Protein Arginine Methyltransferase 2 Inhibits Angiotensin II-Induced Proliferation and Inflammation in Vascular Smooth Muscle Cells.

OBJECTIVES: Protein arginine methyltransferase 2 (PRMT2) protects against vascular injury-induced intimal hyperplasia; however, little is known about the role of PRMT2 in angiotensin II (Ang II)-induced VSMCs proliferation and inflammation. This research aims to determine whether PRMT2 inhibits Ang II-induced proliferation and inflammation of vascular smooth muscle cells (VSMCs). MATERIALS AND METHODS: PRMT2 overexpression was used to elucidate the role of PRMT2 in Ang II-induced VSMCs proliferation and inflammation. Western blotting and reverse transcriptional PCR were adopted to detect protein and mRNA expression severally. Cell viability was evaluated by 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay and cell cycle distribution by flow cytometry. RESULTS: Ang II significantly reduced mRNA and protein levels of PRMT2 in VSMCs in time-dependent and dose-dependent manner. Results of PRMT2 overexpression indicated that PRMT2 inhibited proliferation of VSMCs stimulated with 100 nmol/L Ang II for 24 hours. Furthermore, overexpression of PRMT2 reduced Ang II-induced production of proinflammatory cytokines such as interleukin 6 (IL-6) and interleukin 1ß (IL-1ß) in VSMCs. CONCLUSIONS: These findings suggest that PRMT2 alleviates Ang II-induced VSMCs proliferation and inflammation, providing a new mechanism about how Ang II mediated VSMCs proliferation and inflammation.

[Involvement of the receptor component protein in the regulation of vascular peroxidase-1 expression induced by calcitonin gene-related peptide and angiotensin II in vascular smooth muscle cell].

Angiotensin II (Ang II) and calcitonin gene-related peptide (CGRP) play important roles in vascular injury and protection. In order to determine the role of CGRP receptor component protein (RCP) in signal transduction whereby CGRP and Ang II mediate the expression of vascular peroxidase-1 (VPO1) in vascular smooth muscle cell (VSMC), mouse derived A10 vascular smooth muscle cell line (A10VSMC) was cultured with CGRP or/and Ang II in vitro. RCP-specific small interference RNA (siRNA-RCP) was used to silence oligonucleotide sequence. Western blot and RT-PCR were used to determine the protein and mRNA expressions of RCP and VPO1, respectively. The results showed that the expressions of RCP and VPO1 were increased in the presence of CGRP or Ang II in the quiescent A10VSMC. But the protein expressions of RCP and VPO1 induced by Ang II were decreased by pretreatment of CGRP (P < 0.05). The expressions of VPO1 were decreased in all the groups treated with siRNA-RCP, compared with those of wide-type counterparts. Meanwhile, the expression of VPO1 was significantly induced by CGRP but not Ang II in the siRNA-RCP treated A10VSMCs. Ang II in combination with CGRP increased the protein expression of VPO1 in the siRNA-RCP-transfected cells, compared with Ang II alone, and this effect could be abolished by catalase. The results suggest that RCP may play an important role in the integration of signal transduction whereby CGRP and Ang II receptors jointly regulate VPO1 expression in VSMC.

Effects of eugenol on hepatic glucose production and AMPK signaling pathway in hepatocytes and C57BL/6J mice.

Eugenol is a phenylpropanoid with many pharmacological activities, but its anti-hyperglycemic activity is not yet fully explored. For in vitro study, HepG2 cells and primary rat hepatocytes were used, and glucose production was induced by adding 100 nM of glucagon in the presence of gluconeogenic substrates. In animal study, hyperglycemia was induced by high fat diet (HFD) in male C57BL/6J mice, and eugenol was orally administered at 20 or 40 mg per kg (E20, E40) for 15 weeks. Eugenol significantly inhibited glucagon-induced glucose production and phosphorylated AMPK in the HepG2 and primary rat hepatocytes, and these effects were reversed in the presence of compound C (an AMPK inhibitor) or STO-609 (a CAMKK inhibitor). In addition, the protein and gene expression levels of CREB, CRTC2·CREB complex, PGC-1α, PEPCK and G6Pase were all significantly suppressed. Moreover, inhibition of AMPK by over-expression of dominant negative AMPK prevented eugenol from suppressions of gluconeogenic gene expression and hepatic glucose production. In animal study, plasma glucose and insulin levels of the E40 group were decreased by 31% and 63%, respectively, when compared to those of HFD control. In pyruvate tolerance tests, pyruvate-induced glucose excursions were decreased, indicating that the anti-hyperglycemic activity of eugenol is primarily due to the suppression of hepatic gluconeogenesis. In summary, eugenol effectively ameliorates hyperglycemia through inhibition of hepatic gluconeogenesis via modulating CAMKK-AMPK-CREB signaling pathway. Eugenol or eugenol-containing medicinal plants could represent a promising therapeutic agent to prevent type 2 diabetes.

Beneficial effect of betulinic acid on hyperglycemia via suppression of hepatic glucose production.

The inhibitory effect of betulinic acid (BA) on hepatic glucose production was examined in HepG2 cells and high fat diet (HFD)-fed ICR mice. BA significantly inhibited the hepatic glucose production (HGP) and gene expression levels of PGC-1α, PEPCK, and G6Pase. BA activated AMPK and suppressed the expression level of phosphorylated CREB. These effects were all abolished in the presence of compound C (an AMPK inhibitor). Moreover, inhibition of AMPK by overexpression of dominant negative AMPK prevented BA from suppression of HGP, indicating that the inhibitory effect of BA on HGP is AMPK-dependent. In addition, BA markedly phosphorylated CAMKK, and phosphorylation of AMPK and ACC, and suppression of HGP were all reversed in the presence of STO-609 (a CAMKK inhibitor), suggesting that CAMKK is an upstream kinase for AMPK. In an animal study, HFD-fed ICR mice were orally administered with 5 or 10 mg of BA per kg (B5 and B10) for three weeks. Plasma glucose, triglyceride, and the insulin resistance index of the B10 group were decreased by 34%, 59%, and 38%, respectively. In a pyruvate tolerance test, pyruvate-induced glucose excursion was decreased by 27% when mice were pretreated with 10 mg/kg of BA. In summary, BA effectively ameliorates hyperglycemia through inhibition of hepatic gluconeogenesis via modulating the CAMKK-AMPK-CREB signaling pathway.

Korean red ginseng extract alleviates advanced glycation end product-mediated renal injury.

The effect of Korean red ginseng (KRG) on diabetic renal damage was investigated using streptozotocin (STZ)-induced diabetic rats. The diabetic rats showed loss of body weight gain, and increases in kidney weight and urine volume, whereas the oral administration of KRG at a dose of 100 or 250 mg/kg of body weight per day for 28 d prevented these diabetes-induced physiological abnormalities. Among the kidney function parameters, elevated plasma levels of urea nitrogen and creatinine in diabetic control rats tended to be lowered in KRG-treated rats. In addition, administration of KRG at a dose of 100 mg/kg body weight in the diabetic rats showed significant decreases in serum glucose and tumor necrosis factor-α (TNF-α), implying that KRG might prevent the pathogenesis of diabetic complications caused by impaired glucose metabolism and oxidative stress. KRG also significantly reduced advanced glycation end product (AGE) formation and secretion from kidney of diabetic rats. Furthermore, KRG decreased the levels of N-(carboxymethyl) lysine and expression of AGE receptor. KRG also reduced the overexpression of cyclooxygenase-2 and inducible nitric oxide synthase in the kidney via deactivation of nuclear factor-kappa B. We also found that KRG prevented STZ-induced destruction of glomerular structure and significantly suppressed high glucose-induced fibronectin production. Taken together, KRG ameliorates abnormalities associated with diabetic nephropathy through suppression of inflammatory pathways activated by TNF-α and AGEs. These findings indicate that KRG has a beneficial effect on pathological conditions associated with diabetic nephropathy.

Caffeine attenuates lipid accumulation via activation of AMP-activated protein kinase signaling pathway in HepG2 cells.

The main purpose of this study is to examine the effect of caffeine on lipid accumulation in human hepatoma HepG2 cells. Significant decreases in the accumulation of hepatic lipids, such as triglyceride (TG), and cholesterol were observed when HepG2 cells were treated with caffeine as indicated. Caffeine decreased the mRNA level of lipogenesis-associated genes (SREBP1c, SREBP2, FAS, SCD1, HMGR and LDLR). In contrast, mRNA level of CD36, which is responsible for lipid uptake and catabolism, was increased. Next, the effect of caffeine on AMP-activated protein kinase (AMPK) signaling pathway was examined. Phosphorylation of AMPK and acetyl-CoA carboxylase were evidently increased when the cells were treated with caffeine as indicated for 24 h. These effects were all reversed in the presence of compound C, an AMPK inhibitor. In summary, these data indicate that caffeine effectively depleted TG and cholesterol levels by inhibition of lipogenesis and stimulation of lipolysis through modulating AMPK-SREBP signaling pathways.

Licochalcone A regulates hepatic lipid metabolism through activation of AMP-activated protein kinase.

Licochalcone A (LA) is a major phenolic ingredient of Glycyrrhiza plant. Although multiple pharmacological activities of LA have been reported, effect on hepatic lipid metabolism is unknown yet. The present study showed LA to suppress the hepatic triglyceride accumulation in HepG2 cells and ICR mice fed on a high fat diet (HFD). LA inhibited lipogenesis via suppression of sterol regulatory element-binding protein 1c (SREBP1c) and its target enzymes (stearoyl-CoA desaturase 1, fatty acid synthase and glycerol-3-phosphate acyltransferase) transcription. In addition, LA up-regulated gene expression of proteins such as peroxisome proliferator-activated receptor α (PPARα) and fatty acid transporter (FAT/CD36), which are responsible for lipolysis and fatty acid transport, respectively. These effects were mediated through activation of AMP-activated protein kinase (AMPK), and were abrogated when HepG2 cells were treated with an AMPK inhibitor, compound C. To explore how LA activates AMPK, oxygen consumption rate and ATP levels were measured in HepG2 cells. LA significantly inhibited the mitochondrial respiration and ATP levels, suggesting that LA activated AMPK indirectly. In animal study, LA (5 and 10mg/kg) was orally administered to six-week-old mice once a day for 3 weeks. In vitro results were likely to hold true in vivo experiment, as LA markedly lowered the triglyceride levels and activated AMPK signaling pathway in the liver of ICR mice fed on a HFD. In conclusion, the current study suggests that LA suppressed hepatic triglyceride accumulation through modulation of AMPK-SREBP signaling pathway and thus LA may be a potential therapeutic agent for treating fatty liver disease.

Betulinic acid alleviates non-alcoholic fatty liver by inhibiting SREBP1 activity via the AMPK-mTOR-SREBP signaling pathway.

Non-alcoholic fatty liver disease (NAFLD) is emerging as the most common liver disease in industrialized countries. The discovery of food components that can ameliorate NAFLD is therefore of interest. Betulinic acid (BA) is a triterpenoid with many pharmacological activities, but the effect of BA on fatty liver is as yet unknown. To explore the possible anti-fatty liver effects and their underlying mechanisms, we used insulin-resistant HepG2 cells, primary rat hepatocytes and liver tissue from ICR mice fed a high-fat diet (HFD). Oil Red O staining revealed that BA significantly suppressed excessive triglyceride accumulation in HepG2 cells and in the livers of mice fed a HFD. Ca(+2)-calmodulin dependent protein kinase kinase (CAMKK) and AMP-activated protein kinase (AMPK) were both activated by BA treatment. In contrast, the protein levels of sterol regulatory element-binding protein 1 (SREBP1), mammalian target of rapamycin (mTOR) and S6 kinase (S6K) were all reduced when hepatocytes were treated with BA for up to 24h. We found that BA activates AMPK via phosphorylation, suppresses SREBP1 mRNA expression, nuclear translocation and repressed SREBP1 target gene expression in HepG2 cells and primary hepatocytes, leading to reduced lipogenesis and lipid accumulation. These effects were completely abolished in the presence of STO-609 (a CAMKK inhibitor) or compound C (an AMPK inhibitor), indicating that the BA-induced reduction in hepatic steatosis was mediated via the CAMKK-AMPK-SREBP1 signaling pathway. Taken together, our results suggest that BA effectively ameliorates intracellular lipid accumulation in liver cells and thus is a potential therapeutic agent for the prevention of fatty liver disease.

Licochalcone A prevents adipocyte differentiation and lipogenesis via suppression of peroxisome proliferator-activated receptor γ and sterol regulatory element-binding protein pathways.

Licochalcone A (LA) has been shown to exert multiple pharmacological effects, including anti-inflammatory, antiparasitic, antifungal, anticancer, and osteogenic activities. The present study investigated the ability of LA to suppress the differentiation of 3T3-L1 preadipocytes, and its antiobesity activity was explored using high fat diet (HFD)-fed ICR mice. During the terminal differentiation process, 3T3-L1 preadipocytes were treated with LA, and the lipid contents were quantified along with any changes in the expression of biomarkers associated with adipocyte differentiation and lipogenesis. The results show that LA significantly reduced lipid accumulation and down-regulated the expression of peroxisome proliferator-activated receptor γ, CCAAT/enhancer binding protein α, sterol regulatory element-binding protein 1c, and their target genes (fatty acid binding protein, fatty acid synthase, stearoyl-CoA desaturase 1, and glycerol-3-phosphate acyltransferase). In an animal study, body weight, triglyceride, cholesterol, and nonesterified fatty acid levels in the group given 10 mg/kg LA were significantly decreased by 14.0, 48.2, 58.9, and 73.5%, respectively. Transverse microcomputed tomography indicated that visceral fat depots in LA-treated mice were markedly reduced when compared with those of the HFD control group. In summary, these results suggest that LA exerts an antiobesity effect and that it is a candidate for future clinical trials.

Ginsenoside Re lowers blood glucose and lipid levels via activation of AMP-activated protein kinase in HepG2 cells and high-fat diet fed mice.

Ginsenoside Re is a protopanaxatriol-type saponin isolated from Panax ginseng berry. Although anti-diabetic and anti-hyperlipidemic effects of Re have been reported by several groups, its mechanism of action is largely unknown until now. Here, we examine anti-diabetic and anti-hyperlipidemic activities of Re and action mechanism(s) in human HepG2 hepatocytes and high-fat diet fed C57BL/6J mice. Re suppresses the hepatic glucose production via induction of orphan nuclear receptor small heterodimer partner (SHP), and inhibits lipogenesis via suppression of sterol regulatory element binding protein-1c (SREBP-1c) and its target gene [fatty acid synthase (FAS), stearoyl-CoA desaturase-1 (SCD1)] transcription. These effects were mediated through activation of AMP-activated protein kinase (AMPK), and abolished when HepG2 cells were treated with an AMPK inhibitor, Compound C. C57BL/6J mice were randomly divided into five groups: regular diet fed group (RD), high-fat diet fed group (HFD) and the HFD plus Re (5, 10, 20 mg/kg) groups. Re treatment groups were fed a high-fat diet for 6 weeks, and then orally administered Re once a day for 3 weeks. The in vitro results are likely to hold true in an in vivo experiment, as Re markedly lowered blood glucose and triglyceride levels and protected against hepatic steatosis in high-fat diet fed C57BL/6J mice. In conclusion, the current study suggest that ginsenoside Re improves hyperglycemia and hyperlipidemia through activation of AMPK, and confers beneficial effects on type 2 diabetic patients with insulin resistance and dyslipidemia.

Ginsenoside Rd stimulates the differentiation and mineralization of osteoblastic MC3T3-E1 cells by activating AMP-activated protein kinase via the BMP-2 signaling pathway.

As part of our search for biologically active anti-osteoporotic agents that enhance the differentiation and mineralization of osteoblastic MC3T3-E1 cells, we identified the ginsenoside Rd as the most active compound among ginsenosides. In this study, we showed that Rd stimulates osteoblastic differentiation and mineralization, manifested by the up-regulation of differentiation markers (alkaline phosphatase and osteogenic genes) and von Kossa/Alizarin Red staining, respectively. Rd induces the mRNA expression of bone morphogenetic protein-2 (BMP-2) and the secretion of the corresponding protein into media in a concentration-dependent manner. The mRNA expression and enzyme activity of alkaline phosphatase (ALP) were suppressed when MC3T3-E1 cells were exposed to noggin, a BMP-2 antagonist. The level of phosphorylated AMP-activated protein kinase (pAMPK) protein was also up-regulated by Rd in a time- and concentration-dependent manner. Rd-induced ALP activity, mineralization, and BMP-2 production were all inhibited by either Ara-A (AMPK inhibitor) or siRNA targeting AMPK. In addition, we investigated whether Rd-induced BMP-2 transduces signals through the Smad signaling pathways. Rd induced a significant level of phosphorylation of Smad1/5, and this effect was blocked when the cells were transfected with siRNA targeting Smad4, indicating that Smad1/5 must form complex with Smad4 to translocate into the nucleus and regulate the transcription of osteogenic genes. In summary, these results indicate that Rd induces the differentiation and mineralization of MC3T3-E1 cells through the activation of the AMPK/BMP-2/Smad signaling pathways. These findings provide a molecular basis for the osteogenic effect of Rd in MC3T3-E1 cells.

Ginsenoside Rg2 induces orphan nuclear receptor SHP gene expression and inactivates GSK3ß via AMP-activated protein kinase to inhibit hepatic glucose production in HepG2 cells.

Panax ginseng is known to have anti-diabetic activity, but the active ingredients have not been fully explored yet. Here, we test whether ginsenoside Rg2 has an inhibitory effect on hepatic glucose production and determine its mechanism of action. Rg2 significantly inhibits hepatic glucose production and induces phosphorylations of liver kinase B1 (LKB1), AMP-activated protein kinase (AMPK) and glycogen synthase kinase 3ß (GSK3ß) in time- and concentration-dependent manners in human HepG2 hepatoma cells, and these effects were abolished in the presence of compound C, a selective AMPK inhibitor. In addition, phosphorylated form of cAMP-response element-binding protein (CREB), a key transcription factor for hepatic gluconeogenesis, was decreased in time- and concentration-dependent manners. Next, gene expression of orphan nuclear receptor small heterodimer partner (SHP) was also examined. Rg2 markedly enhanced the gene expression of SHP and its direct interaction with CREB, which results in disruption of CREB·CRTC2 complex. Consequently, expressions of relevant genes such as peroxisome proliferation-activated receptor γ coactivator-1α (PGC-1α), phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) were all significantly suppressed and these effects were also reversed in the presence of compound C. In conclusion, our results propose that ginsenoside Rg2 suppresses the hepatic glucose production via AMPK-induced phosphorylation of GSK3ß and induction of SHP gene expression. Further studies are warranted to elucidate a therapeutic potential of Rg2 for type 2 diabetic patients.

Ginsenoside Rh2(S) induces the differentiation and mineralization of osteoblastic MC3T3-E1 cells through activation of PKD and p38 MAPK pathways.

As part of the search for biologically active anti-osteoporotic agents that enhance differentiation and mineralization of osteoblastic MC3T3-E1 cells, we identified the ginsenoside Rh2(S), which is an active component in ginseng. Rh2(S) stimulates osteoblastic differentiation and mineralization, as manifested by the up-regulation of differentiation markers (alkaline phosphatase and osteogenic genes) and Alizarin Red staining, respectively. Rh2(S) activates p38 mitogen-activated protein kinase (MAPK) in time- and concentration-dependent manners, and Rh2(S)-induced differentiation and mineralization of osteoblastic cells were totally inhibited in the presence of the p38 MAPK inhibitor, SB203580. In addition, pretreatment with Go6976, a protein kinase D (PKD) inhibitor, significantly reversed the Rh2(S)-induced p38 MAPK activation, indicating that PKD might be an upstream kinase for p38 MAPK in MC3T3-E1 cells. Taken together, these results suggest that Rh2(S) induces the differentiation and mineralization of MC3T3-E1 cells through activation of PKD/p38 MAPK signaling pathways, and these findings provide a molecular basis for the osteogenic effect of Rh2(S).

Ginsenoside Rh2(S) induces differentiation and mineralization of MC3T3-E1 cells through activation of the PKD/AMPK signaling pathways.

As part of our search for biologically active anti-osteoporotic agents that enhance differentiation and mineralization of osteoblastic MC3T3-E1 cells, we identified the ginsenoside Rh2(S). Mostly known to exhibit beneficial effects in cancer prevention and metabolic diseases, Rh2(S) is one of the most active ginsenosides. Here, we show that Rh2(S) stimulates osteoblastic differentiation and mineralization, manifested by the up-regulation of differentiation markers (alkaline phosphatase and osteogenic genes) and von Kossa/Alizarin Red staining, respectively. Rh2(S) also activated protein kinase D (PKD) and AMP-activated protein kinase (AMPK) in a time- and concentration-dependent manner, and Rh2(S)-induced differentiation and mineralization of osteoblastic cells were significantly abolished in the presence of specific inhibitors; Go6976 for PKD and Ara-A for AMPK. Furthermore, Go6976 suppressed Rh2(S)-mediated activation of AMPK, indicating that PKD may be an upstream signal for AMPK in Rh2(S)-induced differentiation and mineralization of MC3T3-E1 cells. Taken together, these results indicate that Rh2(S) induces the differentiation and mineralization of MC3T3-E1 cells through activation of PKD/AMPK signaling pathways. These findings provide a molecular basis for the osteogenic effect of Rh2(S).

Cinnamaldehyde prevents adipocyte differentiation and adipogenesis via regulation of peroxisome proliferator-activated receptor-γ (PPARγ) and AMP-activated protein kinase (AMPK) pathways.

Cinnamaldehyde (CA), one of the active components of cinnamon, has been known to exert several pharmacological effects such as anti-inflammatory, antioxidant, antitumor, and antidiabetic activities. However, its antiobesity effect has not been reported yet. This study investigated the antidifferentiation effect of CA on 3T3-L1 preadipocytes, and the antiobesity activity of CA was further explored using high-fat-diet-induced obese ICR mice. During 3T3-L1 preadipocytes were differentiated into adipocytes, 10-40 µM CA was treated and lipid contents were quantified by Oil Red O staining, along with changes in the expression of genes and proteins associated with adipocyte differentiation and adipogenesis. It was found that CA significantly reduced lipid accumulation and down-regulated the expression of peroxisome proliferator-activated receptor-γ (PPAR-γ), CCAAT/enhancer-binding proteins α (C/EBPα), and sterol regulatory element-binding protein 1 (SREBP1) in concentration-dependent manners. Moreover, CA markedly up-regulated AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC), and these effects were blunted in the presence of AMPK inhibitor, compound C. In the animal study, weight gains, insulin resistance index, plasma triglyceride (TG), nonesterified fatty acid (NEFA), and cholesterol levels in the 40 mg/kg of CA-administered group were significantly decreased by 67.3, 55, 39, 31, and 23%, respectively, when compared to the high-fat diet control group. In summary, these results suggest that CA exerts antiadipogenic effects through modulation of the PPAR-γ and AMPK signaling pathways.

Anti-Diabetic Effect of Pectinase-Processed Ginseng Radix (GINST) in High Fat Diet-Fed ICR Mice.

In the present study, we investigate anti-diabetic effect of pectinase-processed ginseng radix (GINST) in high fat diet-fed ICR mice. The ICR mice were divided into three groups: regular diet group, high fat diet control group (HFD), and GINST-treated group. To induce hyperglycemia, mice were fed a high fat diet for 10 weeks, and mice were administered with 300 mg/ kg of GINST once a day for 5 weeks. Oral glucose tolerance test revealed that GINST improved glucose tolerance after glucose challenge. Compared to the HFD control group, fasting blood glucose and insulin levels were decreased by 57.8% (p<0.05) and 30.9% (p<0.01) in GINST-treated group, respectively. With decreased plasma glucose and insulin levels, the insulin resistance index of the GINST-treated group was reduced by 68.1% (p<0.01) compared to the HFD control group. Pancreas of GINST-treated mice preserved a morphological integrity of islets and consequently having more insulin contents. In addition, GINST up-regulated the levels of phosphorylated AMP-activated protein kinase (AMPK) and its target molecule, glucose transporter 4 (GLUT4) protein expression in the skeletal muscle. Our results suggest that GINST ameliorates a hyperglycemia through activation of AMPK/ GLUT4 signaling pathway, and has a therapeutic potential for type 2 diabetes.

20(S)-Ginsenoside Rg3-induced apoptosis in HT-29 colon cancer cells is associated with AMPK signaling pathway.

20(S)-ginsenoside Rg3 [20(S)-Rg3)], one of the main constituents isolated from Panax ginseng, has been shown to have an anti-cancer effect and to induce apoptosis by interfering with several signaling pathways. However, the molecular mechanisms of AMP-activated protein kinase (AMPK) associated with apoptosis in HT-29 colon cancer cells remain unclear. In the present study, we investigated whether 20(S)-Rg3 exerts an anti-proliferative effect and induces apoptosis by modulating the AMPK signaling pathway in HT-29 cells. 20(S)-Rg3-treated cells displayed several apoptotic features, including DNA fragmentation, proteolytic cleavage of poly (ADP-ribose) polymerase (PARP) and morphological changes. 20(S)-Rg3 down-regulated the expression of anti-apoptotic protein B-cell CLL/lymphoma 2 (Bcl2), up-regulated the expression of pro-apoptotic protein of p53 and Bcl-2-associated X protein (Bax), and caused the release of mitochondrial cytochrome c, PARP, caspase-9 and caspase-3. However, 20(S)-Rg3-induced apoptosis was completely abolished in the presence of compound C (AMPK inhibitor) or small interfering RNA for AMPK (siAMPK). In addition, STO-609 (CaMKKß inhibitor) attenuated 20(S)-Rg3-induced AMPK activation and apoptosis. These results suggest that 20(S)-Rg3-induced apoptosis in HT-29 cells is mediated via the AMPK signaling pathway, and that 20(S)-Rg3 is capable of inducing apoptosis in colon cancer.