Ginsenoside Rh1 Inhibits Angiotensin II-Induced Vascular Smooth Muscle Cell Migration and Proliferation through Suppression of the ROS-Mediated ERK1/2/p90RSK/KLF4 Signaling Pathway.

Vascular smooth muscle cell (VSMC) proliferation and migration play key roles in the progression of atherosclerosis and restenosis. A variety of ginsenosides exert various cardiovascular benefits. However, whether and how ginsenoside Rh1 (Rh1) inhibits VSMC dysfunction remain unclear. Here, we investigated the inhibitory effects of Rh1 on rat aortic smooth muscle cell (RASMC) migration and proliferation induced by angiotensin II (Ang II) and the underlying mechanisms. Cell proliferation and migration were evaluated using sulforhodamine B and wound-healing assay. The molecular mechanisms were investigated using Western blotting, quantitative reverse-transcription polymerase chain reaction analysis, immunofluorescence staining, and luciferase assay. Reactive oxygen species (ROS) production was measured using dihydroethidium and MitoSOX staining. We found that Rh1 dose-dependently suppressed Ang II-induced cell proliferation and migration. Concomitantly, Ang II increased protein levels of osteopontin, vimentin, MMP2, MMP9, PCNA, and cyclin D1, while these were reduced by Rh1 pretreatment. Notably, Ang II enhanced both the protein expression and promoter activity of KLF4, a key regulator of phenotypic switching, whereas pretreatment with Rh1 reversed these effects. Mechanistically, the effects of Rh1 on VSMC proliferation and migration were found to be associated with inhibition of ERK1/2/p90RSK signaling. Furthermore, the inhibitory effects of Rh1 were accompanied by inhibition of ROS production. In conclusion, Rh1 inhibited the Ang II-induced migration and proliferation of RASMCs by suppressing the ROS-mediated ERK1/2/p90RSK signaling pathway.

Ginsenoside Rh1 inhibits tumor growth in MDA-MB-231 breast cancer cells via mitochondrial ROS and ER stress-mediated signaling pathway.

Ginsenoside-Rh1 (Rh1) is a ginseng-derived compound that has been reported to exert anticancer effects by regulating cell cycle arrest and apoptosis according to reactive oxygen species (ROS) production. However, the effects of Rh1 on mitochondrial dysfunction are involved in triple negative breast cancer (TNBC) cell apoptosis, and the related molecular mechanisms remain unknown. Rh1 treatment induced cell toxicity less than 50% at 50 µM. In addition, Rh1 induced apoptosis in TNBC cells through cleaved caspase-3 activation and G1/S arrest. The Rh1-treated TNBC cells showed a significant increase in mitochondrial ROS (mtROS), which in turn increased protein expression of mitochondrial molecules, such as Bak and cytochrome C, and caused the loss of mitochondrial membrane potential. Pretreatment with mitochondria-targeted antioxidant Mito-TEMPO alters the Rh1-reduced rate of mito- and glycol-ATP. Furthermore, Rh1 induces ER stress-mediated calcium accumulation via PERK/eIF2α/ATF4/CHOP pathway. Inhibition of ATF4 by siRNA transfection significantly inhibited Rh1-mediated apoptosis and calcium production. Interestingly, Mito-TEMPO treatment significantly reduced apoptosis and ER stress induced by Rh1. Finally, Rh1 at 5 mg/kg suppressed tumor growth through increased levels of ROS production, cleaved caspase-3, and ATF4 more than 5-fluorouracil treated group. Overall, our results suggest that Rh1 has potential for use in TNBC treatment.

Role of mitochondrial dynamics and mitophagy of vascular smooth muscle cell proliferation and migration in progression of atherosclerosis.

Vascular smooth muscle cell (VSMC) proliferation and migration are critical events that contribute to the pathogenesis of vascular diseases such as atherosclerosis, restenosis, and hypertension. Recent findings have revealed that VSMC phenotype switching is associated with metabolic switch, which is related to the role of mitochondria. Mitochondrial dynamics are directly associated with mitochondrial function and cellular homeostasis. Interestingly, it has been suggested that mitochondrial dynamics and mitophagy play crucial roles in the regulation of VSMC proliferation and migration through various mechanisms. Especially, dynamin-related protein-1 and mitofusion-2 are two main molecules that play a key role in regulating mitochondrial dynamics to induce VSMC proliferation and migration. Therefore, this review describes the function and role of mitochondrial dynamics and mitophagy in VSMC homeostasis as well as the underlying mechanisms. This will provide insight into the development of innovative approaches to treat atherosclerosis.

Ginsenoside Rh1 Prevents Migration and Invasion through Mitochondrial ROS-Mediated Inhibition of STAT3/NF-κB Signaling in MDA-MB-231 Cells.

Breast cancer (BC) a very common cancer in women worldwide. Triple negative breast cancer (TNBC) has been shown to have a poor prognosis with a high level of tumor metastatic spread. Here, the inhibitory effects of ginsenoside-Rh1 (Rh1) on BC metastasis, and its underlying signaling pathway in TNBC were investigated. Rh1-treated MDA-MB-231 cells were analyzed for metastasis using a wound healing assay, transwell migration and invasion assay, western blotting, and qRT-PCR. Rh1 treatment significantly inhibited BC metastasis by inhibiting the both protein and mRNA levels of MMP2, MMP9, and VEGF-A. Further, Rh1-mediated inhibitory effect on BC migration was associated with mitochondrial ROS generation. Rh1 treatment significantly eliminated STAT3 phosphorylation and NF-κB transactivation to downregulate metastatic factors, such as MMP2, MMP9, and VEGF-A. In addition, Mito-TEMPO treatment reversed Rh1 effects on the activation of STAT3, NF-κB, and their transcriptional targets. Rh1 further enhanced the inhibitory effects of STAT3 or NF-κB specific inhibitor, stattic or BAY 11-7082 on MMP2, MMP9, and VEGF-A expression, respectively. In summary, our results revealed the potent anticancer effect of Rh1 on TNBC migration and invasion through mtROS-mediated inhibition of STAT3 and NF-κB signaling.

Ginsenoside-Rg2 affects cell growth via regulating ROS-mediated AMPK activation and cell cycle in MCF-7 cells.

BACKGROUND: Ginsenoside-Rg2 (G-Rg2) is a protopanaxatriol-type ginsenoside isolated from ginseng. It has been found to exhibit various pharmacological effects, including antioxidant, anti-inflammatory, and anticancer effects. PURPOSE: This study aimed to investigate the anticancer effects of G-Rg2 on estrogen receptor-positive MCF-7 breast cancer (BC) cells, and the underlying mechanisms involving in reactive oxygen species (ROS) production. STUDY DESIGN/METHODS: Cell viability, cell cycle distribution, apoptosis, and ROS production were measured following exposure to G-Rg2. The protein expression levels of p-ERK1/2, p-Akt, PARP, p-Rb, cyclin D1, CDK6, and p-AMPK were quantified using western blot analysis. The in vivo activity of G-Rg2 was assessed in a xenograft model. Immunohistochemistry staining for p-Rb and p-AMPK was performed in tumor tissues. RESULTS: G-Rg2 significantly decreased cell viability but increased cell apoptosis. In MCF-7 cells, G-Rg2 increased ROS production by inhibiting ERK1/2 and Akt activation. G-Rg2-induced ROS induced G0/G1 cell cycle arrest and AMPK phosphorylation. In the xenograft model, the 5 mg/kg G-Rg2-treated group showed decreased tumor volume and weight, similar to the 5 mg/kg 4-OHT-treated group, compared to the control group. Immunohistochemistry staining showed that G-Rg2 treatment decreased Rb phosphorylation, while increasing AMPK phosphorylation in tumor tissues. CONCLUSION: G-Rg2 has potential anticancer effects by increasing the ROS-AMPK signaling pathway and inhibiting ERK1/2 and Akt activation-mediated cell proliferation and cell cycle progression in MCF-7 BC cells.

Ginsenoside Rh1 Induces MCF-7 Cell Apoptosis and Autophagic Cell Death through ROS-Mediated Akt Signaling.

Breast cancer (BC) is the leading cause of cancer-related deaths among women worldwide. Ginsenosides exhibit anticancer activity against various cancer cells. However, the effects of ginsenoside Rh1 on BC and the underlying mechanisms remain unknown. Here, we investigated the anticancer effects of Rh1 on human BC MCF-7 and HCC1428 cells and the underlying signaling pathways. The anticancer effects of Rh1 in vitro were evaluated using sulforhodamine B (SRB), 3-(4, 5-dimethylthiazole-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), clonogenic assay, propidium iodide (PI)/Hoechst staining, Western blotting, flow cytometry, and immunofluorescence analysis. The in vivo effects of Rh1 were determined using a xenograft model via hematoxylin and eosin and the immunohistochemistry staining of tumor tissues. We found that Rh1 exerted cytotoxicity in the cells by increasing cell apoptosis, autophagy, and cell cycle arrest. These effects were further enhanced by a phosphatidylinositol 3-kinase (PI3K) inhibitor but were rescued by the inhibition of reactive oxygen species (ROS). Moreover, enhanced ROS generation by Rh1 inhibited the activation of the PI3K/Akt pathway. Consistently, Rh1 treatment significantly reduced tumor growth in vivo and increased the ROS production and protein expression of LC3B and cleaved caspase-3 but decreased the phosphorylation of Akt and retinoblastoma (Rb) in tumor tissues. Taken together, Rh1 exerted a potential anticancer effect on BC cells by inducing cell cycle arrest, apoptosis, and autophagy via inhibition of the ROS-mediated PI3K/Akt pathway.

Protective effects of ginsenoside-Rg2 and -Rh1 on liver function through inhibiting TAK1 and STAT3-mediated inflammatory activity and Nrf2/ARE-mediated antioxidant signaling pathway.

Systemic or hepatic inflammation is caused by intraperitoneal application of lipopolysaccharide (LPS). In this study, we investigated anti-inflammatory and antioxidant properties of combination of ginsenoside-Rg2 (G-Rg2) and -Rh1 (G-Rh1) on liver function under LPS challenging. We first confirmed that G-Rg2 and -Rh1 at 100 µg/ml did not show cytotoxicity in HepG2 cells. G-Rg2 and -Rh1 treatment significantly inhibited activation of STAT3 and TAK1, and inflammatory factors including iNOS, TNF-α, and IL-1ß in peritoneal macrophages. In HepG2 cells, G-Rg2 and -Rh1 treatment inhibited activation of STAT3 and TAK1/c-Jun N-terminal kinase, and down-regulated nuclear translocation of NF-κB transcription factor. In addition, LPS-induced mitochondrial dysfunction was restored by treatment with G-Rg2 and -Rh1. Interestingly, pretreatment with G-Rg2 and -Rh1 effectively inhibited mitochondrial damage-mediated ROS production induced by LPS stimulation, and alterations of Nrf2 nuclear translocation and ARE promotor activity were involved in G-Rg2 and -Rh1 effects on balancing ROS levels. In liver tissues of LPS-treated mice, G-Rg2 and -Rh1 treatment protected liver damages and increased Nrf2 expression while reducing CD45 expression. Taken together, G-Rg2 and -Rh1 exerts a protective effect on liver function by increasing antioxidant through Nrf2 and anti-inflammatory activities through STAT3/TAK1 and NF-κB signaling pathways in liver cells and macrophages.

Minor Ginsenoside Rg2 and Rh1 Attenuates LPS-Induced Acute Liver and Kidney Damages via Downregulating Activation of TLR4-STAT1 and Inflammatory Cytokine Production in Macrophages.

Ginsenosides have been reported to have various biological effects, such as immune regulation and anticancer activity. In this study, we investigated the anti-inflammatory role of a combination of Rg2 and Rh1, which are minor ginsenosides, in lipopolysaccharide (LPS)-stimulated inflammation. In vitro experiments were performed using the RAW264.7 cell line, and an in vivo model of inflammation was established using LPS-treated ICR mice. We employed Griess assay, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, quantitative reverse transcriptase-polymerase chain reaction, western blotting, immunofluorescence staining, and hematoxylin and eosin staining to evaluate the effect of Rg2 and Rh1. We found that Rg2 and Rh1 significantly decreased LPS-induced major inflammatory mediator production, inducible-nitric oxide synthase expression, and nitric oxide production in macrophages. Moreover, Rg2 and Rh1 combination treatment inhibited the binding of LPS to toll-like receptor 4 (TLR4) on peritoneal macrophages. Therefore, the combination of ginsenoside Rg2 and Rh1 suppressed inflammation by abolishing the binding of LPS to TLR4, thereby inhibiting the TLR4-mediated signaling pathway. The combined ginsenoside synergistically blocked LPS-mediated PKCδ translocation to the plasma membrane, resulting in p38-STAT1 activation and NF-κB translocation. In addition, mRNA levels of pro-inflammatory cytokines, including TNF-α, IL-1ß, and IFN-ß, were significantly decreased by combined ginsenoside treatment. Notably, the 20 mg/kg ginsenoside treatment significantly reduced LPS-induced acute tissue inflammation levels in vivo, as indicated by the tissue histological damage scores and the levels of biochemical markers for liver and kidney function from mouse serum. These results suggest that the minor ginsenosides Rg2 and Rh1 may play a key role in prevention of LPS-induced acute inflammation and tissue damage.

Therapeutic effects of ginsenosides on breast cancer growth and metastasis.

Breast cancer is the most common cause of cancer-related deaths among women worldwide. Thus, the development of new and effective low-toxicity drugs is vital. The specific characteristics of breast cancer have allowed for the development of targeted therapy towards each breast cancer subtype. Nevertheless, increasing drug resistance is displayed by the changing phenotype and microenvironments of the tumor through mutation or dysregulation of various mechanisms. Recently, emerging data on the therapeutic potential of biocompounds isolated from ginseng have been reported. Therefore, in this review, various roles of ginsenosides in the treatment of breast cancer, including apoptosis, autophagy, metastasis, epithelial-mesenchymal transition, epigenetic changes, combination therapy, and drug delivery system, have been discussed.

Inhibition of p90RSK is critical to abolish Angiotensin II-induced rat aortic smooth muscle cell proliferation and migration.

Angiotensin II (Ang II) has been reported to induce vascular smooth muscle cell (VSMC) proliferation and migration, which are major events that are highly linked to vascular diseases such as atherosclerosis and restenosis. p90 ribosomal S6 kinase (p90RSK), a potential downstream effector of ERK1/2, has been demonstrated to be activated by Ang II in VSMCs. However, the role of p90RSK on Ang II-induced VSMC proliferation and migration and its underlying signaling pathways remain unknown. In this study, we found that the inhibition of p90RSK, using a p90RSK specific inhibitor FMK or transfected cells with a plasmid encoding dominant negative RSK1, inactivated p90RSK kinase action completely and suppressed Ang II-induced rat aortic smooth muscle cell (RASMC) proliferation and migration. Interestingly, inhibition of p90RSK kinase activity abolished the phosphorylation of Akt as well as the protein expression of ICAM-1, VCAM-1, MMP-2, and NF-κB p65 in Ang II-treated RASMCs. Furthermore, the luciferase reporter assay revealed the inhibitory effect of FMK on NF-κB promoter activity induced by Ang II. Notably, using the partial carotid ligation model in mice, FMK was found to attenuate the medial thickness of carotid arteries increased by Ang II. Taken together, these results suggest that p90RSK plays a critical role in Ang II-induced VSMC proliferation and migration by increasing Akt phosphorylation and NF-κB p65 promoter activity associated with up-regulation of adhesion molecules and MMP-2 expression.

Inhibition of p90RSK activation sensitizes triple-negative breast cancer cells to cisplatin by inhibiting proliferation, migration and EMT.

Cisplatin (Cis-DDP) is one of the most widely used anti-cancer drugs. It is applicable to many types of cancer, including lung, bladder, and breast cancer. However, its use is now limited because of drug resistance. p90 ribosomal S6 kinase (p90RSK) is one of the downstream effectors in the extracellular signalregulated protein kinases 1 and 2 (ERK1/2) pathway and high expression of p90RSK is observed in human breast cancer tissues. Therefore, we investigated the role of p90RSK in the Cis-DDP resistance-related signaling pathway and epithelialmesenchymal transition (EMT) in breast cancer cells. First, we discovered that MDA-MB-231 cells exhibited more Cis-DDP resistance than other breast cancer cells, including MCF-7 and BT549 cells. Cis-DDP increased p90RSK activation, whereas the inactivation of p90RSK using a small interfering RNA (siRNA) or dominant-negative kinase mutant plasmid overexpression significantly reduced Cis-DDP-induced cell proliferation and migration via the inhibition of matrix metallopeptidase (MMP)2 and MMP9 in MDA-MB-231 cells. In addition, p90RSK activation was involved in EMT via the upregulation of mRNA expression, including that of Snail, Twist, ZEB1, N-cadherin, and vimentin. We also investigated NF-κB, the upstream regulator of EMT markers, and discovered that Cis-DDP treatment led to NF-κB translocation in the nucleus as well as its promoter activity. Our results suggest that targeting p90RSK would be a good strategy to increase Cis-DDP sensitivity in triple-negative breast cancers. [BMB Reports 2019; 52(12): 706-711].

Therapeutic targets for endothelial dysfunction in vascular diseases.

Vascular endothelial cells are located on the surface of the blood vessels. It has been recognized as an important barrier to the regulation of vascular homeostasis by regulating the blood flow of micro- or macrovascular vessels. Indeed, endothelial dysfunction is an initial stage of vascular diseases and is an important prognostic indicator of cardiovascular and metabolic diseases such as atherosclerosis, hypertension, heart failure, or diabetes. Therefore, in order to develop therapeutic targets for vascular diseases, it is important to understand the key factors involved in maintaining endothelial function and the signaling pathways affecting endothelial dysfunction. The purpose of this review is to describe the function and underlying signaling pathway of oxidative stress, inflammatory factors, shear stress, and epigenetic factors in endothelial dysfunction, and introduce recent therapeutic targets for the treatment of cardiovascular diseases.