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1.
Int J Biol Macromol ; 258(Pt 2): 129123, 2024 Feb.
Article in English | MEDLINE | ID: mdl-38163496

ABSTRACT

Isodon rubescens has garnered much attention due to its anti-tumor or anti-cancer properties. However, little is known about the molecular mechanism of oridonin biosynthesis leveraging the regulatory network between small RNAs and mRNAs. In this study, the regulatory networks of miRNAs and targets were examined by combining mRNA, miRNA, and degradome. A total of 348 miRNAs, including 287 known miRNAs and 61 novel miRNAs, were identified. Among them, 51 miRNAs were significantly expressed, and 36 miRNAs responded to MeJA. A total of 3066 target genes were associated with 228 miRNAs via degradome sequencing. Multi-omics analysis demonstrated that 27 miRNA-mRNA pairs were speculated to be involved in MeJA regulation, and 36 miRNA-mRNA pairs were hypothesized to be involved in the genotype-dependence of I. rubescens. Furthermore, 151 and 7 miRNA-mRNA modules were likely engaged in oridonin biosynthesis as identified by psRNATarget and degradome sequencing, respectively. Some miRNA-mRNA modules were confirmed via RT-qPCR. Moreover, miRNAs targeting plant hormone signal transduction pathway genes were identified, such as miR156, miR167, miR393, and PC-3p-19822_242. Collectively, our results demonstrate for the first time that miRNAs are identified in I. rubescens, and laid a solid foundation for further research on the molecular mechanism of oridonin biosynthesis mediated by miRNA.


Subject(s)
Diterpenes, Kaurane , Isodon , MicroRNAs , MicroRNAs/genetics , Isodon/genetics , Isodon/metabolism , Multiomics , Transcriptome , RNA, Messenger/genetics , Gene Expression Regulation, Plant
2.
Planta ; 257(5): 90, 2023 Mar 29.
Article in English | MEDLINE | ID: mdl-36991182

ABSTRACT

MAIN CONCLUSION: We identify two ferruginol synthases and a 11-hydroxyferruginol synthase from a traditional Chinese medicinal herb Isodon lophanthoides and propose their involvement in two independent abietane diterpenoids biosynthetic pathways. Isodon lophanthoides is a traditional Chinese medicinal herb rich in highly oxidized abietane-type diterpenoids. These compounds exhibit a wide range of pharmaceutical activities, yet the biosynthesis is barely known. Here, we describe the screening and functional characterization of P450s that oxidize the abietane skeleton abietatriene. We mainly focused on CYP76 family and identified 12 CYP76AHs by mining the RNA-seq data of I. lophanthoides. Among the 12 CYP76AHs, 6 exhibited similar transcriptional expression features as upstream diterpene synthases, including root or leaf-preferential expression pattern and highly MeJA inducibility. These six P450s were considered as first-tier candidates and functionally characterized in yeast and plant cells. In yeast assays showed that both CYP76AH42 and CYP76AH43 were ferruginol synthases hydroxylating the C12 position of abietatriene, whereas CYP76AH46 was characterized as a 11-hydroxyferruginol synthase which catalyzes two successive oxidations at C12 and C11 of abietatriene. Heterologous expression of three CYP76AHs in Nicotiana benthamiana resulted in the formation of ferruginol. qPCR analysis showed CYP76AH42 and CYP76AH43 were mainly expressed in the root, which was consistent with the distribution of ferruginol in the root periderms. CYP76AH46 was primarily expressed in the leaves where barely ferruginol or 11-hydroxyferruginol was detected. In addition to distinct organ-specific expression pattern, three CYP76AHs exhibited different genomic structures (w or w/o introns), low protein sequence identities (51-63%) and were placed in separate subclades in the phylogenetic tree. These results suggest that the identified CYP76AHs may be involved in at least two independent abietane biosynthetic pathways in the aerial and underground parts of I. lophanthoides.


Subject(s)
Diterpenes , Isodon , Abietanes , Isodon/chemistry , Isodon/genetics , Isodon/metabolism , Saccharomyces cerevisiae/metabolism , Phylogeny , Diterpenes/metabolism , Cytochrome P-450 Enzyme System/genetics , Cytochrome P-450 Enzyme System/metabolism
3.
Chem Biodivers ; 19(12): e202200748, 2022 Dec.
Article in English | MEDLINE | ID: mdl-36369642

ABSTRACT

Tyrosinase inhibitors can alleviate the harm to the liver caused by tyrosinase. How to effectively screen out natural tyrosinase inhibitors becomes a focus. In this study, Isodon excisoides was first extracted with the ultrasound optimized by response surface methodology. Then, a method combined ultrafiltration with ultra-liquid chromatography mass spectrometry (UHPLC/MS) was built to screen and identify tyrosinase inhibitors. The binding energies of active ingredients to tyrosinase were calculated by molecular docking. The reliability of the results was validated by the IC50 of enzyme inhibition assay. As a result, the binding energies of 7 components including excisanin B, lasiokaurin, rabdophyllin G, rabdoserrin B, rabdosin D, rabdosinate and weisiensin were lower than that of resveratrol. It was indicated that these components had high tyrosinase inhibitory activity. The IC50 values of lasiokaurin and excisanin B were 177 and 142 µmol/mL, which were less than that of resveratrol (183 µmol/mL). It showed that this way was simple, rapid, reliable and effective, which provided a new strategy to screen natural bioactive compounds from plants.


Subject(s)
Isodon , Monophenol Monooxygenase , Molecular Docking Simulation , Chromatography, High Pressure Liquid/methods , Isodon/metabolism , Enzyme Inhibitors/pharmacology , Enzyme Inhibitors/chemistry , Resveratrol , Ultrafiltration/methods , Reproducibility of Results
4.
Plant Cell Physiol ; 62(9): 1423-1435, 2021 Nov 17.
Article in English | MEDLINE | ID: mdl-34133748

ABSTRACT

Isodon lophanthoides var. gerardiana (Lamiaceae), also named xihuangcao, is a traditional Chinese medicinal herb that exhibits a broad range of pharmacological activities. Abietane-type diterpenoids are the characteristic constituents of I. lophanthoides, yet their biosynthesis has not been elucidated. Although the aerial parts are the most commonly used organs of I. lophanthoides, metabolite profiling by gas chromatography-mass spectrometry showed the underground parts also contain large amounts of labdane diterpenoids including abietatriene, miltiradiene and ferruginol, which are distinct from the 13-hydroxy-8(14)-abietene detected in the aerial parts. Comparative transcriptome analysis of root and leaf samples identified a diverse diterpene synthase family including 6 copalyl diphosphate synthase (IlCPS1-6) and 5 kaurene synthase-like (IlKSL1-5). Here we report the functional characterization of six of these enzymes using yeast heterologous expression system. Both IlCPS1 and IlCPS3 synthesized (+)-copalyl diphosphate (CPP), in combination with IlKSL1 resulted in miltiradiene, precursor of abietane-type diterpenoids, while coupling with IlKSL5 led to the formation of hydroxylated diterpene scaffold nezukol. Expression profiling and phylogenetic analysis further support the distinct evolutionary relationship and spatial distribution of IlCPS1 and IlCPS3. IlCPS2 converted GGPP into labda-7,13E-dien-15-ol diphosphate. IlCPS6 was identified as ent-CPS, indicating a role in gibberellin metabolism. We further identified a single residue that determined the water addition of nezukol synthase IlKSL5. Substitution of alanine 513 with isoleucine completely altered the product outcome from hydroxylated nezukol to isopimara-7,15-diene. Together, these findings elucidated the early steps of bioactive abietane-type diterpenoid biosynthesis in I. lophanthoides and the catalytic mechanism of nezukol synthase.


Subject(s)
Alkyl and Aryl Transferases/genetics , Diterpenes/metabolism , Genes, Plant , Isodon/genetics , Multigene Family , Plant Proteins/genetics , Alkyl and Aryl Transferases/chemistry , Alkyl and Aryl Transferases/metabolism , Amino Acid Sequence , Isodon/chemistry , Isodon/metabolism , Plant Proteins/chemistry , Plant Proteins/metabolism , Plants, Medicinal/chemistry , Plants, Medicinal/genetics , Plants, Medicinal/metabolism , Sequence Alignment
5.
Sci Rep ; 10(1): 10043, 2020 06 22.
Article in English | MEDLINE | ID: mdl-32572147

ABSTRACT

The ecological and economic cultivation of Isodon rubescens is currently being carried out. The demand of I. rubescens for light intensity should be made clear to estimate whether the environmental conditions of an area are suitable for cultivating I. Rubescens and improve cultivation techniques. The photosynthetic and chlorophyll fluorescence characteristics of I. rubescens were determined with a Li-6400 photosynthesis system and PAM-2500 portable chlorophyll fluorescence apparatus. The results showed that there was no obvious midday depression of photosynthesis in I. rubescens leaves. The light compensation point and light saturation point of I. rubescens leaves were 21.83482 µmol·m-2·s-1 and 802.262 µmol·m-2·s-1, respectively. The CO2 compensation point and CO2 saturation point of I. rubescens leaves were 101.7199 µmol·mol-1 and 1674.514 µmol·mol-1, respectively. The maximal photochemical efficiency of photosystem II ((Fm-Fo)/Fm) in I. rubescens leaves reached 0.7. The electron transport rate of photosystem II in I. rubescens leaves reached 20 µmol electrons/(m2·s). I. rubescens can tolerate intense light above the light compensation point and utilize low light. I. rubescens leaves have a strong photoprotective capacity. I. rubescens can grow in both sunny and shady places. The most important factor affecting photosynthetic efficiency in I. rubescens leaves is the concentration of CO2 in air.


Subject(s)
Chlorophyll/metabolism , Isodon/physiology , Agriculture , Carbon Dioxide/metabolism , Electron Transport , Fluorescence , Isodon/metabolism , Lithium , Photosynthesis , Plant Leaves/metabolism , Plant Leaves/physiology
6.
Bioorg Med Chem ; 28(11): 115527, 2020 06 01.
Article in English | MEDLINE | ID: mdl-32345458

ABSTRACT

Based on DNA topoisomerase IB (TOP1) and tyrosyl-DNA phosphodiesterase 1 (TDP1) inhibition of the ethanol extract of the roots of Isodon ternifolius (D. Don) Kudo (Labiatae), its secondary metabolites has been studied. Two new compounds, an ent-abietane diterpenoid isodopene A (1) and a 2,3-seco-triterpene isodopene B (13), along with 25 known compounds were isolated. Their structures were elucidated by spectroscopic analysis and theoretical calculations. The enzyme-based assays indicated that 1 and 13 showed strong (+++) and moderate (++) TOP1 inhibition, respectively. Two chalcone derivatives 11 and 12 were firstly found as dual TDP1 and TOP1 natural inhibitors, and showed synergistic effect with the clinical TOP1 inhibitors topotecan in MCF-7 cells. Compounds 8, 16, and 22 acted as TOP1 catalytic inhibitors with equipotent TOP1 inhibition to camptothecin (++++). Compounds 7 and 8 exhibited significant cytotoxicity against MCF-7, A549, and HCT116 cells with GI50 values in the range of 2.2-4.8 µM. This work would provide valuable information that secondary metabolites from I. ternifolius could be developed as anticancer agents.


Subject(s)
Antineoplastic Agents, Phytogenic/pharmacology , DNA Topoisomerases, Type I/metabolism , Isodon/chemistry , Phosphodiesterase Inhibitors/pharmacology , Phosphoric Diester Hydrolases/metabolism , Topoisomerase I Inhibitors/pharmacology , Animals , Antineoplastic Agents, Phytogenic/chemistry , Antineoplastic Agents, Phytogenic/isolation & purification , Cattle , Cell Proliferation/drug effects , Cell Survival/drug effects , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , Humans , Isodon/metabolism , Molecular Structure , Phosphodiesterase Inhibitors/chemistry , Phosphodiesterase Inhibitors/isolation & purification , Structure-Activity Relationship , Topoisomerase I Inhibitors/chemistry , Topoisomerase I Inhibitors/isolation & purification , Tumor Cells, Cultured
7.
Chem Biodivers ; 16(6): e1900206, 2019 Jun.
Article in English | MEDLINE | ID: mdl-31081987

ABSTRACT

Three new 4,5-seco-20(10→5)-abeo-abietane diterpenoids, 16-hydroxysalvilenone (1), 15-hydroxysalprionin (2), and 11ß,15-dihydroxysalprionin-12-one (3), and nine known abietane diterpenoids, 4-12, along with one known sempervirane diterpenoid, hispidanol A (13), were isolated from the aerial parts of Isodon lophanthoides var. graciliflorus. The structures of compounds 1-3 were determined on the basis of spectroscopic methods including extensive analysis of NMR and mass spectroscopic data. All diterpenoids were tested for their TNF-α inhibitory effects on LPS-induced RAW264.7 cells. Compound 9 (16-acetoxyhorminone) was the most potent with an IC50 value of 3.97±0.70 µm.


Subject(s)
Abietanes/chemistry , Anti-Inflammatory Agents/chemistry , Isodon/chemistry , Abietanes/isolation & purification , Abietanes/pharmacology , Animals , Anti-Inflammatory Agents/isolation & purification , Anti-Inflammatory Agents/pharmacology , Isodon/metabolism , Lipopolysaccharides/pharmacology , Macrophages/cytology , Macrophages/drug effects , Macrophages/metabolism , Mice , Molecular Conformation , Plant Components, Aerial/chemistry , Plant Components, Aerial/metabolism , Plant Extracts/chemistry , RAW 264.7 Cells
8.
Int J Mol Sci ; 20(2)2019 Jan 21.
Article in English | MEDLINE | ID: mdl-30669669

ABSTRACT

Isodon rugosus (Wall. ex Benth.) Codd accumulates large amounts of phenolics and pentacyclic triterpenes. The present study deals with the in vitro callus induction from stem and leaf explants of I. rugosus under various plant growth regulators (PGRs) for the production of antioxidant and anti-ageing compounds. Among all the tested PGRs, thidiazuron (TDZ) used alone or in conjunction with α-napthalene acetic acid (NAA) induced highest callogenesis in stem-derived explants, as compared to leaf-derived explants. Stem-derived callus culture displayed maximum total phenolic content and antioxidant activity under optimum hormonal combination (3.0 mg/L TDZ + 1.0 mg/L NAA). HPLC analysis revealed the presence of plectranthoic acid (373.92 µg/g DW), oleanolic acid (287.58 µg/g DW), betulinic acid (90.51 µg/g DW), caffeic acid (91.71 µg/g DW), and rosmarinic acid (1732.61 µg/g DW). Complete antioxidant and anti-aging potential of extracts with very contrasting phytochemical profiles were investigated. Correlation analyses revealed rosmarinic acid as the main contributor for antioxidant activity and anti-aging hyaluronidase, advance glycation end-products inhibitions and SIRT1 activation, whereas, pentacyclic triterpenoids were correlated with elastase, collagenase, and tyrosinase inhibitions. Altogether, these results clearly evidenced the great valorization potential of I. rugosus calli for the production of antioxidant and anti-aging bioactive extracts for cosmetic applications.


Subject(s)
Antioxidants/chemistry , Antioxidants/pharmacology , Isodon/chemistry , Isodon/growth & development , Phytochemicals/chemistry , Plant Extracts/chemistry , Plant Extracts/pharmacology , Cellular Senescence/drug effects , In Vitro Techniques , Isodon/metabolism , Metabolome , Metabolomics/methods , Molecular Structure , Phenols/chemistry , Secondary Metabolism
9.
Phytochemistry ; 158: 96-102, 2019 Feb.
Article in English | MEDLINE | ID: mdl-30496917

ABSTRACT

The Isodon plants (Lamiaceae) have been used in traditional Chinese medicine to alleviate sufferings from inflammations and cancers. This feature has been attributed to the presence of pharmacologically active ent-kaurane diterpenoids such as eriocalyxin B and oridonin. The Isodon eriocalyx (Dunn) Kudô species native to southwest China can accumulate a particularly high content of ent-kaurane diterpenoids (∼1.5% w/w of dried leaves). We previously identified diterpene synthases IeCPS1 and IeCPS2 as ent-copalyl diphosphate synthases (ent-CPS) potentially involved in Isodon ent-kaurane diterpenoids biosynthesis. In this study, analysis of RNA-seq transcriptome of the I. eriocalyx plant revealed three other diterpene synthase genes (IeCPS3, IeKS1, and IeKSL1). Their functional characterization through coupled in vitro enzyme assays has confirmed that IeCPS3 is an ent-CPS specifically producing ent-copalyl diphosphate (ent-CPP). IeKS1 accepted ent-CPP to produce exclusively ent-kaurene and may thus be defined as an ent-kaurene synthase (ent-KS). When IeKSL1 was combined with IeCPS2 or IeCPS3, no product was detected. Based on tissue-specific expression and metabolic localization studies, the IeCPS3 and IeKS1 transcripts were significantly accumulated in leaves where the ent-kaurane diterpenoid eriocalyxin B dominates, whereas weak expression of both were observed in germinating seeds in which gibberellin biosynthetic pathway is normally active. Our findings suggest that both IeCPS3 and IeKS1 possess dual roles in general (gibberellins) and specialized diterpenoid metabolism, such as that of the Isodon ent-kaurane diterpenoids.


Subject(s)
Alkyl and Aryl Transferases/metabolism , Diterpenes/metabolism , Isodon/metabolism , Plant Proteins/metabolism , Alkyl and Aryl Transferases/genetics , Cloning, Molecular , Diterpenes/chemistry , Diterpenes, Kaurane/metabolism , Gibberellins/biosynthesis , Isodon/chemistry , Isodon/genetics , Phylogeny , Plant Leaves/metabolism , Plant Proteins/genetics , Plants, Medicinal/metabolism
10.
BMC Plant Biol ; 18(1): 272, 2018 Nov 08.
Article in English | MEDLINE | ID: mdl-30409115

ABSTRACT

BACKGROUND: Isodon amethystoides (Ben-th) Cy Wu et Hsuan is an important traditional medicinal plant endowed with pharmacological properties effective in the treatment of various diseases, including pulmonary tuberculosis. The tetracyclic diterpenoids, Wangzaozins (Wangzaozin A, glaucocalyxin A, glaucocalyxin B), are the major bioactive compounds of I. amethystoides. However, the molecular information about the biosynthesis of these compounds still remains unclear. RESULTS: An examination of the accumulated levels of Wangzaozins in I. amethystoides revealed considerable variations in the root, stem, and leaf tissues of this plant, indicating possible differences in metabolite biosynthesis and accumulation among various tissues. To better elucidate the tetracyclic diterpenoid biosynthesis pathway, we generated transcriptome sequences from the root, stem, and leaf tissues, and performed de novo sequence assembly, yielding 230,974 transcripts and 114,488 unigenes, with average N50 lengths of 1914 and 1241 bp, respectively. Putative functions could be assigned to 73,693 transcripts (31.9%) based on BLAST searches against annotation databases, including GO, KEGG, Swiss-Prot, NR, and Pfam. Moreover, the candidate genes involving in the diterpenoid biosynthesis, such as CPS, KSL, were also analyzed. The expression profiles of eight transcripts, involving the tetracyclic diterpenoid biosynthesis, were validated in different I. amethystoides tissues by qRT-PCR, unraveling the gene expression profile of the pathway. The differential expressions of ISPD, ISPF and ISPH (MEP pathway), and IaCPS and IaKSL (diterpenoid pathway) candidate genes in leaves and roots, may contribute to the high accumulation of Wangzaozins in I. amethystoides leaves. CONCLUSION: The genomic dataset and analyses reported here lay the foundations for further research on this important medicinal plant.


Subject(s)
Isodon/genetics , Plant Leaves/genetics , Plant Roots/genetics , Plant Stems/genetics , Transcriptome/genetics , Isodon/metabolism , Molecular Sequence Annotation , Plant Leaves/metabolism , Plant Roots/metabolism , Plant Stems/metabolism
11.
PLoS One ; 12(4): e0176507, 2017.
Article in English | MEDLINE | ID: mdl-28445526

ABSTRACT

Plants produce an immense diversity of natural products (i.e. secondary or specialized metabolites) that offer a rich source of known and potentially new pharmaceuticals and other desirable bioproducts. The Traditional Chinese Medicinal plant Isodon rubescens (Lamiaceae) contains an array of bioactive labdane-related diterpenoid natural products. Of these, the ent-kauranoid oridonin is the most prominent specialized metabolite that has been extensively studied for its potent antimicrobial and anticancer efficacy. Mining of a previously established transcriptome of I. rubescens leaf tissue identified seven diterpene synthase (diTPSs) candidates. Here we report the functional characterization of four I. rubescens diTPSs. IrTPS5 and IrTPS3 were identified as an ent-copalyl diphosphate (CPP) synthase and a (+)-CPP synthase, respectively. Distinct transcript abundance of IrTPS5 and the predicted ent-CPP synthase IrTPS1 suggested a role of IrTPS5 in specialized ent-kaurene metabolism possibly en route to oridonin. Nicotiana benthamiana co-expression assays demonstrated that IrTPS4 functions sequentially with IrTPS3 to form miltiradiene. In addition, IrTPS2 converted the IrTPS3 product (+)-CPP into the hydroxylated tricyclic diterpene nezukol not previously identified in I. rubescens. Metabolite profiling verified the presence of nezukol in I. rubescens leaf tissue. The proposed IrTPS2-catalyzed reaction mechanism proceeds via the common ionization of the diphosphate group of (+)-CPP, followed by formation of an intermediary pimar-15-en-8-yl+ carbocation and neutralization of the carbocation by water capture at C-8 to yield nezukol, as confirmed by nuclear magnetic resonance (NMR) analysis. Oxygenation activity is rare for the family of class I diTPSs and offers new catalysts for developing metabolic engineering platforms to produce a broader spectrum of bioactive diterpenoid natural products.


Subject(s)
Alkyl and Aryl Transferases/metabolism , Diterpenes/metabolism , Isodon/metabolism , Plant Proteins/metabolism , Alkyl and Aryl Transferases/chemistry , Alkyl and Aryl Transferases/classification , Biocatalysis , Cloning, Molecular , Diterpenes/chemistry , Diterpenes, Kaurane/biosynthesis , Diterpenes, Kaurane/chemistry , Gas Chromatography-Mass Spectrometry , Gene Expression , Isodon/chemistry , Isodon/genetics , Magnetic Resonance Spectroscopy , Metabolome , Phylogeny , Plant Leaves/chemistry , Plant Leaves/metabolism , Plant Proteins/chemistry , Plant Proteins/classification , Plants, Medicinal/chemistry , Plants, Medicinal/metabolism , Nicotiana/chemistry , Nicotiana/metabolism
12.
Cell Biochem Biophys ; 73(2): 559-564, 2015 Nov.
Article in English | MEDLINE | ID: mdl-27352353

ABSTRACT

Colorectal cancer is one of the most commonly diagnosed cancers in the world. Currently, drug resistance of cancer cell to chemotherapy is a major cause for cancer recurrence and death of the patients; therefore, new therapeutic strategy is required to improve the care of colorectal cancer patients. The Chinese herb, Isodon eriocalyx, has been used a therapeutic for a long time in China. In this study, we showed that Epieriocalyxin A (EpiA), a diterpenoid isolated from I. eriocalyx, suppressed Caco-2 colon cancer cell growth. EpiA induced annexin V flipping in cell membrane and DNA fragment. We also showed that EpiA induced the generation of ROS in cells, as well as damage of the mitochondrial membrane. Western blot results showed that both JNK and ERK1/2 activation was decreased after EpiA treatment in a dose-dependent manner. EpiA increased the expression of caspase 3 and Bax, and decreased Bcl2 expression. Our results suggest that EpiA is a novel compound that induces colon cancer apoptosis. EpiA could be a potential drug for colon cancer therapy in the future.


Subject(s)
Apoptosis/drug effects , Diterpenes/toxicity , JNK Mitogen-Activated Protein Kinases/metabolism , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , Blotting, Western , Caco-2 Cells , Caspase 3/metabolism , Colonic Neoplasms/metabolism , Colonic Neoplasms/pathology , DNA Fragmentation/drug effects , Diterpenes/chemistry , Down-Regulation/drug effects , Humans , Isodon/chemistry , Isodon/metabolism , Membrane Potential, Mitochondrial/drug effects , Proto-Oncogene Proteins c-bcl-2/metabolism , Reactive Oxygen Species/metabolism , Signal Transduction/drug effects , bcl-2-Associated X Protein/metabolism
13.
PLoS One ; 9(10): e109951, 2014.
Article in English | MEDLINE | ID: mdl-25333664

ABSTRACT

Rabdosia serra is well known for its antibacterial, anti-inflammatory and antitumor activities, but no information has been available for the active compounds derived from this plant in inhibiting human nasopharyngeal carcinoma (NPC) cell growth. In this study, we isolated and purified a natural diterpenoid from Rabdosia serra and identified its chemical structure as effusanin E and elucidated its underlying mechanism of action in inhibiting NPC cell growth. Effusanin E significantly inhibited cell proliferation and induced apoptosis in NPC cells. Effusanin E also induced the cleavage of PARP, caspase-3 and -9 proteins and inhibited the nuclear translocation of p65 NF-κB proteins. Moreover, effusanin E abrogated the binding of NF-κB to the COX-2 promoter, thereby inhibiting the expression and promoter activity of COX-2. Pretreatment with a COX-2 or NF-κB-selective inhibitor (celecoxib or ammonium pyrrolidinedithiocarbamate) had an additive effect on the effusanin E-mediated inhibition of proliferation, while pretreatment with an activator of NF-κB/COX-2 (lipopolysaccharides) abrogated the effusanin E-mediated inhibition of proliferation. Effusanin E also significantly suppressed tumor growth in a xenograft mouse model without obvious toxicity, furthermore, the expression of p50 NF-κB and COX-2 were down-regulated in the tumors of nude mice. These data suggest that effusanin E suppresses p50/p65 proteins to down-regulate COX-2 expression, thereby inhibiting NPC cell growth. Our findings provide new insights into exploring effusanin E as a potential therapeutic compound for the treatment of human nasopharyngeal carcinoma.


Subject(s)
Antineoplastic Agents/pharmacology , Cyclooxygenase 2/metabolism , Diterpenes/pharmacology , NF-kappa B/metabolism , Signal Transduction/drug effects , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/therapeutic use , Apoptosis/drug effects , Carcinoma/drug therapy , Carcinoma/metabolism , Carcinoma/pathology , Caspase 3/metabolism , Caspase 9/metabolism , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Cyclooxygenase 2/genetics , Diterpenes/chemistry , Diterpenes/therapeutic use , Humans , Isodon/chemistry , Isodon/metabolism , Male , Mice , Mice, Nude , Nasopharyngeal Neoplasms/drug therapy , Nasopharyngeal Neoplasms/metabolism , Nasopharyngeal Neoplasms/pathology , Poly(ADP-ribose) Polymerases/metabolism
14.
Cell Death Dis ; 5: e1400, 2014 Sep 04.
Article in English | MEDLINE | ID: mdl-25188510

ABSTRACT

Adenanthin, a natural diterpenoid isolated from the leaves of Isodon adenanthus, has recently been reported to induce leukemic cell differentiation by targeting peroxiredoxins (Prx) I and II. On the other hand, increasing lines of evidence propose that these Prx proteins would become potential targets to screen drugs for the prevention and treatment of solid tumors. Therefore, it is of significance to explore the potential activities of adenanthin on solid tumor cells. Here, we demonstrate that Prx I protein is essential for the survival of hepatocellular carcinoma (HCC) cells, and adenanthin can kill these malignant liver cells in vitro and xenografts. We also show that the cell death-inducing activity of adenanthin on HCC cells is mediated by the increased reactive oxygen species (ROS) levels. Furthermore, the silencing of Prx I or Prx II significantly enhances the cytotoxic activity of adenanthin on HCC, whereas the ectopic expression of Prx I and Prx II but not their mutants of adenanthin-bound cysteines can rescue adenanthin-induced cytotoxicity in Prxs-silenced HCC cells. Taken together, our results propose that adenanthin targets Prx I/II to kill HCC cells and its therapeutic significance warrants to be further explored in HCC patients.


Subject(s)
Antineoplastic Agents, Phytogenic/toxicity , Apoptosis/drug effects , Diterpenes, Kaurane/toxicity , Peroxiredoxins/metabolism , Animals , Antineoplastic Agents, Phytogenic/therapeutic use , Carcinoma, Hepatocellular/drug therapy , Carcinoma, Hepatocellular/pathology , Cell Line, Tumor , Diterpenes, Kaurane/therapeutic use , Hep G2 Cells , Humans , Isodon/chemistry , Isodon/metabolism , Liver Neoplasms/drug therapy , Liver Neoplasms/pathology , Male , Mice , Mice, Inbred BALB C , Mice, Nude , Oxidative Stress/drug effects , Peroxiredoxins/antagonists & inhibitors , Peroxiredoxins/genetics , Plant Leaves/chemistry , Plant Leaves/metabolism , RNA Interference , RNA, Small Interfering/metabolism , Reactive Oxygen Species/metabolism , Transplantation, Heterologous
15.
Angew Chem Int Ed Engl ; 53(11): 2988-91, 2014 Mar 10.
Article in English | MEDLINE | ID: mdl-24519748

ABSTRACT

The total synthesis of sculponeatin N, a bioactive polycyclic diterpene isolated from Isodon sculponeatus, is reported. Key features of the synthesis include diastereoselective Nazarov and ring-closing metathesis reactions, and a highly efficient formation of the bicyclo[3.2.1]octane ring system by a reductive radical cyclization.


Subject(s)
Isodon/chemistry , Terpenes/chemical synthesis , Bridged Bicyclo Compounds/chemistry , Cell Line, Tumor , Cell Survival/drug effects , Cyclization , Diterpenes/chemical synthesis , Diterpenes/chemistry , Free Radicals/chemistry , Hep G2 Cells , Humans , Isodon/metabolism , Stereoisomerism , Terpenes/chemistry , Terpenes/pharmacology
16.
Oncol Rep ; 31(2): 975-82, 2014 Feb.
Article in English | MEDLINE | ID: mdl-24297112

ABSTRACT

Oridonin, an active component isolated from Rabdosia rubescens, has been reported to exhibit antitumor effects. In the present study, we evaluated the antitumor activity and the mechanisms of action of oridonin in pancreatic cancer. Oridonin treatment significantly induced apoptotic cell death in SW1990 pancreatic cancer cells in a dose-dependent manner. Additionally, cell apoptosis was markedly inhibited by PFT α (pifithrin α), a p53-specific inhibitor, which was applied to evaluate the function of p53, showing that p53 was responsible for the cytotoxity of oridonin. Moreover, oridonin increased the expression of p-p53 with a concomitant increase in p21 in the SW1990 cells. Following treatment with mitogen-activated protein kinase (MAPK) inhibitors, PD98059 (ERK inhibitor), SP600125 (JNK inhibitor) and SB203580 (p38 inhibitor), the cytotoxity of oridonin was not influenced by JNK (SP600125) and ERK (PD98059), but these effects were opposite to the cytotoxity of oridonin observed with SP203580 treatment. These findings confirmed that orodonin-induced apoptosis was p38-dependent, but JNK- and ERK-independent. Furthermore, the activation of the p38 kinase promoted the activation of p53 and its downstream target p21, and further caused caspase-9 and -3 activation, as demonstrated by evidence showing that the p38 inhibitor SB203580 not only blocked the phosphorylation of p38 but also reduced the activation of p53, p21 and caspase-9 and -3. Collectively, these results suggest that p53-dependent and caspase-dependent induction of p38 MAPK directly participates in apoptosis induced by oridonin.


Subject(s)
Apoptosis/drug effects , Diterpenes, Kaurane/pharmacology , Pancreatic Neoplasms/drug therapy , Tumor Suppressor Protein p53/metabolism , p38 Mitogen-Activated Protein Kinases/metabolism , Anthracenes/pharmacology , Benzothiazoles/pharmacology , Caspase 3/genetics , Caspase 9/genetics , Cell Line, Tumor , Cyclin-Dependent Kinase Inhibitor p21/biosynthesis , Cyclin-Dependent Kinase Inhibitor p21/genetics , Enzyme Activation , Extracellular Signal-Regulated MAP Kinases/antagonists & inhibitors , Flavonoids/pharmacology , G1 Phase Cell Cycle Checkpoints , Humans , Imidazoles/pharmacology , Isodon/metabolism , JNK Mitogen-Activated Protein Kinases/antagonists & inhibitors , Phosphorylation/drug effects , Plant Preparations/pharmacology , Pyridines/pharmacology , RNA, Messenger/biosynthesis , Toluene/analogs & derivatives , Toluene/pharmacology , Tumor Suppressor Protein p53/antagonists & inhibitors , Tumor Suppressor Protein p53/biosynthesis , Tumor Suppressor Protein p53/genetics , p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors , p38 Mitogen-Activated Protein Kinases/genetics
17.
Food Chem ; 135(2): 730-7, 2012 Nov 15.
Article in English | MEDLINE | ID: mdl-22868152

ABSTRACT

Water-soluble polysaccharides of Rabdosia serra leaf and stem were fractionated by ultrafiltration and DEAE-Sepharose fast flow chromatogram to obtain water (RSLP-I and RSSP-I), 0.1M NaCl (RSLP-II and RSSP-II) and 0.2M NaCl (RSLP-III and RSSP-III) eluates. Their molecular weights were determined by high performance gel permeation chromatography. Monosaccharide composition analysis indicated that the water eluates comprised of rhamnose, arabinose, xylose, mannose, glucose and galactose. The ascending percentage of galactose and descending percentage of glucose in the eluates were observed with the increase of NaCl concentration. The branched RSLP-I and RSSP-I were composed mainly of →6)-Glcp-(1→, →6)-Galp-(1→, and →5)-Araf-(1→ residues with the ratio of 15.0:4.2:3.8 and 5.5:6.4:3.5, respectively. However, a low level of 1,6-linked glucosyl was observed in RSLP-II, RSSP-II, RSLP-III and RSSP-III. The structural characteristics were further analysed by infrared spectrophotometry. The purified leaf and stem polysaccharides possessed moderate antioxidant capacities.


Subject(s)
Antioxidants/chemistry , Isodon/metabolism , Plant Extracts/chemistry , Plant Leaves/metabolism , Plant Stems/metabolism , Polysaccharides/chemistry , Antioxidants/isolation & purification , Antioxidants/metabolism , Carbohydrate Sequence , Isodon/chemistry , Molecular Sequence Data , Molecular Structure , Molecular Weight , Plant Extracts/isolation & purification , Plant Extracts/metabolism , Plant Leaves/chemistry , Plant Stems/chemistry , Polysaccharides/isolation & purification , Polysaccharides/metabolism , Solubility
18.
Int J Mol Sci ; 13(4): 4880-4888, 2012.
Article in English | MEDLINE | ID: mdl-22606016

ABSTRACT

Here we investigated the potential immune-enhancing activity of Isodon japonicus on murine splenocyte and natural-killer (NK) cells in vitro. The ethanol extract of I. japonicus significantly enhanced the proliferation of splenocyte and induced the significant enhancement of NK cells' activity against tumor cells (YAC-1). In addition, I. japonicus increased the production of interferon (IFN)-γ and tumor necrosis factor (TNF)-α, suggesting that the increase in NK cell cytotoxicity could be due to the enhancement of the NK cell production of both cytokines. Taken together, I. japonicus extract inhibited the growth of human leukemia cells (K562) by 74%. Our observation indicated that the anti-tumor effects of I. japonicus may be attributed to its ability to serve as a stimulant of NK anti-tumor activity. In addition, our results support the development of functional food studies on I. japonicus.


Subject(s)
Interferon-gamma/biosynthesis , Isodon/metabolism , Killer Cells, Natural/immunology , Plant Extracts/pharmacology , Tumor Necrosis Factor-alpha/biosynthesis , Animals , B-Lymphocytes/immunology , Cell Line, Tumor , Cell Proliferation/drug effects , HT29 Cells , Humans , Immunologic Factors/pharmacology , Immunomodulation/drug effects , Leukemia/drug therapy , MCF-7 Cells , Male , Medicine, Korean Traditional , Mice , Mice, Inbred C57BL , Spleen/cytology , Spleen/immunology , T-Lymphocytes/immunology
19.
Mol Cancer Ther ; 4(4): 578-86, 2005 Apr.
Article in English | MEDLINE | ID: mdl-15827331

ABSTRACT

This study found that oridonin, a natural diterpenoid purified from Rabdosia rubescens, inhibited growth of multiple myeloma (MM; U266, RPMI8226), acute lymphoblastic T-cell leukemia (Jurkat), and adult T-cell leukemia (MT-1) cells with an effective dose that inhibited 50% of target cells (ED50) ranging from 0.75 to 2.7 microg/mL. Terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling staining showed that oridonin caused apoptosis of MT-1 cells in a time-dependent manner. We explored effects of oridonin on antiapoptotic Bcl-2 family members and found that it down-regulated levels of Mcl-1 and BCL-x(L), but not Bcl-2 protein, in both MT-1 and RPMI8226 cells. Further studies found that oridonin inhibited nuclear factor-kappa B (NF-kappa B) DNA-binding activity in these cells as measured by luciferase reporter gene, ELISA-based, and electrophoretic mobility shift assays. Oridonin also blocked tumor necrosis factor-alpha- and lipopolysaccharide-stimulated NF-kappa B activity in Jurkat cells as well as RAW264.7 murine macrophages. Of note, oridonin decreased survival of freshly isolated adult T-cell leukemia (three samples), acute lymphoblastic leukemia (one sample), chronic lymphocytic leukemia (one sample), non-Hodgkin's lymphoma (three samples), and MM (four samples) cells from patients in association with inhibition of NF-kappa B DNA-binding activity. On the other hand, oridonin did not affect survival of normal lymphoid cells from healthy volunteers. Taken together, oridonin might be useful as adjunctive therapy for individuals with lymphoid malignancies, including the lethal disease adult T-cell leukemia.


Subject(s)
Cell Proliferation/drug effects , Diterpenes/pharmacology , Isodon/metabolism , NF-kappa B/metabolism , Phytotherapy/methods , Plant Extracts/pharmacology , Adult , Aged , Aged, 80 and over , Animals , Apoptosis , Blotting, Western , Cell Line , Cell Line, Tumor , Diterpenes, Kaurane , Dose-Response Relationship, Drug , Enzyme-Linked Immunosorbent Assay , Female , Genes, Reporter , Human T-lymphotropic virus 1/genetics , Human T-lymphotropic virus 1/metabolism , Humans , In Situ Nick-End Labeling , Jurkat Cells , Leukemia/drug therapy , Leukemia/pathology , Lipopolysaccharides/metabolism , Male , Mice , Middle Aged , Models, Chemical , Multiple Myeloma/drug therapy , Multiple Myeloma/pathology , Proto-Oncogene Proteins c-bcl-2/metabolism , Signal Transduction , T-Lymphocytes/metabolism , T-Lymphocytes/virology , Thymidine/chemistry , Thymidine/metabolism , Time Factors , Transfection , Trypan Blue/pharmacology , bcl-X Protein
20.
J Int Med Res ; 32(6): 617-25, 2004.
Article in English | MEDLINE | ID: mdl-15587755

ABSTRACT

Oridonin, an extract from the Chinese herb Rabdosia rubescens, is currently one of the most important traditional Chinese herbal medicines. We investigated the anti-proliferative effect of oridonin on the lung cancer cell line SPC-A-1 and its mechanism of action. Growth inhibition was measured using a microculture tetrazolium assay and apoptosis was measured by several standard methods. Western blot analysis measured the expression of bcl-2 and bax proteins. Oridonin (> 28 micromol/l) inhibited the growth of SPC-A-1 cells and induced apoptosis. Marked morphological changes indicative of apoptosis were observed, especially in cells treated with oridonin for 48 - 60 h. Western blot analysis revealed downregulation of bcl-2 and upregulation of bax proteins following treatment with oridonin for 48 h. We conclude that oridonin demonstrated anti-proliferative and apoptosis-inducing effects on SPC-A-1 cells in vitro, and that changes in bcl-2 and bax protein levels may play an important role in its mechanism of action.


Subject(s)
Diterpenes/pharmacology , Isodon/metabolism , Plant Extracts/pharmacology , Apoptosis , Bisbenzimidazole/pharmacology , Blotting, Western , Cell Line, Tumor , Coloring Agents/pharmacology , DNA Fragmentation , Diterpenes, Kaurane , Dose-Response Relationship, Drug , Down-Regulation , Electrophoresis, Agar Gel , Flow Cytometry , Humans , Microscopy, Electron , Proto-Oncogene Proteins c-bcl-2/metabolism , Tetrazolium Salts/pharmacology , Thiazoles/pharmacology , Time Factors , Up-Regulation , bcl-2-Associated X Protein
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