Arisaema heterophyllum Blume is one of the three medicinal plants known as traditional Chinese medicine Rhizoma Arisaematis(RA). RA has been popularly used to treat patients with convulsions, inflammation, and cancer ...Arisaema heterophyllum Blume is one of the three medicinal plants known as traditional Chinese medicine Rhizoma Arisaematis(RA). RA has been popularly used to treat patients with convulsions, inflammation, and cancer for a long time. However, the underlying mechanisms for RA effects are still unclear. The present study was designed to determine the cytotoxicity of agglutinin isolated from Arisema heterophyllum Blume(AHA) and explore the possible mechanisms in human non-small-cell lung cancer A549 cells. AHA with purity up to 95% was isolated and purified from Arisaema heterophyllum Blume using hydrophobic interaction chromatography. AHA dose-dependently inhibited the proliferation of A549 cells and induced G_1 phase cell cycle arrest. AHA induced apoptosis by up-regulating pro-apoptotic Bax, decreasing anti-apoptotic Bcl-2, and activating caspase-9 and caspase-3. In A549 cells treated with AHA, the PI3K/Akt pathway was inhibited. Furthermore, AHA induced increase in the levels of ER stress markers such as phosphorylated eukaryotic initiation factor 2α(p-eIF2α), C/EBP-homologous protein(CHOP), inositol-requiring enzyme 1α(IRE1α), and phosphorylated c-Jun NH_2-terminal kinase(p-JNK). AHA also induced autophagy in A549 cells. Staining of acidic vesicular organelles(AVOs) and increase in the levels of LC3II and ATG7 were observed in AHA-treated cells. These findings suggested that AHA might be one of the active components with anti-cancer effects in Arisaema heterophyllum Blume. In conclusion, cytotoxicity of AHA on cancer cells might be related to its effects on apoptosis and autophagy through inhibition of PI3K/Akt pathway and induction of ER stress.展开更多
Gambogic acid （GA） is an anticancer agent in phase IIb clinical trial in China but its mechanism of action has not been fully clarified. The present study was designed to search the possible target-related proteins ...Gambogic acid （GA） is an anticancer agent in phase IIb clinical trial in China but its mechanism of action has not been fully clarified. The present study was designed to search the possible target-related proteins of GA in cancer cells using proteomic method and establish possible network using bioinformatic analysis. Cytotoxicity and anti-migration effects of GA in MDA-MB-231 cells were checked using MTT assay, flow cytometry, wound migration assay, and chamber migration assay. Possible target-related proteins of GA at early （3 h） and late stage （24 h） of treatment were searched using a proteomic technology, two-dimensional electro- phoresis （2-DE）. The possible network of GA was established using bioinformatic analysis. The intracellular expression levels of vimentin, keratin 18, and calumenin were determined using Western blotting. GA inhibited cell proliferation and induced cell cycle arrest at G2/M phase and apoptosis in MDA-MB-231 cells. Additionally, GA exhibited anti-migration effects at non-toxic doses. In 2-DE analysis, totally 23 possible GA targeted proteins were found, including those with functions in cytoskeleton and transport, regulation of redox state, metabolism, ubiquitin-proteasome system, transcription and translation, protein transport and modification, and cytokine. Network analysis of these proteins suggested that cytoskeleton-related proteins might play important roles in the effects of GA. Results of Western blotting confirmed the cleavage of vimentin, increase in keratin 18, and decrease in calumenin levels in GA-treated cells. In summary, GA is a multi-target compound and its anti-cancer effects may be based on several target-related pro- teins such as cytoskeleton-related proteins.展开更多
The sea dragon Solenognathus hardwickii has long been used as a traditional Chinese medicine for the treatment of various diseases, such as male impotency. To gain a comprehensive insight into the protein components o...The sea dragon Solenognathus hardwickii has long been used as a traditional Chinese medicine for the treatment of various diseases, such as male impotency. To gain a comprehensive insight into the protein components of the sea dragon, shotgun proteomic analysis of its protein expression profiling was conducted in the present study. Proteins were extracted from dried sea dragon using a trichloroacetic acid/acetone precipitation method and then separated by SDS-PAGE. The protein bands were cut from the gel and digested by trypsin to generate peptide mixture. The peptide fragments were then analyzed using nano liquid chromatography tandem mass spectrometry(nano-LC-ESI MS/MS). 810 proteins and 1 577 peptides were identified in the dried sea dragon. The identified proteins exhibited molecular weight values ranging from 1 900 to 3 516 900 Da and p I values from 3.8 to 12.18. Bioinformatic analysis was conducted using the DAVID Bioinformatics Resources 6.7 Gene Ontology(GO) analysis tool to explore possible functions of the identified proteins. Ascribed functions of the proteins mainly included intracellular non-membrane-bound organelle, non-membrane-bounded organelle, cytoskeleton, structural molecule activity, calcium ion binding and etc. Furthermore, possible signal networks of the identified proteins were predicted using STRING(Search Tool for the Retrieval of Interacting Genes) database. Ribosomal protein synthesis was found to play an important role in the signal network. The results of this study, to best of our knowledge, were the first to provide a reference proteome profile for the sea dragon, and would aid in the understanding of the expression and functions of the identified proteins.展开更多
基金supported by the National Nature Science Foundation of China(No.81373964)Shanghai Science&Technology Support Program(No.13431900401)+1 种基金Shanghai Science&Technology Innovation Action Program(No.15140904800)the National Science&Technology Major Project of China(No.2014 ZX09301-306-03)
文摘Arisaema heterophyllum Blume is one of the three medicinal plants known as traditional Chinese medicine Rhizoma Arisaematis(RA). RA has been popularly used to treat patients with convulsions, inflammation, and cancer for a long time. However, the underlying mechanisms for RA effects are still unclear. The present study was designed to determine the cytotoxicity of agglutinin isolated from Arisema heterophyllum Blume(AHA) and explore the possible mechanisms in human non-small-cell lung cancer A549 cells. AHA with purity up to 95% was isolated and purified from Arisaema heterophyllum Blume using hydrophobic interaction chromatography. AHA dose-dependently inhibited the proliferation of A549 cells and induced G_1 phase cell cycle arrest. AHA induced apoptosis by up-regulating pro-apoptotic Bax, decreasing anti-apoptotic Bcl-2, and activating caspase-9 and caspase-3. In A549 cells treated with AHA, the PI3K/Akt pathway was inhibited. Furthermore, AHA induced increase in the levels of ER stress markers such as phosphorylated eukaryotic initiation factor 2α(p-eIF2α), C/EBP-homologous protein(CHOP), inositol-requiring enzyme 1α(IRE1α), and phosphorylated c-Jun NH_2-terminal kinase(p-JNK). AHA also induced autophagy in A549 cells. Staining of acidic vesicular organelles(AVOs) and increase in the levels of LC3II and ATG7 were observed in AHA-treated cells. These findings suggested that AHA might be one of the active components with anti-cancer effects in Arisaema heterophyllum Blume. In conclusion, cytotoxicity of AHA on cancer cells might be related to its effects on apoptosis and autophagy through inhibition of PI3K/Akt pathway and induction of ER stress.
基金supported by the Twelfth Five-Year National Science & Technology Support Program(No.2012BAI 29B06)Shanghai Science & Technology Support Program(No.13431900 401)+4 种基金China Postdoctoral Science Foundation Funded Project(No.2012M5 10907)Shanghai Postdoctoral Scientific Program(No.13R21417800)the Postdoctor Research Program of Shanghai Institutes for Biological Sciences,Chinese Academy of Sciences(No.2012KIP516)the Sanofi-Aventis-Shanghai Institutes for Biological Sciences Scholarship Programthe National Nature Science Foundation(Nos.81302809 and 81373964)
文摘Gambogic acid （GA） is an anticancer agent in phase IIb clinical trial in China but its mechanism of action has not been fully clarified. The present study was designed to search the possible target-related proteins of GA in cancer cells using proteomic method and establish possible network using bioinformatic analysis. Cytotoxicity and anti-migration effects of GA in MDA-MB-231 cells were checked using MTT assay, flow cytometry, wound migration assay, and chamber migration assay. Possible target-related proteins of GA at early （3 h） and late stage （24 h） of treatment were searched using a proteomic technology, two-dimensional electro- phoresis （2-DE）. The possible network of GA was established using bioinformatic analysis. The intracellular expression levels of vimentin, keratin 18, and calumenin were determined using Western blotting. GA inhibited cell proliferation and induced cell cycle arrest at G2/M phase and apoptosis in MDA-MB-231 cells. Additionally, GA exhibited anti-migration effects at non-toxic doses. In 2-DE analysis, totally 23 possible GA targeted proteins were found, including those with functions in cytoskeleton and transport, regulation of redox state, metabolism, ubiquitin-proteasome system, transcription and translation, protein transport and modification, and cytokine. Network analysis of these proteins suggested that cytoskeleton-related proteins might play important roles in the effects of GA. Results of Western blotting confirmed the cleavage of vimentin, increase in keratin 18, and decrease in calumenin levels in GA-treated cells. In summary, GA is a multi-target compound and its anti-cancer effects may be based on several target-related pro- teins such as cytoskeleton-related proteins.
基金supported in part by the National High Technology Research and Development Program of China(No.2013AA093002)Shanghai Science&Technology Support Program(No.13431900401)+2 种基金Shanghai Science&Technology Innovation Action Program(No.15140904800)the National Nature Science Foundation of China(No.81373964)the National Science&Technology Major Project of China(No.2014ZX09301-306-03)
文摘The sea dragon Solenognathus hardwickii has long been used as a traditional Chinese medicine for the treatment of various diseases, such as male impotency. To gain a comprehensive insight into the protein components of the sea dragon, shotgun proteomic analysis of its protein expression profiling was conducted in the present study. Proteins were extracted from dried sea dragon using a trichloroacetic acid/acetone precipitation method and then separated by SDS-PAGE. The protein bands were cut from the gel and digested by trypsin to generate peptide mixture. The peptide fragments were then analyzed using nano liquid chromatography tandem mass spectrometry(nano-LC-ESI MS/MS). 810 proteins and 1 577 peptides were identified in the dried sea dragon. The identified proteins exhibited molecular weight values ranging from 1 900 to 3 516 900 Da and p I values from 3.8 to 12.18. Bioinformatic analysis was conducted using the DAVID Bioinformatics Resources 6.7 Gene Ontology(GO) analysis tool to explore possible functions of the identified proteins. Ascribed functions of the proteins mainly included intracellular non-membrane-bound organelle, non-membrane-bounded organelle, cytoskeleton, structural molecule activity, calcium ion binding and etc. Furthermore, possible signal networks of the identified proteins were predicted using STRING(Search Tool for the Retrieval of Interacting Genes) database. Ribosomal protein synthesis was found to play an important role in the signal network. The results of this study, to best of our knowledge, were the first to provide a reference proteome profile for the sea dragon, and would aid in the understanding of the expression and functions of the identified proteins.
