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Polyphyllin I (PPI) purified from Polyphyllarhizomes displays puissant cytotoxicity in many kinds of cancers. Several researches investigated its anti-cancer activity. But novel mechanisms are still worth investigation. This study aimed to explore PPI-induced endoplasmic reticulum (ER) stress as well as the underlying mechanism in non-small cell lung cancer (NSCLC). Cell viability or colony-forming was detected by MTT or crystal violet respectively. Cell cycle, apoptosis, reactive oxygen species (ROS) and mitochondrial membrane potential were assessed by flow cytometry. Gene and protein levels were evaluated by qRT-PCR and immunoblotting respectively. Protein interaction was determined by immunoprecipitation or immunofluorescence assay. Gene overexpression or silencing was carried out by transient transfection with plasmids or small interfering RNAs. The Cancer Genome Atlas (TCGA) database was used for Gene Set Enrichment Analysis (GSEA), survival analysis, gene expression statistics or pathway enrichment assay. PPI inhibited the propagation of NSCLC cells, increased non-viable apoptotic cells, arrested cell cycle at G2/M phase, induced ROS levels but failed to decrease mitochondrial membrane potential. High levels of GRP78 indicates poor prognosis in NSCLC patients. PPI selectively suppressed unfolded protein response (UPR)-induced GRP78 expression, subsequently protected CHOP from GRP78-mediated ubiquitination and degradation. We demonstrated that the natural product PPI, obtained from traditional herbal medicine, deserves for further study as a valuable candidate for lead compound in the chemotherapy of NSCLC.
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Objective: To investigate the molecular mechanism of polyphyllin (PP) inhibiting proliferation and inducing apoptosis of human gl i oma U251 cel l s by di rectl y targeti ng si gnal transducer and activator of transcription 3 (STAT3). Methods: U251 cells were treated with different concentrations of PP, then the effect of PP on the proliferation of glioma U251 cells was detected by MTT assay, trypan blue dyeing and clone forming assay, respectively. Apoptosis of glioma U251 cells was determined by flow cytometry after Annexin-FITC/PI double staining. The change of mitochondrial membrane potential was detected by flow cytometry after fluorescent probe JC-1 staining. SwissTargetPrediction online prediction software was used to detect the targeting proteins of PP. Western blotting was used to detect the expressions of STAT3 signaling pathway-related proteins. Molecular docking simulated the binding and action modes of PP and STAT3. Results: PPinhibited the proliferation of U251 cells in a time and concentration-dependent manner (all P 0.05). The hydrogen atom of PP was capable of forming hydrogen bonds with the oxygen atom of aspartic acid (Asp)-334 on STAT3, thereby enhancing its ability of targeting STAT3. Conclusion: PP can inhibit the proliferation of glioma U251 cells and induce apoptosis by directly targeting STAT3.
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Objective: To observe the effect of polyphyllin I (PPI) on osteoblasts injuries induced by tricalcium phosphate (TCP) wear particles in vitro, and explain its regulation mechanisms. Methods: Primary osteoblasts obtained from the calvaria of neonatal SD rat by the series of digestion were identified with ALP staining. The osteoblasts were treated with TCP wear particles (TCP, 0.1 mg/mL) for 48 h to establish an in vitro injuries model of the calvarial osteoblasts. The experiment was randomly divided into control group, model (TCP, 0.1 mg/mL) group, PPI (30 μg/mL) group and PPI (100 μg/mL) group. CCK-8 and chemical colorimetry were used to examine cell viability and lactic dehydrogenase (LDH) content in culture media; Real-time PCR was performed to detect mRNA levels of ALP, Collagen I and RUNX2 in osteoblasts; The flow cytometry was used to examine apoptosis of osteoblasts using Annexin V/PI double staining; When the osteoblasts were treated for 14 d, mineral nodules formation was observed with alizarin S staining; Western blot was applied to examine proteins expression of Bax, Bcl-2, cleaved Caspase-3, Atg5, p62, and microtubule associated protein 1 light chain3 (LC-3). Results: Compared with control group, model group showed that the cell viability of osteoblasts, mRNA levels of ALP, Collagen I and RUNX2, and mineral nodules formation were significantly decreased; LDH content, percentage of apoptosis and proteins expression of Bax, cleaved Caspase-3, Atg5, LC-3 and the ratio of LC-3II/LC-3I were obviously increased in calvarial osteoblasts, whereas proteins expression of Bcl-2 and p62 was remarkably decreased. Compared with model group, PPI groups indicated that cell viability of osteoblasts, mRNA levels of ALP, Collagen I and RUNX2, and mineral nodules formation were dramatically increased; LDH content, percentage of apoptosis, protein expressions of Bax, cleaved Caspase-3, Atg5, and LC-3 and the ratio of LC-3II/LC-3I were obviously decreased, but Bcl-2 and p62 expression were obviously increased. Conclusion: PPI alleviates osteoblasts injuries induced by TCP wear particles via inhibition of autophagy.
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Cancerous inhibitor of protein phosphatase 2A (CIP2A) is a human oncoprotein that is overexpressed in multiple kinds of cancers including non-small cell lung cancer (NSCLC). CIP2A plays an 'oncogenic nexus' to participate in the tumorigenesis and chemoresistance in several cancer types. AKT and mTORC1 overactivation are detected in NSCLC and many other cancers. Previous studies found that the CIP2A/AKT/mTOR pathway controls cell growth, apoptosis, autophagy process. Polyphyllin I (PPI) and polyphyllin VII (PPVII) are natural components extracted from Paris polyphylla that display anti-cancer properties. In the present study, we investigated whether PPI and PPVII can be used in the cisplatin (DDP)-resistant human NSCLC cell line A549/DDP. Results demonstrated that PPI and PPVII treatment significantly suppressed A549/DDP cell proliferation, migration, invasion and EMT, induced apoptosis and autophagy. Further examination of the mechanism revealed that the PPI and PPVII significantly upregulated the p53, induced caspase-dependent apoptosis and suppressed the CIP2A/AKT/mTOR pathway. The activation of autophagy was mediated through PPI and PPVII induced inhibition of mTOR. We propose that PPI and PPVII might be developed as candidate drugs for DDP-resistant NSCLC.
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Objective Mining Paris resources with medicinal value could underlay the breeding selection and relieve the pressure on wild resources. Methods Twenty-one Paris resources in the mountain area of Sichuan Basin were collected and selected according to their phenotype and rhizome features. Two resources GQ, MH with bigger rhizome and one resource (GK) with polygemmic feature were screened. After preliminary identification, based on Kimura-2-parameter model, molecular phylogenetic trees were constructed based on the different sequence of ITS and psbA-trnH between the screened resources and the homologous sequences from NCBI using the Maximum Like (ML) method. Main active saponin was determinated by HPLC method to predict its potential medicinal value. Results GQ, GK, and MH were special resources of P. polyphylla var. chinensis, P. polyphylla var. yunnanensis, and P. forrestii, respectively, in mountains around Sichuan Basin. The content and proportion of polyphyllin I, II, VI, VII in GQ, GK, MH were different. The total content was GQ > MH > CK > GK. The proportion of polyphyllin I in GQ and MH was 67.61% and 73.25% higher than CK, respectively. While the proportion of polyphyllin VII was most in GK (56.38%). Conclusion This study specified three Sichuan local Paris resources with excellent rhizome features. And they performed well after introduced to Chengdu Plain providing the material basis for the follow-up breeding study of Paris. Three resources have medicinal potential, especially the polyphyllin I and polyphyllin content in MH (P. forrestii) is higher, which can provide a new choice for screening substitution materials of P. polyphylla var. chinensis and P. polyphylla var. yunnanensis.
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Objective: To prepare pH-dependent Paridis Rhizoma saponin (PRS) and Astragali Radix polysaccharide (ARP) colon targeting pellets for the treatment of colon cancer and finish its in vitro release performance evaluation. Methods: The colon targeted pellets were prepared with extrusion-spheronization and air-flow coating method and the the process parameters were optimized by orthogonal design. The coating fluid prescription was investigated by single factor test. In vitro release performance evaluation of the pellets was evaluated with polyphyllin I and II as the indexes. Results: The optimum technologic parameters of extrusion spheronization equipment were as follows: the rate of extrusion was 60 r/min, the rate of spheronization was 1 200 r/min, and the time of spheronization was 5 min. The optimum coating liquid formulation of pH-dependent colon targetting pellets was 15% weight gains of Eudrugit S100, 1.5% anti-plastering aid amount of Glycerin monostearate, and 5% plasticizer amount of TEC. In vitro release test showed that cumulative release rate of berberine hydrochloride was close to 0% in artificial gastric juice after 2 h and less than 10% in artificial intestinal fluid after 4 h, but the cumulative release rate in artificial colon juice after 2 h was more than 90%. Conclusion: The preparation method can be applied to the preparation of colon targeted pellets and the pellets can achieve the targeted release in the colon.