the regulation of adipogenesis.

Shikonin inhibits fat accumulation in 3T3-L1 adipocytes.

Shikonin, 5,6‐dihydroxyflavone‐7‐glucuronic acid, is the main ingredient of Lithospermum erythrorhizon Sieb. et Zucc, and was reported to have various biological activities including antiinflammatory, anticancer, antimicrobial and others. This study aimed to elucidate, for the first time, the antiobesity activity of shikonin and its mechanism of action. Shikonin was found to inhibit fat droplet formation and triglyceride accumulation in 3T3‐L1 adipocytes. The half inhibitory concentration, IC50, for the inhibition of triglyceride accumulation was found to be 1.1 μM. The expression of genes involved in lipid metabolism, such as FABP4 and LPL, were significantly inhibited following shikonin treatment. Shikonin also inhibited the ability of PPARγ and C/EBPα, the major transcription factors of adipogenesis, to bind to their target DNA sequences. The expressions of mRNA and protein of PPARγ and C/EBPa were significantly down‐regulated following shikonin treatment. Among the upstream regulators of adipogenesis, only SREBP1C was found to be down‐regulated by shikonin. The results of this study suggest that shikonin down‐regulates the expression of SREBP1C and subsequently the expression of PPARγ and C/EBPα. Together, these changes result in the down‐regulation of lipid metabolizing enzymes and reduced fat accumulation. Copyright

Shikonin inhibits adipogenesis by modulation of the WNT/β-catenin pathway

AIM Our previous study showed for the first time that shikonin, a natural compound isolated from Lithospermun erythrorhizon Sieb. Et Zucc, inhibits adipogenesis and fat accumulation. This study was conducted to investigate the molecular mechanism of the anti-adipogenic effects of shikonin. MAIN METHODS Gene knockdown experiments using small interfering RNA (siRNA) transfection were conducted to elucidate the crucial role of β-catenin in the anti-adipogenic effects of shikonin. KEY FINDINGS Shikonin prevented the down-regulation of β-catenin and increased the level of its transcriptional product, cyclin D1, during adipogenesis of 3T3-L1 cells, preadipocytes originally derived from mouse embryo. β-catenin was a crucial mediator of the anti-adipogenic effects of shikonin, as determined by siRNA-mediated knockdown. Shikonin-induced reductions of the major transcription factors of adipogenesis including peroxisome proliferator-activated receptor γ and CCAAT/enhancer binding protein α, and lipid metabolizing enzymes including fatty acid binding protein 4 and lipoprotein lipase, as well as intracellular fat accumulation, were all significantly recovered by siRNA-mediated knockdown of β-catenin. Among the genes located in the WNT/β-catenin pathway, the levels of WNT10B and DVL2 were significantly up-regulated, whereas the level of AXIN was down-regulated by shikonin treatment. SIGNIFICANCE This study clearly shows that shikonin inhibits adipogenesis by the modulation of WNT/β-catenin pathway in vitro, and also suggests that WNT/β-catenin pathway can be used as a therapeutic target for obesity and related diseases using a natural compound like shikonin, even though the in vivo effects of shikonin and its clinical significance remain to be elucidated.