Myeong Hun Yeom

20‐O‐β‐d‐Glucopyranosyl‐20(S)‐Protopanaxadiol Suppresses UV‐Induced MMP‐1 Expression Through AMPK‐Mediated mTOR Inhibition as a Downstream of the PKA‐LKB1 Pathway

Various health effects have been attributed to the ginsenoside metabolite 20‐O‐β‐D‐glucopyranosyl‐20(S)‐protopanaxadiol (GPD); however, its effect on ultraviolet (UV)‐induced matrix metalloproteinase (MMP)‐1 expression and the mechanism underlying this effect are unknown. We examined the inhibitory effect of GPD on UV‐induced MMP‐1 expression and its mechanisms in human dermal fibroblasts (HDFs). GPD attenuated UV‐induced MMP‐1 expression in HDFs and suppressed the UV‐induced phosphorylation of mammalian target of rapamycin (mTOR) and p70S6K without inhibiting the activity of phosphatidylinositol 3‐kinase and Akt, which are well‐known upstream kinases of mTOR. GPD augmented the phosphorylation of liver kinase B1 (LKB1) and adenosine monophosphate‐activated protein kinase (AMPK), which are inhibitors of mTOR, to a greater extent than UV treatment alone. Similar to GPD, 5‐aminoimidazole‐4‐carboxamide‐1‐β‐d‐ribofuranosyl 5′‐monophosphate (AICAR), an activator of AMPK, augmented UV‐induced AMPK phosphorylation to a greater extent than UV treatment alone, resulting in the inhibition of MMP‐1 expression. AICAR also decreased the phosphorylation of mTOR and p70S6K. However, compound C, an antagonist of AMPK, increased MMP‐1 expression. In HDF cells with AMPK knock‐down using shRNA, MMP‐1 expression was increased. These results indicate that AMPK activation plays a key role in MMP‐1 suppression. Additionally, the cAMP‐dependent protein kinase (PKA) inhibitor, H‐89, antagonized GPD‐mediated MMP‐1 suppression via the inhibition of LKB1. Our results suggest that the suppressive activity of GPD on UV‐induced MMP‐1 expression is due to the activation of AMPK as a downstream of the PKA‐LKB1 pathway. J. Cell. Biochem. 115: 1702–1711, 2014.

20-O-β-D-glucopyranosyl-20(S)-protopanaxadiol, a metabolite of ginsenoside Rb1, enhances the production of hyaluronic acid through the activation of ERK and Akt mediated by Src tyrosin kinase in human keratinocytes

The aim of the present study was to determine the mechanisms through which 20-O-β-D-glucopyranosyl-20(S)-protopanaxadiol (20GPPD) promotes the production of hyaluronic acid (HA) in human keratinocytes. 20GPPD is the primary bioactive metabolite of Rb1, a major ginsenoside found in ginseng (Panax ginseng). We sought to elucidate the underlying mechanisms behind the 20GPPD-induced production of HA. We found that 20GPPD induced an increase in HA production by elevating hyaluronan synthase 2 (HAS2) expression in human keratinocytes. The phosphorylation of extracellular signal-regulated kinase (ERK) and Akt was also enhanced by 20GPPD in a dose-dependent manner. The pharmacological inhibition of ERK (using U0126) or Akt (using LY294002) suppressed the 20GPPD-induced expression of HAS2, whereas treatment with an epidermal growth factor receptor (EGFR) inhibitor (AG1478) or an intracellular Ca2+ chelator (BAPTA/AM) did not exert any observable effects. The increased Src phosphorylation was also confirmed following treatment with 20GPPD in the human keratinocytes. Following pre-treatment with the Src inhibitor, PP2, both HA production and HAS2 expression were attenuated. Furthermore, the 20GPPD-enhanced ERK and Akt signaling decreased following treatment with PP2. Taken together, our results suggest that Src kinase plays a critical role in the 20GPPD-induced production of HA by acting as an upstream modulator of ERK and Akt activity in human keratinocytes.

The ginsenoside 20-O-β-D-glucopyranosyl-20(S)-protopanaxadiol induces autophagy and apoptosis in human melanoma via AMPK/JNK phosphorylation.

Studies have shown that a major metabolite of the red ginseng ginsenoside Rb1, called 20-O-β-D-glucopyranosyl-20(S)-protopanaxadiol (GPD), exhibits anticancer properties. However, the chemotherapeutic effects and molecular mechanisms behind GPD action in human melanoma have not been previously investigated. Here we report the anticancer activity of GPD and its mechanism of action in melanoma cells. GPD, but not its parent compound Rb1, inhibited melanoma cell proliferation in a dose-dependent manner. Further investigation revealed that GPD treatment achieved this inhibition through the induction of autophagy and apoptosis, while Rb1 failed to show significant effect at the same concentrations. The inhibitory effect of GPD appears to be mediated through the induction of AMPK and the subsequent attenuation of mTOR phosphorylation. In addition, GPD activated c-Jun by inducing JNK phosphorylation. Our findings suggest that GPD suppresses melanoma growth by inducing autophagic cell death and apoptosis via AMPK/JNK pathway activation. GPD therefore has the potential to be developed as a chemotherapeutic agent for the treatment of human melanoma.

MLK3 is a direct target of biochanin A, which plays a role in solar UV-induced COX-2 expression in human keratinocytes

Solar UV (sUV) is an important environmental carcinogen. Recent studies have shown that sUV is associated with numerous human skin disorders, such as wrinkle formation and inflammation. In this study, we found that the isoflavone, biochanin A, inhibited the expression of sUV-induced COX-2, which is a well-characterized sUV-induced enzyme, in both human HaCaT keratinocytes and JB6 P+ mouse skin epidermal cells. Several studies have demonstrated the beneficial effects of biochanin A. However, its direct molecular target is unknown. We found that biochanin A inhibited sUV-induced phosphorylation of MKK4/JNK/c-Jun and MKK3/6/p38/MSK1. Mixed-lineage kinase 3 (MLK3) is an upstream kinase of MKK4 and MKK3/6. Thus, we evaluated the effect of biochanin A on MLK3. We found that sUV-induced MLK3 phosphorylation was not affected, whereas MLK3 kinase activity was significantly suppressed by biochanin A. Furthermore, direct binding of biochanin A in the MLK3 ATP-binding pocket was detected using pull-down assays. Computer modeling supported our observation that MLK3 is a novel target of biochanin A. These results suggest that biochanin A exerts chemopreventive effects by suppressing sUV-induced COX-2 expression mediated through MLK3 inhibition.

Rutin inhibits B(a)PDE-induced cyclooxygenase-2 expression by targeting EGFR kinase activity

Rutin is a well-known flavonoid that exists in various natural sources. Accumulative studies have represented the biological effects of rutin, such as anti-oxidative and anti-inflammatory effects. However, the underlying mechanisms of rutin and its direct targets are not understood. We investigated whether rutin reduced B[a]PDE-induced-COX-2 expression. The transactivation of AP-1 and NF-κB were inhibited by rutin. Rutin also attenuated B[a]PDE-induced Raf/MEK/ERK and Akt activation, but had no effect on the phosphorylation of EGFR. An in vitro kinase assay revealed rutin suppressed EGFR kinase activity. We also confirmed direct binding between rutin and EGFR, and found that the binding was regressed by ATP. The EGFR inhibitor also inhibited the B[a]PDE-induced MEK/ERK and Akt signaling pathways and subsequently, suppressed COX-2 expression and promoter activity, in addition to suppressing the transactivation of AP-1 and NF-κB. In EGFR(-/-)mouse embryonic fibroblast cells, B[a]PDE-induced COX-2 expression was also diminished. Collectively, rutin inhibits B[a]PDE-induced COX-2 expression by suppressing the Raf/MEK/ERK and Akt signaling pathways. EGFR appeared to be the direct target of rutin.