Mechanism of gypenosides of Gynostemma pentaphyllum inducing apoptosis of renal cell carcinoma by PI3K/AKT/mTOR pathway.
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.
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.
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.
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.
Gypenosides induced apoptosis of RCC cells by modulating PI3K/Akt/mTOR signaling pathway.
Liu H
，Li X
，Duan Y
，Xie JB
，Piao XL
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A network pharmacology approach and experimental validation to investigate the anticancer mechanism and potential active targets of ethanol extract of Wei-Tong-Xin against colorectal cancer through induction of apoptosis via PI3K/AKT signaling pathway.
Wei-Tong-Xin (WTX), derives from the Chinese herbal decoction (CHD) of Wan-Ying-Yuan in ancient China, has been shown to be effective therapeutic herbal decoction for treating gastrointestinal diseases. Present studies have demonstrated that WTX had potential to alleviate the symptoms of gastrointestinal inflammation, gastric ulcer and improve gastric motility.
The study primarily focused on exploring the therapeutic effect and possible pharmacological mechanism of WTX on colorectal cancer (CRC) based on network pharmacology, in vitro and in vivo experiments.
Firstly, colorectal cancer and WTX associated with targets were searched from GeneCards database and TCM Systems Pharmacology Database and Analysis Platform (TCMSP) respectively. The protein-protein interaction (PPI) network also was constructed to screening key targets. In addition, the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were applied to predict the underlying biological function and mechanism involving in the anti-colorectal cancer effect of WTX. Next, CCK-8, colony formation and transwell assays were performed to verify the influence of proliferation and metastasizing ability of HCT116 cells after treated with WTX. Cell cycle, apoptosis and reactive oxygen species (ROS) were analysis by flow cytometry. Hoechst 33258 staining was conducted to observe nuclear morphology changes. Protein expression of apoptosis and PI3K/AKT signaling as well as mRNA expression of ferroptosis and apoptosis were determined by Western Blotting and RT-qPCR. The effects of WTX and LY294002 combination on the PI3K/Akt/mTOR signaling pathway were measured by Western Blotting. Finally, the xenograft tumor mouse model was established by subcutaneous injection of CT26 cells to measure tumors volume and weight. Hematoxylin and eosin (HE) staining and immunohistochemical analysis were used to observe the pathological changes and the protein expression in tumor tissues.
There were 286 potential treatment targets from 130 bioactive compounds in WTX, 1349 CRC-related targets were identified. Eleven core targets (TP53, AKT1, STAT3, JUN, TNF, HSP90AA1, IL-6, MAPK3, CASP3, EGFR, MYC) were found by PPI network analysis constructed of 142 common targets. The results of KEGG enrichment displayed PI3K/AKT signaling pathway as core pathway. After the treatment of WTX, the inhibitory of viability, metastases and cell cycle arrest at G2/M phase were observed in HCT116 cells. Moreover, WTX induced an increase in the expression of apoptosis proteins (Bak, cytochrome c, cleaved caspase-9/caspase-9 and cleaved caspase-3/caspase-3) and the levels of ROS and MDA, a decrease in the expression of PI3K/AKT signaling related proteins (PI3K, p-PI3K, p-AKT/AKT and p-mTOR/mTOR) and the level of SOD. WTX treatment significantly reduced the tumor weight, increased cleaved caspase-3 positive area and decreased that of ki67 in xenograft mouse model.
Through a network pharmacology approach and in vitro experiments, we predicted and verified the effect of WTX on colorectal cancer cells mainly depended on the regulation of intrinsic apoptosis via PI3K/AKT signaling pathway, and further animal experiments proved that WTX has a good anti-colon cancer effect in vivo.
Lin F
，Zhang G
，Yang X
，Wang M
，Wang R
，Wan M
，Wang J
，Wu B
，Yan T
，Jia Y
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Scutellarin inhibits human renal cancer cell proliferation and migration via upregulation of PTEN.
Scutellarin is a naturally flavone glycoside that has been shown to exhibit anti-proliferative and anti-apoptotic activities among various human malignancies. However, the anti-cancer effect of Scutellarin in Renal cell carcinoma (RCC) and the underlying mechanism remains unclear.
RCC cell lines ACHN and 786-O were treated with different concentrations (0-210 μM) of Scutellarin in vitro. Cell viability and proliferation were investigated by MTT and colony formation assays. Cell invasion and migration were detected by Transwell assays. Cell apoptosis and cell cycle distribution was measured by flow cytometry. Western blot was used to investigate the expression levels of crucial proteins. Xenograft tumor model was established to evaluate tumor growth in vivo.
Scutellarin significantly inhibited RCC cell proliferation in a dose- and time- dependent manner. Treatment of RCC cells with Scutellarin (30, 60, and 90 μM) markedly induced apoptosis and cell cycle arrested at G0/G1 phase in a concentration-dependent characteristic. Cell invasion and migration capacities of RCC cells were also dose-dependently suppressed by Scutellarin treatment. Western blot assays revealed that the crucial proteins including cyclin D1, CDK2, Bcl2, MMP-2, and MMP-9 were significantly reduced while Bax, cleaved caspase 3 and p21 were increased by Scutellarin in RCC cells. In vivo assay indicated that Scutellarin possessed anti-cancer effect on xenograft without triggering toxic effect. Mechanically, Scutellarin dramatically increased the protein level of phosphatase and tensin homologue (PTEN) and inhibited the activity of P13K/AKT/mTOR signaling. Ectopic expression of PTEN enhanced the inhibitory effect of Scutellarin on RCC proliferation while knockdown of PTEN abrogated it through regulating its downstream P13K/AKT/mTOR signaling pathway.
Scutellarin inhibited RCC cell proliferation and invasion partially by enhancing the expression of PTEN through inhibition of P13K/AKT/mTOR pathway, suggesting that Scutellarin might serve as a potential therapeutic agent in RCC treatment.
Deng W
，Han W
，Fan T
，Wang X
，Cheng Z
，Wan B
，Chen J
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Apoptosis induced by the methanol extract of Salvia miltiorrhiza Bunge in non-small cell lung cancer through PTEN-mediated inhibition of PI3K/Akt pathway.
Salvia miltiorrhiza Bunge, a well-known traditional Chinese medicinal (TCM) plant, has been used to treat cardiovascular diseases since thousands of years. Many studies reported that the active component tanshinones displayed a variety of biological activities: anti-thrombous, anti-allergic, anti-inflammatory, antioxidant and anti-tumor promoting. But the mechanism of how the active components working still need to be clarified. The anti-tumor effect of compounds of tanshinone (CTN), the methanol extract of Salvia miltiorrhiza Bunge roots, was investigated. The aim of this study was to investigate the effects of CTN on the growth inhibition, apoptosis and molecular targets of human non-small cell lung cancer (NSCLC).
CTN-induced cytotoxicity was determined by MTT assay. The cell survival was evaluated using clonogenic survival assay. The morphology of Glc-82 cells after treatment with CTN was determined by fluorescence microscopy. Cell cycle distribution was revealed by flow cytometry. The apoptotic cells were quantified with annexin V-FITC/PI staining and flow cytometry, and observed using Hoechst 33258 staining and TUNEL assays. The expression levels of proteins were analyzed using western blot. Tumor growth was assessed by subcutaneous inoculation of cells into BALB/c nude mice.
CTN inhibited the proliferation of NSCLC in a dose-dependent manner and induced both early and late apoptosis. Treatment of Glc-82 cells with CTN (5-80μg/ml) significantly (p<0.05) suppressed the cell proliferation in a concentration and time-dependent manner. CTN induced significant (p<0.05) and dose-dependent apoptosis of Glc-82 cells. Cell cycle assay showed that CTN induced a G2/M phase arrest, and significantly (p<0.05) increased expression of p53 and p21, actived caspase-3/9 and PARP1, which suggest the involvement of the mitochondria in the apoptotic signals. In addition, CTN decreased expression of the anti-apoptotic protein Bcl-2, Bcl-xl and increased expression of the pro-apoptotic protein Bax. Result also showed that CTN could increase expression levels of PTEN, and reduce the phosphorylated levels of Akt (protein kinase B) on Thr 308 and Ser 473 domain. In vivo assay showed that the antitumor effect of CTN was significantly augmented without increasing toxicity in nude mice bearing Glc-82 xenograft.
The PTEN/Akt signaling axis is defined as a critical pathway regulated by PTEN in NSCLC. CTN, the methanol extract of Salvia miltiorrhiza Bunge, are the active compounds as shown by their ability to induce apoptosis through the mitochondrial pathway of apoptosis and PTEN-mediated inhibition of PI3K/Akt pathway. CTN could inhibit tumor growth more efficiently, which supports the ethno-medicinal use of this herb as an alternative or complementary therapy for NSCLC.
Ye YT
，Zhong W
，Sun P
，Wang D
，Wang C
，Hu LM
，Qian JQ
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ResearchGate
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