Ginsenoside Rk3 is a novel PI3K/AKT-targeting therapeutics agent that regulates autophagy and apoptosis in hepatocellular carcinoma

Hepatocellular carcinoma(HCC)is the third leading cause of cancer death worldwide.Ginsenoside Rk3,an important and rare saponin in heat-treated ginseng,is generated from Rg1 and has a smaller molecular weight.However,the anti-HCC efficacy and mechanisms of ginsenoside Rk3 have not yet been characterized.Here,we investigated the mechanism by which ginsenoside Rk3,a tetracyclic triterpenoid rare ginsenoside,inhibits the growth of HCC.We first explored the possible potential targets of Rk3 through network pharmacology.Both in vitro(HepG2 and HCC-LM3 cells)and in vivo(primary liver cancer mice and HCC-LM3 subcutaneous tumor-bearing mice)studies revealed that Rk3 significantly inhibits the proliferation of HCC.Meanwhile,Rk3 blocked the cell cycle in HCC at the G1 phase and induced autophagy and apoptosis in HCC.Further proteomics and siRNA experiments showed that Rk3 regulates the phosphatidylinositol 3-kinase(PI3K)/protein kinase B(AKT)pathway to inhibit HCC growth,which was validated by molecular docking and surface plasmon resonance.In conclusion,we report the discovery that ginsenoside Rk3 binds to PI3K/AKT and promotes autophagy and apoptosis in HCC.Our data strongly support the translation of ginsenoside Rk3 into novel PI3K/AKT-targeting therapeutics for HCC treatment with low toxic side effects.

Ginsenoside Rg_3 inhibit hepatocellular carcinoma growth via intrinsic apoptotic pathway

AIM:To investigate the anti-tumor function of ginsenoside Rg3 on hepatocellular carcinoma(HCC) in vitro and in vivo,and its mechanism.METHODS:Hep1-6 and HepG2 cells were treated by Rg3 in different concentrations(0,50,100 and 200 μg/mL) in vitro.After incubation for 0,6,12,24 and 48 h,cell viability was measured by 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide assay.Apoptosis was identified by terminal deoxynucleotidyl transferasemediated dUTP-biotin nick end labeling.Caspase-3 activity was measured by chromophore p-nitroanilide and flow cytometry.Bcl-2 family proteins were ascertained by Western-blotting.Mitochondria membrane potentialwas detected by 5,5',6' 6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide.Forty liver tumor-bearing C57Bl6 mice were divided randomly into 4 groups for intra-tumor injection of saline,ginsenoside Rg3,cyclophosphamide(CTX) and ginsenoside Rg3 + CTX combination.RESULTS:The survival time was followed up to 102 d.The mice in the Rg3 + CTX group showed significant increased survival time compared with those in the control group(P < 0.05).Rg3 could inhibit HCC cell proliferation and induce cell apoptosis in vitro in the concentration and time dependent manner.It also induced mitochondria membrane potential to decrease.Caspase-3 activation can be blocked by the inhibitor z-DEVD-FMK.Bax was up-regulated while Bcl-2 and Bcl-XL were down-regulated after Rg3 treatment.CONCLUSION:Our data suggested that Rg3 alone or combined with CTX inhibited tumor growth in vivo and prolonged mouse survival time by inducing HCC cell apoptosis via intrinsic pathway by expression alterations of Bcl-2 family proteins.

Preparation and Characterization of Biodegradable Polylactide(PLA) Microspheres Encapsulating Ginsenoside Rg3

In this study, the process of a biodegradable polylactide（PLA） microsphere encapsulating ginsenoside Rg3 was first studied by the emulsion solvent evaporation method, for enhancing solubility and stability of ginsenoside Rg3. Alabum was also first used as a modifier in this method. The mean diameter of the prepared PLA microspheres containing Rg3 was 40 μm. Ginsenoside Rg3 released from the microspheres was studied by HPLC and detected by UV. It was found that the drug release curve fitted the Model Heller-Baker best.

A mini-review on pharmacological effects of ginsenoside Rb3,a marked saponin from Panax genus

Ginsenoside Rb3(G-Rb3)is one of the primary active compounds isolated from Panax ginseng Meyer,which belongs to protopanaxadiol ginsenosides(PPD).Based on the structure-activity relationship(SAR)of ginsenosides,the pentose structure of G-Rb3 limited itself to possess more pharmacological activity to a certain extent.However,pharmacokinetics show that G-Rb3 is processed through deglycosylation in the intestinal tract and converted into more active rare saponins,such as Compound K,F2,etc.A series of studies focused on neuroprotection and the cardiovascular system demonstrating its therapeutic potentials,which was achieved by diminishing oxidative stress and apoptosis.Therefore,more systematic and in-depth studies are needed to complete the pharmaceutical value and to promote its clinical applications.This article highlights the multiple pharmacological effects and mechanisms of G-Rb3 and prospects for its development.

Enrichment of the less polar ginsenoside (Rg3) from ginseng grown in New Zealand by post-harvest processing and extraction

Background:Previous studies showed that New Zealand-grown ginseng contains an abundance of ginsenosides and that rare less polar ginsenosides,such as Rg3,exhibit more pharmacological activities than polar ginsenosides,which are the major components of ginseng.Methods:The ginsenoside profile of New Zealand-grown Panax ginseng was manipulated by treatment with acetic acid,sodium hydroxide,pH,and high temperature.The abundance of 23 ginsenosides extracted by different treatments was quantified using high-performance liquid chromatography.Results:Treatment with 0.5 mol/L acetic acid can stimulate the degradation of polar ginsenosides to less polar ginsenosides(5.6%Rg3 was accumulated,P<0.0001).Furthermore,when ginseng root was treated at 121℃ for 100 min in a pH 3.0 acetic acid aqueous solution,the majority of the polar ginsenosides were converted into less polar ginsenosides.Specifically,83.46±3.69%(P=0.0360)of the less polar ginsenosides and 41.01±2.39%(P=0.0412)of Rg3 were enriched.In contrast,alkali treatment did not convert the polar ginsenosides into less polar ginsenosides at mild temperature and less conversion was observed compared with acid treatment at high temperature.Conclusion:This is the first attempt to manipulate the ginsenoside profile of New Zealand-grown ginseng.The conditions(high temperature with low pH)may be modified to produce and enrich the less polar ginsenoside fraction(especially Rg3)from the total ginseng extract.

Ginsenoside 3β-O-Glc-DM(C3DM) Enhances the Antitumor Activity of Taxol on Lewis Lung Cancer by Targeting the Interleukin-6/Jak2/STAT3 and Interleukin-6/AKT Signaling Pathways

Objective:Nonsmall-cell lung cancer(NSCLC)is an aggressive,highly chemoresistant disease.Taxol is an effective chemotherapeutic drug widely used for the treatment of NSCLC.However,the clinical use of Taxol is limited due to the occurrence of adverse side effects under high therapeutic doses.Therefore,it is desirable to explore combination therapy to reduce the dose of chemotherapeutic drugs and achieve excellent outcomes.A biosynthetic ginsenoside,3-O-β-D-glucopyranosyl-dammar-24-ene-3β,20 S-diol(3β-O-Glc-DM,C3 DM)is obtained from microbial fermentation by metabolic engineering.Based on previous study findings,we aimed to explore the mechanism of combination therapy with C3 DM and Taxol and its increasing antitumor effect on Lewis lung cancer(LLC)in this study.Materials and Methods:A thiazolyl blue tetrazolium bromide(MTT)assay was performed to evaluate cell viability;the apoptotic effect was studied using cell apoptosis assay.The Lewis tumor xenograft experiment was performed to determine the effects of C3 DM combined with Taxol on tumor growth in vivo,and western blotting was performed to analyze protein expressions.Results:C3 DM effectively inhibited the proliferation of NSCLC cells.Moreover,C3 DM increased the antiproliferative activity of Taxol and significantly enhanced cell apoptosis induced by Taxol in Lewis lung cancer cells.C3 DM alone also suppressed Lewis tumor growth and enhanced the antitumor activity of Taxol in vivo.Western blot analysis revealed that the effects of the antiproliferation and apoptosis induction of C3 DM treatment alone or in combination with Taxol on Lewis lung cancer were mediated by inhibiting the interleukin-6(IL-6)/Jak2/STAT3 and IL-6/AKT signaling pathways.Conclusions:The results showed that C3 DM has the potential to be used in combination therapy with Taxol against NSCLC.

THE CLINICAL EVALUATION OF ANGIOGENESIS INHIBITOR-20(R)-GINSENOSIDE RG_3 IN LUNG CANCER

Objective The aim of the study was to make a further evaluation of Ginsenoside Rg3. Methods The clinical effects of the drug on moderate and advanced lung cancer, including side effects, were observed. Results Ginsenoside Rg3 improved chemotherapy significantly. The clinical relief rate of patients treated with antiangiogenic agent 20 (R) Ginsenoside Rg3 was 36.6%, which was higher than that of the patients not treated with it (16.7%)( P <0.05). It had no significantly different effects on lung cancers of different types of tissues ( P >0.05). It provided better treatment on the cancer at early stage than that at advanced stage ( P <0.05). Moreover the living qualities of the patients were improved notably ( P <0.05). Conclusion Combined with chemotherapy, angiogenesis inhibitor 20(R) Ginsenoside Rg3 can improve clinical therapeutic efficacy and the living qualities of patients significantly.

Ginsenoside rg3 reduces body weight by regulating fat content and browning in obese mice

Objective:To determine the effects of ginsenoside rg3 on the body weight of C57BL/6J obese mice and to investigate its underlying weight loss mechanisms with a focus on white fat browning-related factors.Methods:Eight-week-old C57BL/6J male mice were fed a high-fat diet for 12 successive weeks to construct the obese model.C57BL/6J male mice were fed a standard chow diet to construct normal control group.After 8 weeks of intervention with ginsenoside rg3,the food intake,body weight,body fat mass,blood sugar,and lipid profiles of the mice in each group were detected.Hematoxylin and eosin(HE)staining was used to observe the histological morphology of the adipose tissues.Real-time polymerase chain reaction(RT-PCR)and Western blotting(WB)were applied to detect the gene and protein expression levels of peroxisome proliferators-activated receptor gama(PPARg),Peroxisome proliferatoractivated receptor-gamma coactivator-1alpha(PGC-1a),PR domain containing 16(PRDM16),and uncoupling protein 1(UCP-1).Results:Compared to normal control group mice,the body weight,food intake,body fat composition,and blood lipid levels of model group mice increased significantly.After 8 weeks of intervention with ginsenoside rg3,body weight,body fat composition,food intake,and blood lipid profiles decreased.HE staining showed that ginsenoside rg3 can improve white adipocyte hypertrophy to a certain extent.RTPCR and WB demonstrated that ginsenoside rg3 can increase the mRNA and protein expression levels of PPARg,PGC-1a,PRDM16,and UCP-1 in the adipose tissues of obese mice.Conclusion:The weight reduction effect of ginsenoside rg3 may be related to the promotion of white fat browning.

Apoptosis Induced by Ginsenoside Rg3 in a Human Bladder Carcinoma Cell Line

OBJECTIVE This study was conducted to explore the effect of Rg3 on inhibition of proliferation and induction of apoptosis in bladder cancer cells. METHODS The EJ bladder cancer cell line was treated with Rg3 at various concentrations. Cell proliferation was measured by the MTT assay. Morphological changes in the cells were observed by fluorescent staining using Hoechst 33258. The cell cycle and apoptotic rate were analyzed by flow cytometry （FCM） and the expression of caspase-3 in cells was detected by immunocytochemistry. DNA ladder analysis was conducted by agarose gel electrophoresis. RESULTS Rg3 inhibited proliferation of EJ cells in a concentration-dependent manner, resulting in an IC50 for Rg3 at 48 h of 125.5 μg/ml. When treated with 150 μg/ml of Rg3 for 24 h and 48 h, the cells showed apoptotic morphological characteristics including condensed chromatin, nuclear fragmentation, apoptotic bodies and bright fluorescent granules as well as a higher caspase-3 expression. The FCM assay indicated that Rg3 altered the cell cycle and induced apoptosis of the EJ cells, when treated for 24 h and 48 h with 75 μg/ml of Rg3 as well as for 48 h with 150 μg/ml. The percentages of cells in the S phase and the GJM transition were increased, whereas the percentages of cells in the G0-G1 transition were decreased. The apoptotic rates were increased from （1.05±0.17）% in the control group cells to （8.41 ±0.98）%, （18.57±2.20）% and （33.98±1,64）% respectively. Significant changes in the DNA ladders, showed that the effects of Rg3 were displayed in a dose and time dependent manner. CONCLUSION The results suggest that Ginsenoside Rg3 exerts an inhibitory effect on proliferation of EJ cells by inducing apoptosis.

Ginsenoside Rg3 Promotes the Survival and Migration of Trophoblast Cells in a Rat Model of Gestational Hypertension by Regulating miR-100a/Insulin Growth Factor-2 Axis

Objective:Preeclampsia(PE)is a common complication during pregnancy.miR-100a is expressed in the placenta and regulates the survival and development of placental cells.Insulin growth factor-2(IGF-2)may serve as its downstream target.This study investigated the protective mechanisms of ginsenoside Rg3 against PE in rat model.Materials and Methods:LPS-induced rat PE models were suitable for intravenous administration of the highly expressed miR-100a ginsenoside Rg3 lentiviral vector.Human trophoblasts were cultured in vitro for JEG-3,and PE cell models were constructed.In vivo effects on tumor growth and apoptosis were observed.Ginsenoside Rg3 was treated with different concentrations of shRNA,miR-100a analogs,inhibitors,or IGF-2.Autophagy and the expression of autophagy-related proteins were examined.Trophoblast activity and migration were determined using Cell Counting Kit-8 and Transwell assays.Both drugs strongly inhibited trophoblasts under normal conditions with some synergy between them.Double-luciferase return assay confirmed the binding affinity of miR-100a for IGF-2.Results:In response to Rg3,autophagy and the expression of autophagy-related proteins LC3-I/II,Beclin1,and SQSTM1 were reduced in PE rat placental trophoblasts.Rg3 inhibited autophagy in JEG-3 cells and promoted JEG-3 survival and migration in a concentration-dependent manner.miR-100a upregulated PE expression.These results suggested that autophagy was a vital signaling system.Rg3 intervention inhibited miR-100a expression and miR-100a downregulated IGF-2 expression in placental tissues and promoted autophagy,thereby inhibiting JEG-3cell survival and migration.In rats,Rg3 inhibited PE development by regulating the activity of the miR-100a-IGF-2 signaling axis.Conclusion:Ginsenoside Rg3 positively regulates the miR-100a-IGF-2 axis and protects PE rats by inhibiting trophoblastic autophagy and promoting trophoblastic cell survival and migration.

Preparation, Release-control and Cell Apoptosis of C_6 Glioma Cells in PEG-PLGA-Rg3 Nanoparticles

With biodegradable material poly（ethylene glycol）-poly（lactide-co-glycolide） （PEG-PLGA） as substrate, the size distribution of Rg3-NPs was approved by the scanning electron microscopy. MTT assay was used to detect the effects of Rg3-NPs on the growth rate of C6 cells at various concentrations and flow cytometry（FCM） was applied to assay the cell cycle and cell apoptosis of C6 glioma cells. Western blot analysis was used to measure the protein level of PCNA. The results show that Rg3-NPs are slick and uniformity, the average diameter of the nanoparticles is about 75-90 nm, entrapment efficiency is （89.7±1.7）%. MTT assay shows the growth of C6 Glioma Cells can be significantly inhibited by Rg3-NPs in a dose-dependence manner. FCM and Western blot analysis show Rg3 can be released from the conjugated nanoparticles to function in the cell nuclei so as to lead to the changes in the growth cycle of the cells, which results in the arrest of G0-G1 cell cycle and induces the apoptosis of C6 cells. Therefore, Rg3-NPs may be used for the auxiliary therapy of brain glioma.

Engineering of triterpene metabolism and overexpression of the lignin biosynthesis gene PAL promotes ginsenoside Rg3 accumulation in ginseng plant chassis

The ginsenoside Rgfound in Panax species has extensive pharmacological properties,in particular anti-cancer effects.However,its natural yield in Panax plants is limited.Here,we report a multimodular strategy to improve yields of Rgin a Panax ginseng chassis,combining engineering of triterpene metabolism and overexpression of a lignin biosynthesis gene,phenylalanine ammonia lyase(PAL).We first performed semi-rational design and site mutagenesis to improve the enzymatic efficiency of Pq3-O-UGT2,a glycosyltransferase that directly catalyzes the biosynthesis of Rgfrom Rh.Next,we used clustered regularly interspaced palindromic repeats(CRISPR)/CRISPR-associated protein 9(Cas9)gene editing to knock down the branch pathway of protopanaxatriol-type ginsenoside biosynthesis to enhance the metabolic flux of the protopanaxadiol-type ginsenoside Rg.Overexpression of PAL accelerated the formation of the xylem structure,significantly improving ginsenoside Rgaccumulation(to 6.19-fold higher than in thecontrol).Wecombinedoverexpression of the ginsenoside aglycon synthetic genes squalene epoxidase,Pq3-O-UGT2,and PAL with CRISPR/Cas9-based knockdown of CYP716A53v2 to improve ginsenoside Rgaccumulation.Finally,we produced ginsenoside Rgat a yield of 83.6 mg/L in a shake flask(7.0 mg/g dry weight,21.12-fold higher than with wild-type cultures).The highproduction system established in this study could be a potential platform to produce the ginsenoside Rgcommercially for pharmaceutical use.

Ginsenoside Rg3 inhibits colon cancer cell migration by suppressing nuclear factor kappa B activity

OBJECTIVE: To study the mechanism of the inhibitory effect of ginsenoside Rg3 on colon cancer cell migration.METHODS: Transwell migration assays were performed to investigate the inhibitory effect of ginsenoside Rg3 on SW480 cell migration. Electrophoretic mobility shift assays(EMSAs) and dual luciferase reporter assays were used to study the suppression capability of Rg3 on nuclear factor kappa B(NF-κB) activity. Western blotting was adopted to determine protein levels.RESULTS: Two-hundred micromolar ginsenoside Rg3 significantly inhibited SW480 cell migration(P < 0.05). EMSA showed that Rg3 suppressed the DNA binding ability of NF-κB. Dual luciferase reporter assay showed that Rg3 decreased NF-κB-regulated gene transcription(P < 0.01). Western blots indicated that Rg3 down-regulated expression of the NF-κB-regulated matrix metalloproteinase 9,cyclooxygenase-2 and C-Myc. An NF-κB inhibitor,pyrrolidine dithiocarbamate,enhanced the inhibitory effect of Rg3 on SW480 cell migration.CONCLUSION: Ginsenoside Rg3 has a strong antitumor migration capability by suppressing NF-κB activity and expression of NF-κB-regulated gene products. It could be a good adjuvant for colon cancer patients during the course of chemotherapy.

A novel preparation of Panax japonicas with high bioequivalence of ginsenoside Rg3

In the present study,we aimed to prepare Panax japonicus tablets and carry out quality inspection.Panax japonicus tablets were prepared by ultrafine pulverization-wet granulation,and quality inspection was carried out according to Pharmacopoeia regulations.The plasma concentration of animals with self-made Panax japonicus tablets or ginsenoside Rg3 in single-dose intragastric administration was determined by high-performance liquid chromatography(HPLC).The pharmacokinetic parameters and relative bioavailability were calculated by DAS 2.0 software.The quality inspection of self-made Panax japonicus tablets met the requirements of Chinese Pharmacopoeia(2015 edition),and this preparation had high bioequivalence of ginsenoside Rg3.The preparation of Panax japonicus tablets was reasonable and highly qualified.Moreover,this new Panax japonicus preparation showed better profiles in oral absorption and utilization.This study provided evidence for the industrial production and clinical application of Panax japonicus tablets.

Investigation on the mechanism of ginsenoside Rg3 in treating murine primary mammary tumor

A murine primary mammary tumor model was established to investigate the treatment with ginsenosides Rg3.The relationship between ginsenosides Rg3 and primary mammary tumor was explored.Mammary tumor was induced by using the 7,12-dimethybenz(a)anthracene(DMBA).Ginsenoside Rg3 was employed for treatment.The incidence of mammary tumor in every group was compared,and the expressions of vascular endothelial growth factor(VEGF)and microvessel density(MVD)were detected by immunohistochemical method.The cell cycle and apoptosis percentage were determined by means offlow cytometry.The incidence of tumor in treatment group was significantly lower than that in control group(60.00%vs 33.33%,P<0.05).The average diameter of mammary tumor was(0.86�0.27)cm in control group and(0.39�0.09)cm in treatment group,with the difference being significant between control and treatment groups(P<0.01).The MVD value was(31.9�5.3)in control group and(20.1�4.9)in treatment group,respectively.There was a significant difference between the two groups(P<0.05).The apoptosis percentage in control group was significantly lower than that in treatment group[(2.47�0.69)%vs(5.67�0.99)%,P<0.05].Ginsenoside Rg3 can play an antitumor role in primary mammary tumor model by inhibiting angiogenesis,cell cycle progression,and promoting cell apoptosis.