Gypenosides suppress hepatocellular carcinoma cells by blocking cholesterol biosynthesis through inhibition of MVA pathway enzyme HMGCS1.

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors with high morbidity and mortality. Targeting abnormal cholesterol metabolism is a potential therapeutic direction. Therefore, more natural drugs targeting cholesterol in HCC need to be developed. Gypenosides (Gyp), the major constituent of Gynostemma pentaphyllum, has been demonstrated to have pharmacological properties on anti-cancer, anti-obesity, and hepatoprotective. We investigated whether Gyp, isolated and purified by our lab, could inhibit HCC progression by inhibiting cholesterol synthesis. The present research showed that Gyp inhibited proliferation and migration, and induced apoptosis in Huh-7 and Hep3B cells. Metabolomics, transcriptomics, and target prediction all suggested that lipid metabolism and cholesterol biosynthesis were the mechanisms of Gyp. Gyp could limit the production of cholesterol and target HMGCS1, the cholesterol synthesis-related protein. Downregulation of HMGCS1 could suppress the progression and abnormal cholesterol metabolism of HCC. In terms of mechanism, Gyp suppressed mevalonate (MVA) pathway mediated cholesterol synthesis by inhibiting HMGCS1 transcription factor SREBP2. And the high expression of HMGCS1 in HCC human specimens was correlated with poor clinical prognosis. The data suggested that Gyp could be a promising cholesterol-lowering drug for the prevention and treatment of HCC. And targeting SREBP2-HMGCS1 axis in MVA pathway might be an effective HCC therapeutic strategy.

Comprehensive serum metabolomics and network analysis to reveal the mechanism of gypenosides in treating lung cancer and enhancing the pharmacological effects of cisplatin.

Gypenosides (GYP) exerted anticancer activity against various cancers. However, the mechanism of GYP against lung cancer (LC) in vivo remains unclear. This study aims to reveal the potential mechanism of GYP against LC and enhancing cisplatin efficacy using a comprehensive analysis of metabolomics, network analysis. Pharmacodynamic results showed that GYP inhibited tumor growth, reduced tumor volume and tumor weight, and alleviated pathological symptoms in Lewis tumor-bearing mice, and GYP could enhance the anti-LC effects of cisplatin. Using serum metabolomics methods, 53 metabolites were found to be significantly altered in the model group, and the levels of 23 biomarkers were significantly restored after GYP treatment. GYP-related metabolic pathways involved six pathways, including alpha-linolenic acid metabolism, glutathione metabolism, sphingolipid metabolism, glycerophospholipid metabolism, tryptophan metabolism, and primary bile acid biosynthesis. 57 genes associated with differential metabolites of GYP recovery and 7 genes of 11 saponins of GYP against LC were screened by network analysis, the STRING database was used to find the association between 57 genes and 7 genes, and a compound-intersection gene-metabolite related gene-metabolite-pathway network was constructed, and STAT3, MAPK14, EGFR and TYMS might be the crucial targets of GYP against LC. Western blot results showed that GYP restored the levels of STA3, MAPK14, EGFR, and TYMS in the model group, and GYP also restored the levels of STAT3 and MAPK14 in the cisplatin group, indicating that GYP might exert anti-LC effects and enhance the pharmacological effects of cisplatin through MAPK14/STAT3 signaling pathway. Our method revealed the effect and mechanism of GYP on LC and the pharmacological effects of GYP-enhanced chemotherapeutic agent cisplatin, which provided some reference for the development of anti-cancer drugs.

Uncovering the anti-NSCLC effects and mechanisms of gypenosides by metabolomics and network pharmacology analysis.

ETHNOPHARMACOLOGICAL RELEVANCE: Lung cancer is the chief reason of cancer death worldwide, and non-small cell lung cancer (NSCLC) make up the majority of lung cancers. Gypenosides are the main active constituents from Gynostemma pentaphyllum. Previous studies showed that they were used to remedy many cancers. The effect of gypenosides on NSCLC has never been studied from the perspective of network pharmacology and metabolomics. The mechanism is still not clear and remains to be explored. AIM OF THE STUDY: To explore the anti-NSCLC activity and mechanism of gypenosides in A549 cells. MATERIAL/METHODS: Gypenosides of G. pentaphyllum were detected by HPLC-MS. The cytotoxicity was detected by MTT assay. The migration, cell cycle and apoptosis of gypenosides were studied by wound healing assay, JC-1 assay and flow cytometry. The mechanism of gypenosides on NSCLC was studied by metabolomics and network pharmacology. Some key proteins and pathways were further confirmed by Western blot. RESULTS: Eleven gypenosides were detected by HPLC-MS. Gypenosides could suppress the proliferation of A549 cells, inhibit the migration of A549 cells, induce apoptosis and arrest cell cycle in G0/G1 phase. Metabolomics and network pharmacology approach revealed that gypenosides might affect 17 metabolite related proteins by acting on 9 candidate targets (STAT3, VEGFA, EGFR, MMP9, IL2, TYMS, FGF2, HPSE, LGALS3), thus resulting in the changes of two metabolites (uridine 5'-monophosphate, D-4'-Phosphopantothenate) and two metabolic pathways (pyrimidine metabolism; pantothenate and CoA biosynthesis). Western blotting indicated that gypenosides might inhibit A549 cells through MMP9, STAT3 and TYMS to indirectly affect the pathways of pyrimidine metabolism, pantothenate and CoA biosynthesis. CONCLUSIONS: This study revealed that metabolomics combined with network pharmacology was conducive to understand the anti-NSCLC mechanism of gypenosides.

Gypenoside LI arrests the cell cycle of breast cancer in G0/G1 phase by down-regulating E2F1.

ETHNOPHARMACOLOGICAL RELEVANCE: Gynostemma pentaphyllum (Thunb.) Makino, a traditional medicine in China, has been widely used for the treatment of various diseases. Gypenoside LI (Gyp LI) is a major constituent from steamed G. pentaphyllum. Previous studies have shown that gypnenoside LI possess inhibitory effect on the growth of many cancer cells. However, its pharmacological effect in breast cancer and the mechanism have not been reported yet. AIM OF THE STUDY: To investigate the anti-breast cancer activity of gypenoside LI and underlying mechanisms of gypenoside LI in MDA-MB-231 and MCF-7 cells. MATERIAL/METHODS: The cytotoxicity of gypenoside LI was determined by MTT, colony-formation and three-dimensional spheroid assay. The migration, cell apoptosis and the cell cycle were investigated through cell morphology observation, flow cytometry analysis and key proteins detection. The anticancer mechanisms of gypenoside LI were detected by RNA sequencing (RNA-seq) and Gene Set Enrichment Analysis (GSEA) transcriptome analysis. RESULTS: Gypenoside LI inhibited cell proliferation, migration, induced cell apoptosis and cell cycle arrest. Gypenoside LI arrested cell cycle at G0/G1 phase by regulating E2F1. It also inhibited tumor proliferation by regulating the expression of ERCC6L. Interestingly, we found that E2F1 siRNA also down-regulated the expression of ERCC6L. Gypenoside LI showed potential anti-breast cancer cells activity, especially on triple-negative breast cancer cells. CONCLUSIONS: These data indicate that gypenoside LI could inhibit human breast cancer cells through inhibiting proliferation and migration, inducing apoptosis, arresting cell cycle at G0/G1 phase by regulating E2F1. It could be used as potential multi-target chemopreventive agents for cancer.

Mechanism of gypenosides of Gynostemma pentaphyllum inducing apoptosis of renal cell carcinoma by PI3K/AKT/mTOR pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Gynostemma pentaphyllum (Thunb.) Makino is a traditional medicine commonly used in China, East Asia and Southeast Asia. In clinic, it is mainly used for hyperlipidemia and antitumor. Its antitumor activity was first recorded in "Illustrated Catalogue of Plants". Gypenosides were the main active ingredients of G. pentaphyllum. The anticancer activity of gypenosides in vivo and in vitro had been widely reported. However, the mechanism of gypenosides in renal cell carcinoma (RCC) still unclear. AIM OF THE STUDY: In this study, we tried to investigate the active constituents from G. pentaphyllum and potential mechanisms in RCC treatment through network pharmacology and in vitro experiments. MATERIAL/METHODS: Active compounds and their targets were evaluated and screened through TCMSP and Swiss Target Prediction database. Notably, nine preliminary screened components obtained from database were identified by LC-MS and LC-MS/MS. The targets associated with RCC were obtained from OMIM, TTD and GeneCards database. The PPI network and active component/target/pathway networks were constructed to identify the potential drug targets using String database and Cytoscape software. The functions and pathways of targets were analyzed through DAVID database. Finally, AutoDockTools 1.5.6 was used for molecular docking to assess the binding ability between compounds and targets. To support our prediction, we then explore the antitumor effect and mechanism of gypenosides by vitro experiments. CCK8 and flow cytometry were performed to evaluate cell death treated with gypenosides. Quantitative real-time PCR and Western blot were conducted to detect the changes of PI3K/AKT/mTOR signaling pathway. RESULTS: Nine saponins and 68 targets have been screened. The hub targets covered PIK3CA, VEGFA, STAT3, JAK2, CCND1 and MAPK3. Enrichment analysis showed that the pathways mainly contained PI3K/Akt/mTOR, HIF-1, TNF, JAK-STAT and MAPK signaling pathways. Gypenosides extracted from G. pentaphyllum showed strong activity against 786-O and Caki-1 cells, and cell apoptosis were detected through Annexin V/PI dual staining assay. RT-qPCR showed that gypenosides downregulated the levels of PIK3CA, Akt and mTOR in Caki-1 and 786-O cells. Mechanistically, gypenosides induced apoptosis of RCC cells through regulating PI3K/Akt/mTOR signaling pathway which was implemented though decreasing the phosphorylation level of Akt and mTOR. CONCLUSIONS: Gypenosides induced apoptosis of RCC cells by modulating PI3K/Akt/mTOR signaling pathway.

Inflammation induced by lipopolysaccharide advanced androgen receptor expression and epithelial-mesenchymal transition progress in prostatitis and prostate cancer.

BACKGROUND: To explore the mechanism of prostatic inflammation on prostate cancer (PCa) by comparing the changes of prostate epithelial cells and PCa cells in an inflammatory environment. METHODS: First, immunohistochemistry (IHC) was used to compare the level of expression of TNF-α, IL-1ß, IL-6, and TGF-ß between benign prostatic hyperplasia (BPH), prostatitis, and PCa. Then primary prostate epithelial cells were sampled from patients who were suspected of PCa and had histological prostatitis (HP) confirmed by pathological biopsy. Lipopolysaccharide (LPS) or BAY11-7082 were used to investigate the change of androgen receptor (AR) and AR-mediated transcription, epithelial-mesenchymal transition (EMT) in primary prostate epithelial cells, and lymph node carcinoma of the prostate (LNCap) cells. RESULTS: TNF-α, IL-1ß, IL-6, and TGF-ß were significantly increased in HP and PCa compared with those in BPH patients. The proliferation of primary prostate epithelial cells and LNCap cells got the inflection point at LPS 10 µg/mL. In an inflammatory environment with 10 µg/mL LPS, both primary prostate epithelial cell and LNCap cell viability increased, and AR, AR-mediated transcription, and EMT processes were significantly increased. Inhibitors of NF-κB with 10 nM BAY11-7082 decreased AR, AR-mediated transcription, and EMT processes. CONCLUSIONS: NF-κB regulates AR expression and EMT in prostatitis and PCa, and NF-κB inhibitors may have potential therapeutic value.

Extraperitoneal laparoscopic adenomectomy (Madigan) versus bipolar transurethral resection of the prostate for benign prostatic hyperplasia greater than 80 ml: complications and functional outcomes after 3-year follow-up.

PURPOSE: To compare the performance of voluminous benign prostatic hyperplasia patients who have received laparoscopic simple prostatectomy (LSP) with the patients who have received bipolar transurethral resection of the prostate (B-TURP) in their perioperative and 3-year follow-up period. METHODS: Ninety patients with prostate volumes >80 mL (range 80-130 mL) were randomly assigned to either LSP or B-TURP surgery type. The patients were followed up at 1, 3, 6, 12, 24, and 36 months postoperatively. Perioperative and follow-up characteristics were then recorded and compared. RESULTS: More blood loss, greater resected adenoma volume, and shorter catheterization duration were recorded in LSP group than that of B-TURP group (140.1±81.5 vs 93.1±54.0 mL; 65.3±13.8 vs 49.0±12.7 mL; 3.3±1.2 vs 3.8±1.0 days; p<0.05). None of the patients in LSP group reported complications out of 30 days, while 1 case of urethral stricture, 36 cases of retrograde ejaculation, 1 case of bladder neck contracture, and 2 cases of recurrence were recorded in B-TURP group. At 1, 3, 6, and 12 months postoperatively, there were no significant differences in terms of postvoid residual urine volume, maximal urinary flow rate (Qmax), and International Prostate Symptom Score between the two groups (p>0.05). In contrast, the differences became significant at 24 and 36 months (p<0.05). CONCLUSIONS: Compared with B-TURP, LSP with Madigan technique is accompanied by less residual adenoma, shorter catheterization time, and more blood loss. Further, the risk of late complications is lower with LSP and, in terms of functional outcomes, LSP appears to be better than B-TURP beyond 2 years.

[Expression of a protein peak(3144 m/z) in serum and its clinical significance in patients with gastric cancer].

OBJECTIVE: To investigate the expression of protein peak (3144 m/z) in serum and of its association with clinical pathological characteristics and prognosis in patients with gastric cancer. METHODS: Three hundred and twenty seven pathologically confirmed gastric cancer patients were recruited from February 2006 to October 2008 in the Zhejiang Cancer Hospital. SELDI-TOF-MS was employed to detect the expression of protein peak (3144 m/z) in preoperative serum. RESULTS: The positive rate of 3144 m/z protein peak was 33.9% (111/327), significantly higher than that of CEA (21.1%,69/327), and the difference was statistically significant (P<0.01). The positive rate of combined detection of protein peak (3144 m/z)and CEA was 45.6% (149/327). The expression of protein peak (3144 m/z) was associated with clinical staging (P<0.01), nervous invasion (P<0.01), tumor size (P<0.01), vascular invasion (P<0.05), lymph node metastasis (P<0.05), expression of CEA (P<0.05), and depth of infiltration (P<0.05). Significant difference was observed in 3-year survival rate between the patients with protein peak and patients without protein peak (44.7% vs. 64.4%, P<0.01). However, 3144 m/z protein peak was not an independent prognostic factor on multivariate Cox regression analysis (P=0.057). CONCLUSION: Protein peak (3144 m/z) may be used as a diagnostic or prognostic marker of gastric cancer.

[Clinicopathologic and prognostic significance of serum levels of cytokines in patients with advanced serous ovarian cancer prior to surgery].

OBJECTIVE: To study the clinicopathologic and prognostic significance of serum levels of six cytokines (IFN-γ, TNF-α, IL-10, IL-5, IL-4, IL-2) in patients with advanced serous ovarian cancer prior to surgery. METHODS: The serum levels of six cytokines were detected in 51 patients with advanced serous ovarian cancer and 46 healthy controls, using cytometric bead arrays. RESULTS: The serum levels of IFN-γ (20.68±11.45), IL-2 (4.54±1.18), IL-4 (5.66±2.25), IL-5 (2.72±0.86) µg/L and IL-10 (5.93±7.92) µg/L were higher (P<0.01, P<0.05) and the serum level of TNF-α (7.53±8.47) was lower (P<0.01) in patients with advanced serous ovarian cancer than those in the healthy controls. The IFN-γ/IL-4 ratio (3.93±2.34) of the patients was lower than that of the controls (P<0.01). Kaplan-Meier analysis revealed that patient's age (P=0.016), menopausal status (P=0.001) and serum IL-10 level (P=0.010) correlated significantly with patient's survival. Cox regression analysis showed that serum IL-2 (P=0.045) and IL-10 levels (P=0.007) were the independent prognostic factors. CONCLUSIONS: Patients with advanced serous ovarian cancer have Th1/Th2 imbalance and immune function disturbance. The age of patients and menopausal status are important prognostic factors. IL-2 and IL-10 level are also independent predictors of survival.