This study aims to investigate the molecular mechanism of polyphyllin Ⅰ(PPⅠ) inhibiting proliferation of human breast cancer cells. Human breast cancer BT474 and MDA-MB-436 cells were treated with different concentrations of PPⅠ, and then the effect of PPⅠ on cell proliferation was detected by MTT assay, trypan blue dye exclusion assay, real-time cell analysis, and clone forming assay, respectively. The apoptosis was detected by Annexin V-FITC/PI staining and then analyzed by flow cytometry. The change of mitochondrial membrane potential was detected by flow cytometry after fluorescent probe JC-1 staining. Western blot was used to detect protein expression and phosphorylation. Molecular docking was performed to detect the binding between PPⅠ and EGFR. The affinity between PPⅠ and EGFR was determined by drug affinity responsive target stability assay. The results indicated that PPⅠ inhibited the proliferation and colony formation of BT474 and MDA-MB-436 cells in a time-and concentration-dependent manner. The PPⅠ treatment group showed significantly increased apoptosis rate and significantly decreased mitochondrial membrane potential. PPⅠ down-regulated the expression of pro-caspase-3 protein, promoted the cleavage of PARP, and significantly reduced the phosphorylation levels of EGFR, Akt, and ERK. Molecular docking showed that PPⅠ bound to the extracellular domain of EGFR and formed hydrogen bond with Gln366 residue. Drug affinity responsive target stability assay confirmed that PPⅠ significantly prevented pronase from hydrolyzing EGFR, indicating that PPⅠ and EGFR have a direct binding effect. In conclusion, PPⅠ inhibited the proliferation and induced apoptosis of breast cancer cells by targeting EGFR to block its downstream signaling pathway. This study lays a foundation for the further development of PPⅠ-targeted drugs against breast cancer.
Apoptosis ; Breast Neoplasms/genetics* ; Cell Line, Tumor ; Cell Proliferation ; Diosgenin/analogs & derivatives* ; ErbB Receptors ; Female ; Humans ; Molecular Docking Simulation

ObjectiveTo explore the inhibitory effect of polyphyllin Ⅰ (PPⅠ) on the proliferation of castration-resistant prostate cancer PC3 cells and its molecular mechanism.

METHODSWe cultured human prostate cancer PC3 cells in vitro and treated them with PPⅠ at the concentrations of 0 (blank group), 0.4, 0.8, 1.2, 1.6, 2.0, and 2.4 μmol/L for 24, 48, and 72 hours, respectively. Then we detected the proliferation of the cells by MTT assay, measured their apoptosis by flow cytometry, and determined the expressions of p-ERK1/2, ERK1/2, NF-κB/p65 and DNMT1 proteins as well as the level of NF-κB/p65 in the cells additionally treated with the ERK1/2 inhibitor SP600125 by Western blot.

RESULTSCompared with the blank control group, the PPⅠ-treated PC3 cells showed a concentration- and time-dependent reduction of the survival rate (1.00 ± 0.00 vs 0.85 ± 0.05, P < 0.01) at 0.4 μmol/L after 48 hours of intervention, concentration-dependent early apoptosis at 0.8 μmol/L (4.83 ± 0.95 vs 13.83 ± 2.97, P < 0.01), time-dependent increase of the expressions of p-ERK1/2 (1.00 ± 0.00 vs 1.73 ± 0.17, P < 0.01) and ERK1/2 (1.00 ± 0.00 vs 1.36 ± 0.12, P < 0.01) at 2 hours, and concentration-dependent decrease of the expressions of NF-κB/p65 and DNMT1 at 1.2 μmol/L (1.00 ± 0.00 vs 0.78 ± 0.10 and 0.63 ± 0.06, P < 0.01) and 1.6 μmol/L (1.00 ± 0.00 vs 0.67 ± 0.11 and 0.52 ± 0.09, P<0.01). Inhibition of ERK1/2 phosphorylation with PD98059 markedly reversed PPⅠ-induced decrease of the NF-κB/p65 expression as compared with that in the PPⅠ group (0.86 ± 0.18 vs 0.43 ± 0.09, P < 0.05).

CONCLUSIONSPPⅠ induces the early apoptosis and suppresses the proliferation of PC3 cells, probably by activating the ERK1/2 pathway and inhibiting the expressions of the NF-κB/p65 and DNMT1 proteins.

Apoptosis ; Cell Proliferation ; drug effects ; DNA (Cytosine-5-)-Methyltransferase 1 ; metabolism ; Diosgenin ; analogs & derivatives ; pharmacology ; Flavonoids ; metabolism ; Humans ; MAP Kinase Signaling System ; Male ; Mitogen-Activated Protein Kinase 1 ; metabolism ; Mitogen-Activated Protein Kinase 3 ; metabolism ; NF-kappa B ; metabolism ; PC-3 Cells ; Phosphorylation ; Prostatic Neoplasms, Castration-Resistant ; drug therapy ; metabolism ; pathology ; Signal Transduction ; Transcription Factor RelA ; metabolism

Dioscin, a typical saponin, is widely present in the family of Dioscoreaceae, Liliaceae, Caryophyllaceae and Rosaceae, especially in Dioscoreaceae, including Discorea nipponica Makino, Dioscorea zingiberensis C. H. Wright and Dioscorea panthaica Prain et Burkill. Traditional Chinese medicine reported that dioscin plays a role in expectorant, relaxing the muscles and stimulating the blood circulation, aiding digestion and diuresis. With the development of science and technology in recent years, some new extraction and separation techniques and methods have been applied to the study of dioscin, and more and more pharmacological effects were found. Modern pharmacology studies have confirmed that dioscin had some activities on desensitization, anti-inflammatory, lipid-lowering, anti-tumor, hepatoprotection and anti-viral. After oral administration, dioscin is metabolized to diosgenin, which is the true active ingredient and is an important raw material to synthesize steroid hormone drugs. Therefore, the studies on dioscin are valueable and promising. In this review, we make a summary on the researches of dioscin including the extraction technology, separation and prepara- tion, chemical synthesis, drug metabolism, determination and pharmacological researches.
Animals ; Biological Products ; adverse effects ; chemistry ; pharmacology ; Diosgenin ; adverse effects ; analogs & derivatives ; chemistry ; pharmacology ; Humans ; Plant Extracts ; adverse effects ; chemistry ; pharmacology

Dioscin has a wide range of biological effects and broad application prospects. However the studies concerning the toxicology and mechanism of dioscin is small. This article is to study the hepatotoxicity of dioscin and the effect of dioscin treatment on expression of aryl hydrocarbon receptor (AhR) mRNA and CYP1A mRNA and protein in HepG2 cells in vitro. Dioscin 0.5-32 µmol · L(-1) exposed to HepG2 cells for 12 h, cell viability was examined by CCK-8 assay and the release rate of lactate dehydrogenase (LDH) was to evaluate cell membrane damage. HepG2 cells morphologic changes were quantified by inverted Microscope, and the effect on production of reactive oxygen species (ROS) was detected by flow cytometry. The mRNA expression of CYP1A and AhR was evaluated by RT-RCR. The protein expression of CYP1A1 was detected by western blot. The cell viability was significantly inhibited after HepG2 cells were exposed to dioscin 0.5-32 µmol · L(-1). Compared with the control, the LDH release rate and ROS were significantly increased. The expression of CYPlA and AhR mRNA was increased. The expression of CYP1Al protein was increased after dioscin treatment, and resveratrol, an AhR antagonist, could downregulate the expression of CYP1A1. It follows that large doses dioscin has potential hepatotoxicity. The possible mechanism may be dioscin can active aryl hydrocarbon receptor (AhR) and induce the expression of CYP1A.
Cell Survival ; drug effects ; Chemical and Drug Induced Liver Injury ; etiology ; Cytochrome P-450 CYP1A1 ; genetics ; Diosgenin ; analogs & derivatives ; toxicity ; Hep G2 Cells ; Humans ; L-Lactate Dehydrogenase ; secretion ; RNA, Messenger ; analysis ; Reactive Oxygen Species ; metabolism ; Receptors, Aryl Hydrocarbon ; genetics

To breed a new yam cultivar of Dioscorea alata, the different and excellent germplasm resources were investigated within artificially cultivated population and some superior individuals, with a higher yield and medicinal properties, were selected. Considering results of the yield and medicinal properties during 2006-2013 cropping season, strains and lines were established and selected. As a result, the yield of the new developed cultivar (Wenshanyao No. 1, WSY01-1) reached 2217. 0 kg per 667 m2 (fresh weight) and 348.3 kg per 667 m2 (dry weight), and increased 23.8% and 23.9% comparing with control cultivars (landraces). Comparing with control cultivars, the level of polysaccharide, allantoin, and dioscin increased 36.9%, 48.3%, 20.9%, and reached 12.2%, 1.30%, 579.7 µg · g(-1), respectively. This result showed that the systematic selection method can significantly improve yield and medicinal properties of D. alata, and the developed " Wenshanyao No. 1" exhibits wide spreading prospects.
Allantoin ; analysis ; Breeding ; Dioscorea ; chemistry ; genetics ; growth & development ; Diosgenin ; analogs & derivatives ; analysis ; Polysaccharides ; analysis

OBJECTIVEThe aim of this study was to observe the effects of dioscin on apoptosis and on expression of PRDX1 in pancreatic cancer MiaPaCa-2 cells in vitro.

METHODSMTT assay was used to detect the growth rate among the medication groups treated with different concentrations of dioscin. The apoptosis rate was determined by annexin V-fluorescein isothiocyanate/propidium iodide double staining and flow cytometry. Western blot analysis was used to assay the expression of PRDX1 and apoptotic proteins in the cells. Reactive oxygen species (ROS) formation was measured by 2'7'-dichlorofluorescein diacetate (DCFH-DA).

RESULTSDioscin considerably inhibited the proliferation of MiaPaCa-2 cells in vitro. The inhibitory action was enhanced in a dose-dependent manner. The levels of intracellular ROS detected with DCFH-DA were highly increased after dioscin treatment. The flow cytometry analysis using annexin V-PI staining showed that compared with the apoptotic rate of control group [(3.5 ± 0.7)%], 2.5 µmol/L and 5 µmol/L dioscin induced apoptosis in (28.4 ± 0.9)% and (49.6 ± 2.7)% MiaPaCa-2 cells, and Western blot analysis showed that apoptotic proteins Bax and cleaved caspase-3 expressions were increased and antiapoptotic protein Bcl-2 expression was decreased. In addition, these effects could be blocked by antioxidant N-acetylcysteine (NAC) administration, and the apoptotic rates decreased to (10.8 ± 2.3)% and (18.8 ± 3.0)%, respectively. We further observed the decrease of PRDX1 expression after dioscin treatment. Moreover, after PRDX1 overexpression, dioscin treatment no longer induced high levels of ROS and apoptosis, and the apoptotic rate was decreased to (21.3 ± 5.9)%.

CONCLUSIONDioscin can down-regulate the PRDX1 expression, and then induces ROS-mediated apoptosis in cancer cells.

Apoptosis ; drug effects ; Diosgenin ; analogs & derivatives ; pharmacology ; Humans ; Pancreatic Neoplasms ; pathology ; Peroxiredoxins ; drug effects ; Reactive Oxygen Species ; metabolism

Dioscin is a natural steroid saponin derived from several plants, showing potent anti-cancer effect against a variety of tumor cell lines. In the present study, we investigated the anti-cancer activity of dioscin against human LNCaP cells, and evaluated the possible mechanism involved in its antineoplastic action. It was found that dioscin (1, 2 and 4 μmol/L) could significantly inhibit the viability of LNCaP cells in a time- and concentration-dependent manner. Flow cytometry revealed that the apoptosis rate was increased after treatment of LNCaP cells with dioscin for 24 h, indicating that apoptosis was an important mechanism by which dioscin inhibited cancer. Western blotting was employed to detect the expression of caspase-3, Bcl-2 and Bax in LNCaP cells. The expression of cleaved caspase-3 was significantly increased, and meanwhile procaspase-3 was markedly decreased. The expression of anti-apoptotic protein Bcl-2 was down-regulated, whereas the pro-apoptotic protein Bax was up-regulated. Moreover, the Bcl-2/Bax ratio was drastically decreased. These results suggested that dioscin possessed potential anti-tumor activity in human LNCaP cells through the apoptosis pathway, which might be associated with caspase-3 and Bcl-2 protein family.
Apoptosis ; drug effects ; Blotting, Western ; Caspase 3 ; metabolism ; Cell Line, Tumor ; Cell Survival ; drug effects ; Diosgenin ; analogs & derivatives ; chemistry ; pharmacology ; Dose-Response Relationship, Drug ; Enzyme Activation ; drug effects ; Flow Cytometry ; Humans ; Male ; Molecular Structure ; Prostatic Neoplasms ; metabolism ; pathology ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Time Factors ; bcl-2-Associated X Protein ; metabolism

OBJECTIVETo observe the effects of Dioscornin Tablet (DT) containing serum on nuclear factor of kappa B (NF-kappaB) p65, signal transducer and activator of transcription 3 (STAT3), and vascular endothelial growth factor (VEGF) mRNA expressions in rats' synovial cell strain 364 (RSC-364) induced by interleukin-17 (IL-17) and tumor necrosis factor-alpha (TNF-alpha), and to investigate the underlying mechanisms for DT to inhibit angiogenesis of rheumatoid arthritis (RA).

METHODSIn this experiment, the vehicle control group, the cell model group, the DT containing serum group, and the positive control group (Tripterygium containing serum) were set up. The DT containing serum and the Tripterygium containing serum were prepared. The RA cell model was established by IL-17 combined TNF-alpha induced injury in RSC-364. The RA cells were intervened by DT containing serum and Tripterygium containing serum respectively. The DNA binding activity of NF-kappaB p65 was detected using TransAM NF-kappaB p65. The expression of STAT3 was observed using Western blot. The VEGF mRNA expressions were detected by real-time quantitative PCR.

RESULTSCompared with the vehicle control group, the NF-kappaB p65 activity, the expressions of STAT3 and VEGF mRNA increased significantly in RSC-364 induced by IL-17 +TNF-alpha (P < 0.01, P < 0.05). Compared with the model group, the NF-kappaB p65 activity, the expressions of STAT3 and VEGF mRNA decreased significantly in the DT containing serum group and the positive control group (P < 0.01, P < 0.05). There was no statistical difference between the two groups (P > 0.05).

CONCLUSIONDT inhibited the VEGF mRNA expression through inhibiting the NF-kappaB p65 activity and the STAT3 protein expression in the Janus kinase (JAK)-signal transducer and activating transcription factor pathway, thus inhibiting the angiogenesis of RA.

Animals ; Arthritis, Rheumatoid ; pathology ; Cells, Cultured ; Diosgenin ; analogs & derivatives ; pharmacology ; Interleukin-17 ; adverse effects ; Male ; Neovascularization, Pathologic ; pathology ; RNA, Messenger ; pharmacology ; Rats ; Rats, Wistar ; STAT3 Transcription Factor ; metabolism ; Serum ; Signal Transduction ; Synovial Membrane ; cytology ; drug effects ; metabolism ; Transcription Factor RelA ; metabolism ; Tumor Necrosis Factor-alpha ; adverse effects ; Vascular Endothelial Growth Factor A ; metabolism

Diosgenin can inhibit the growth of A375 and K562 cell lines and induce their apoptosis with an effect on pro-apoptotic members of Bcl-2 family. To study the SAR of diosgenin derivatives, and to improve the anti-tumor activity of diosgenin, a series of novel diosgenin derivatives were designed and synthesized. Their anti-tumor activities in vitro were evaluated. The results revealed that most of the new derivatives had potent effects against K562, A375 and A549 (three tumor cell lines) in vitro, and had no or less effect against H293 and L02 (two normal cell lines). Particularly, some compounds (e.g. 1, 6-8) showed excellent activities on K562 with IC50 values ranging from 1.96 to 4.35 micromol x L(-1).
Antineoplastic Agents ; chemical synthesis ; chemistry ; pharmacology ; Cell Line, Tumor ; Diosgenin ; analogs & derivatives ; chemical synthesis ; chemistry ; pharmacology ; Drug Design ; Humans

The purpose of this paper is to clarify the structure-activity relationship of anti-tumor activity of diosgenin derivatives in vitro. Study has found that diosgenin can inhibit the reproduction of tumor cells by inducing apoptosis and the main target spot of this effect is Bcl-2. Based on the characteristics of pharmacophoric points' of the three-dimensional pharmacophore for Bcl-2 inhibitors, we have docked lots of diosgenin derivatives with Bcl-2, then synthesized 31 compounds of them, finally assessed the anti-tumor activity of the diosgenin derivatives in vitro against A375, A549, HepG-2 and K562. Preliminary studies of SAR have indicated that the aliphatic esters, and aromatic esters of diosgenin without F ring have no anti-tumor activity in vitro. The triazole bromides of diosgenin all achieve fairly good anti-tumor activity in vitro, and those with larger hydrophobic group have the better activity. The stronger is the hydrogen bonding interaction and dipole-dipole interaction of the heterocyclic of diosgenin and diosgenin without F ring and the acid ester of diosgenin without F ring, the better is the activity of derivatives.
Antineoplastic Agents, Phytogenic ; chemical synthesis ; chemistry ; pharmacology ; Apoptosis ; drug effects ; Cell Line, Tumor ; Diosgenin ; analogs & derivatives ; chemical synthesis ; chemistry ; pharmacology ; Humans ; Proto-Oncogene Proteins c-bcl-2 ; antagonists & inhibitors ; Structure-Activity Relationship