Inhibition of DNA Topoisomerase Ι by Flavonoids and Polyacetylenes Isolated from Bidens pilosa L.

Human DNA topoisomerase I (Topo I) is an essential enzyme in regulating DNA supercoiling during transcription and replication, and it is an important therapeutic target for anti-tumor agents. Bidens pilosa L. is a medicinal herb that is used as a folk medicine for cancers in China. A new flavonoid (1) and a new polyacetylene (20), along with eighteen flavonoids (2-19) and nine polyacetylenes (21-29), were isolated and identified from the methanol extract of the whole plant of B. pilosa, and some of the compounds (4, 5, 6 and 7) exhibited potent cytotoxicity against a panel of five human cancer cell lines. The DNA relaxation assay revealed that some flavonoids and polyacetylenes exerted inhibitory activities on human DNA Topo I, among them compounds 1, 2, 5, 6, 7, 8, 15, 19, 20, 22, and 24 were the most active ones, with IC50 values of 393.5, 328.98, 145.57, 239.27, 224.38, 189.84, 89.91, 47.5, 301.32, 178.03, and 218.27 µM, respectively. The structure-activity analysis of flavonoids was performed according to the results from the Topo I inhibition assay. The DNA content analysis revealed that 5, 6, and 7 potently arrested cell cycle at the G1/S and G2/M phases in human colon cancer cell DLD-1 depending on the concentration of the inhibitors. The levels of protein expression related to the G1/S and G2/M cell cycle checkpoints were in accordance with the results from the DNA content analysis. These findings suggest that flavonoids are one of the key active ingredients accounting for the anti-tumor effect of B. pilosa.

Cantharidin analogue alleviates dextran sulfate sodium-induced colitis in mice by inhibiting the activation of NF-κB signaling.

Ulcerative colitis is a chronic inflammatory disease with a remitting-relapsing clinical course, it has evolved into a global burden given its high incidence worldwide. Cantharidin (CTD) derivatives are a class of compounds whose structures characterized with a 7-oxabicyclo [2.2.1]heptane core. Though potent cytotoxicity CTD and its derivatives showed, their clinical usage as anti-cancer drugs was limited by the toxicity in organs. In order to find new CTD analogues with good activity and lower toxicity, 21 CTD analogues with or without alkynyl substitution at C5 position of 7-oxabicyclo [2.2.1]heptane core were synthesized, some compounds showed better in vitro anti-inflammatory activity compared to CTD and norcantharidin (NCTD). Based on the structure-activity relationship results of in vitro experiment, analogue 3i was chosen for further study. Results from the acute toxicity in mice showed that 3i was hypotoxic with the single-dose MTD (maximum tolerated dose) for oral administration is over 1852 mg/kg, at least 35-fold lower than that of NCTD. Mechanism study indicated that 3i could potently inhibit TNF-α induced activation of NF-κB signaling by down-regulation the expression levels of phosphor- IKK, IκBα, and NF-κB p65, and alleviated dextran sulfate sodium-induced colitis in mice. This study indicated that CTD analogues with alkynyl substitution at C5 position of 7-oxabicyclo [2.2.1]heptane core is a kind of new compounds with good anti-inflammatory activity and lower toxicity in vivo, and might be used as therapeutic agents for inflammatory diseases.

Photocatalytic synthesis of alkynylsulfones and alkenylsulfones using sulfonylhydrazides and alkynes.

An efficient and energy saving photocatalytic coupling reaction of benzenesulfonyl hydrazide with bromoacetylene has been reported. A series of alkynylsulfones were obtained in up to 98% yield. In addition, changing the base from KHCO3 to KOAc can give the alkenylsulfone product. In addition, we tested the biological activity of some alkynylsulfone compounds and found that they exhibited excellent in vitro antioxidant activity by activating the Nrf2/ARE pathway, up to 8 fold.

Associations of continuous anionic gap detection with the mortality in critically ill patients receiving renal replacement therapy.

PURPOSE: To investigate the associations of anion gap (AG) levels before and 1-day after hemodialysis as well as anion gap changes with the mortality in critically ill patients receiving renal replacement therapy (RRT). METHODS: Totally, 637 patients from MIMIC-III were included in this cohort study. The associations between AG (T0), AG (T1), or ∆AG [AG (T0) - AG (T1)], and the risk of 30-day or 1-year mortality were examined by Cox restricted cubic spline regression models. Univariate and multivariate Cox proportional-hazards model was applied to assess the associations between AG (T0), AG (T1), ∆AG with 30-day and 1-year mortality, respectively. RESULTS: The median follow-up time was 18.60 (8.53, 38.16) days and 263 (41.3%) patients were survived. There was a linear relationship between AG (T0), AG (T1) or ∆AG and the risk of 30-day or 1-year mortality, respectively. The risk of 30-day mortality was higher in AG (T0) > 21 group (HR = 1.723, 95% CI 1.263-2.350), and AG (T1) > 22.3 group (HR = 2.011, 95% CI 1.417-2.853), while lower in AG > 0 group (HR = 0.664, 95% CI 0.486-0.907). The risk of 1-year mortality was increased in AG (T0) > 21 group (HR = 1.666, 95% CI 1.310-2.119), and AG (T1) > 22.3 group (HR = 1.546, 95% CI 1.159-2.064), while decreased in AG > 0 group (HR = 0.765, 95% CI 0.596-0.981). Patients with AG (T0) ≤ 21 had higher 30-day and 1-year survival probability than those with AG (T0) > 21. CONCLUSION: AG before and after dialysis as well as the changes of AG were important factors associated with the risk of 30-day and 1-year mortality in critically ill patients receiving RRT.

3-Deoxysappanchalcone isolated from Caesalpinia sinensis shows anticancer effects on HeLa and PC3 cell lines: invasion, migration, cell cycle arrest, and signaling pathway.

To study the antitumor activity of compound 3-desoxysulforaphane (3-DSC) isolated from Caesalpinia sinensis, SRB assay, clone formation assay, flow cytometric cell cycle assay, scratch assay, transwell assay, and molecular docking were used to investigate the inhibitory effect of 3-DSC on HeLa and PC3 cells. The results showed that 3-DSC inhibited the cell migration and invasion by down-regulating expression of N-cadherin, Vimentin, MMP-2, and MMP-9 in HeLa and PC3 cells; It also inhibits cell proliferation by promoting the expression of CDK1 (cyclin-dependent kinases 1) and CDK2 (cyclin-dependent kinases 2), which arrests the tumor cell cycle at G2 phase. 3-DSC inhibits phosphorylation of AKT and ERK and upregulates the expression of the tumor suppressor gene p53. Molecular docking results confirmed that 3-DSC could bind firmly to AKT. In conclusion, 3-DSC inhibited the proliferation, migration and invasion of HeLa and PC3 cells.

Mechanisms for Bile Acids CDCA- and DCA-Stimulated Hepatic Spexin Expression.

Spexin (SPX) is a novel peptide involved in glucose and lipid metabolism and suppresses hepatic total bile acid levels by inhibiting hepatic cholesterol 7α-hydroxylase 1 expression. As important mediators for glycolysis/gluconeogenesis and lipid metabolism, the effects of bile acids on SPX expression is yet to be understood. By using SMMC7721 and BEL-7402 cell lines, we screened the effects of bile acids and found that chenodeoxycholic acid (CDCA) and deoxycholic acid (DCA) can stimulate SPX gene transcription. Both CDCA and DCA were able to stimulate SPX mRNA expression in the liver but not colon and ileum in mice. In SMMC7721 and BEL-7402 cells, CDCA- and DCA-induced SPX promoter activity was mimicked by bile acid receptor FXR and TGR5 activation and suppressed by FXR and TGR5 silencing. Adenylate cyclase (AC)/cyclic adenosine monophosphate (cAMP) activators significantly increased SPX promoter activity whereas the inhibitors for AC/CAMP/protein kinase A (PKA) and mitogen-activated protein kinases (MAPK) pathway attenuated CDCA- and DCA-induced SPX transcription. Thus, CDCA and DCA stimulate SPX expression at the hepatic level through FXR and TGR5 mediated AC/cAMP/PKA and MAPK cascades.

Alkaloid from Alstonia yunnanensis diels root against gastrointestinal cancer: Acetoxytabernosine inhibits apoptosis in hepatocellular carcinoma cells.

Liver cancer belongs to Gastrointestinal (GI) malignancies which is a common clinical disease, a thorny public health problem, and one of the major diseases that endanger human health. Molecules from natural products (NPs) or their derivatives play an increasingly important role in various chronic diseases such as GI cancers. The chemical composition of the Alstonia yunnanensis Diels roots was studied using silica column chromatography, gel chromatography, recrystallization, and HPLC, and the compounds were structurally identified by modern spectral analysis using mass spectrometry (MS) and nuclear magnetic resonance (1H-, 13C-, HMQC-, HMBC-, and 1H-1HCOSY-NMR), ultraviolet and visible spectrum (UV), and electronic Circular Dichroism (ECD). Acetoxytabernosine (AC), an indole alkaloid with antitumor activity, was isolated from Alstonia yunnanensis Diels root. The current study aimed to investigate the influence of AC on the cell proliferation of BEL-7402 and SMMC7721 and to elucidate the underlying mechanism. The absolute configuration of AC was calculated by ECD (electronic circular dichroism). The effects of AC on the viability of different tumor cell lines were studied by the SRB method. The death mode of human hepatoma cells caused by AC was studied by TUNEL cell apoptosis detection and AnnexinV-FITC/PI double staining image. Mitochondrial membrane potential was detected by JC-1. The effects of AC on the expression of apoptosis-related proteins (Caspase9, Caspase3, and Parp-1) in SMMC7721 and BEL-7402 cells were detected by western blot. It was found that the absolute configuration of AC is 19(s), 20(s)-Acetoxytabernosine. AC could induce apoptosis of SMMC7721 and BEL-7402, and block the replication of DNA in the G1 phase. Under the treatment of AC, the total protein expression of apoptosis-related proteins (Caspase9, Caspase3, and Parp-1) significantly decreased in SMMC7721 and BEL-7402. The results suggested that AC induced apoptosis through a caspase-dependent intrinsic pathway in SMMC7721 and BEL-7402, and natural product-based drug development is an important direction in antitumor drug discovery and research.

Study on the chemical composition of Caesalpinia sinensis.

Five compounds were isolated from the methanolic extract of Caesalpinia sinensis stems and leaves including a new cassane-type butenolide norditerpenoid compound (1) and a new type of biphenyl compound (2); the compounds were identified as Norcaesalpin-one (1), 4'-hexyl 3-methyl 6-methoxy-[1,1'-biphenyl]-3,4'-dicarboxylate (2), rhapontigenin (3), 3-deoxysappanchalcone (4), isoliquiritigenin (5). Compounds 1-5 were first isolated from C. sinensis. Their structures were elucidaded on the basis of MS, IR, NMR spectroscopic, X-ray diffraction data analyses. The NGF-induced PC12 differentiation assay was performed on compound 1, and the results showed that compound 1 had a promotive effect on PC12 cell differentiation, with a differentiation rate of 12.32%. In addition, compounds 1-5 were evaluated for their cytotoxic activities against four human cancer cell lines (including A-549, BGC-823, MDA-MB-231, HepG2), and the results showed that compounds 3-5 showed inhibitory activity against these cancer cell lines with IC50 values ranging from 22.96 to 74.92 µmol/L, compound 4 showing the best activity against human malignant melanoma cells A375 with an IC50 value of 22.96 µmol/L.

Soft elastic hydrogel couplants for ultrasonography.

Couplants play significant roles in ultrasonography. To ensure imaging quality, it is critical to maintain conformal contact of the couplant with both the skin surface and the ultrasound probe in clinical applications. In addition, either the probe or the couplant should not deform the skin surface significantly, which will result in an overestimated modulus of the tissue for elastography imaging. However, existing liquid gel couplants cannot bear external compressive force, while existing solid gel couplants cannot maintain a conformal contact with skin surface. Especially, the nonconformal contacts and deformation become more severe on body parts of locally high curvatures such as skin tumors, fingers, and elbows. Here we report a bilayer design of couplant for ultrasonography, composing of a stiff layer and a compliant layer of hydrogels. The bilayer hydrogel pad enables it to bear external compression, allowing the probe to move smoothly, conforming high curvature parts and releasing stress concentration. Our clinical experiments further show high quality imaging of thyroid nodules, skin tumors in elbows and fingers using the bilayer hydrogel pad, which represents a promising alternative for a range of applications in ultrasonic diagnosis.

Cloning and Expression of the Tibetan Pig Interleukin-23 Gene and Its Promotion of Immunity of Pigs to PCV2 Vaccine.

Vaccines against Porcine circovirus type 2 (PCV2) have been studied intensely and found to be effective in decreasing mortality and improving growth in swine populations. In this study, interleukin-23 (IL-23) gene was cloned from peripheral blood mononuclear cells (PBMCs) of Tibetan pigs and inserted into a eukaryotic VR1020 expression vector-VRIL23. Coated with chitosan (CS), the VRIL23-CS was intramuscularly injected into 3-week-old piglets with PCV2 vaccine. The blood was collected after vaccination at 0, 1, 2, 4, 8, and 12 weeks, respectively, to detect the immunological changes. The IgG2a and specific PCV2 antibodies were detected using ELISA, and blood CD4+ and CD8+ T cells were quantified by flow cytometry. Quantitative fluorescence PCR was used to evaluate the expression of immune genes. The results indicate that leukocytes, erythrocytes, and CD4+ and CD8+ T cells increased significantly in the blood of VRIL23-CS inoculated piglets in comparison with the control (p < 0.05) and so did the IgG2a and PCV2 antibodies. In addition, the expressions of Toll-like receptor (TLR) 2, TLR7, cluster of differentiation (CD) 45, IL-15, IL-12, signal transducer and activator of transcription (STAT)1, STAT2, STAT3, STAT4, and B-cell lymphoma (Bcl)-2 genes were also obviously higher in the VRIL23-CS inoculated pigs at different time points (p < 0.05). Overall, the results demonstrated that VRIL23-CS can enhance the comprehensive immune responses to PCV2 vaccine in vivo and has the promising potential to be developed into a safe and effective adjuvant to promote the immunity of pig against PCV disease.

Spexin as an anxiety regulator in mouse hippocampus: Mechanisms for transcriptional regulation of spexin gene expression by corticotropin releasing factor.

Spexin (SPX) acts as a neuropeptide with pleiotropic functions that can participate in anxiety regulation. Corticotropin releasing factor (CRF) is widely expressed in brain tissues and associated with depression and anxiety and addiction. With the anxious mice under chronic unpredictable stress, we found SPX mRNA expression level in the hippocampus of the brain was significantly reduced, while local CRF mRNA expression level was increased. Furthermore, CRF injection in the hippocampus could also decrease SPX mRNA expression levels in hippocampus and other brain tissues, including pituitary and hypothalamus. With the primary mouse hippocampal cell model, CRF treatment could decrease SPX mRNA expression at hippocampal cell level and this inhibitory effect was mediated only by corticotropin releasing factor receptor 2 (CRFR2) but not corticotropin releasing factor receptor 1 (CRFR1). In HEK293 cells with CRFR2 over-expression, CRF could also inhibit SPX promoter activity coupling with AC/cAMP/PKA and MEK1/2/Erk1/2 cascades. In addition, Epac was also involved with the CRF-repressed SPX promoter activity and cross-talked with MEK1/2/Erk1/2 pathway. CRF could inhibit SPX gene expression in mouse hippocampus via transcriptional activation at the promoter level with coupling of AC/cAMP and MEK1/2/Erk1/2 signaling, which will be relevant to the anxiety response mediated by SPX in central nervous system.

Artesunate activates the ATF4-CHOP-CHAC1 pathway and affects ferroptosis in Burkitt's Lymphoma.

Currently, there is no effective treatment for Burkitt's lymphoma in patients aged above 60 years, and thus research on effective treatment options for Burkitt's lymphoma has been gaining increasing attention. Artesunate has been identified as a novel effective growth suppressor in Burkitt's lymphoma. Here, we utilized molecular biology, transcriptome analysis, and other techniques to study artesunate-induced death of the Burkitt's lymphoma cells DAUDI and CA-46, the effect of artesunate on gene expression in DAUDI and CA-46 cells, and the effect of artesunate-induced ATF4-CHOP-CHAC1 pathway on ferroptosis. We also studied the inhibitory effects and ferroptosis induction of artesunate on CA-46 cells in mouse xenografts. Results showed that artesunate induced ferroptosis in DAUDI and CA-46 cells, as evidenced by the protective effect of liproxstatin-1, ferrostatin-1, and desferoxamine, resulting in an endoplasmic reticulum stress response, activation of the ATF4-CHOP-CHAC1 pathway enhanced ferroptosis in DAUDI and CA-46 cells. A mouse-transplanted tumor model showed that artesunate can inhibit the proliferation and induce ferroptosis of CA-46 cells in vivo. This study provides a novel perspective for the development of drugs against different types of Burkitt's lymphomas.

[A new lupane-type triterpenoid from Dichroa hirsuta].

The chemical constituents from methanol extract of Dichroa hirsuta were separated by silica gel and Sephadex LH-20 column chromatography,high pressure preparative liquid chromatography( HPLC) and recrystallization. Their structures were elucidated by NMR and MS. Nine compounds were obtained and their structures were identified as 3ß,21α-O-diacetyl-lup-9( 11)-en-7ß-ol( 1),( Z)-methyl p-hydroxycinnamate( 2),cis-p-coumaric acid ethyl ester( 3),( E)-methyl p-hydroxycinnamate( 4),trans-p-coumaric acid ethyl ester( 5),4( 3 H)-quinazolinone( 6),7-hydroxycoumarin( 7),hydrangenol( 8) and thunberginol C( 9). Compound 1 is a new lupane-type triterpenoid,and compounds 1-5,8-9 were firstly isolated from this plant. Dual reporter assay results showed that compounds 2-5 could activate the Nrf2-ARE signaling pathway.

[Chemical constituents of Cassia siamea].

A total of ten compounds were isolated from the 90% Et OH extract of Cassia siamea by using various chormatographic techniques,and their structures were established as( 2' S)-2-( propan-2'-ol)-5,7-dihydroxy-benzopyran-4-one( 1),chrobisiamone( 2), 2-( 2'-hydroxypropyl)-5-methyl-7-hydroxychromone( 3), 2,5-dimethyl-7-hydroxychromone( 4), 2-methyl-5-acetonyl-7-hydroxychromone( 5),3-O-methylquercetin( 6),3,5,7,3',4'-pentahydroxyflavonone( 7),luteolin-5,3'-dimethylether( 8),4-( trans)-acetul-3,6,8-trihydroxy-3-methyl-dihydronapht halenone( 9) and 6-hydroxymellein( 10) based on the spectroscopic data.Compound 1 was a new compound,and 3,4,6,8 were isolated from this plant for the first time.

The Cyclopeptide Astin C Specifically Inhibits the Innate Immune CDN Sensor STING.

cGAS-STING signaling is essential for innate immunity. Its misregulation promotes cancer or autoimmune and autoinflammatory diseases, and it is imperative to identify effective lead compounds that specifically downregulate the pathway. We report here that astin C, a cyclopeptide isolated from the medicinal plant Aster tataricus, inhibits cGAS-STING signaling and the innate inflammatory responses triggered by cytosolic DNAs. Moreover, mice treated with astin C are more susceptible to HSV-1 infection. Consistently, astin C markedly attenuates the autoinflammatory responses in Trex1-/- BMDM cells and in Trex1-/- mouse autoimmune disease model. Mechanistically, astin C specifically blocks the recruitment of IRF3 onto the STING signalosome. Collectively, this study characterizes a STING-specific small-molecular inhibitor that may be applied for potentially manipulating the STING-mediated clinical diseases.

[Chemical constituents from roots and rhizomes of Rubia oncotricha and their cytotoxic activities].

Fourteen compounds, including rubiprasin D (1), rubiprasin B (2), rubiprasin C (3), oleanolic acid (4), methyl-5-hydroxy-dinaphtho[1, 2-2'3']furan-7, 12-dione-6-carboxylate (5), rubioncolin C (6), mollugin (7), furomollugin (8), 3-amino-2-methoxycarbonyl-1, 4-naphthoquinone (9), 1-hydroxy-2-methyl-9, 10-anthraquinone (10), 2-hydroxy-6-methyl-9, 10-anthraquinone (11), 1, 4-dihydroxy-2-hydroxymethyl-9, 10-anthraquinone (12), 2-hydroxy-1-methoxy-9, 10-anthraquinone (13), and 1-hydroxy-2-methoxy-6-methyl-9, 10-anthraquinone(14), were isolated from the methanol extract of the roots and rhizomes of Rubia oncotricha using various column chromatographies. Their structures were mainly determined on basis of NMR and MS spectroscopic data analyses. Among them, 1 is a new oleanane triterpene, and compounds 2-5, 9 and 11-13 were obtained from this plant for the first time. Cytotoxic and nematicidal activities of all these compounds were evaluated, and the results showed that only 4, 6, 11 and 12 exhibited cytotoxicities against A549, SGC-7901 and HeLa cancer cell lines. The IC50 of 6 were 19.42, 2.74, 8.07 µmol·L⁻¹, respectively.

Design and synthesis of plant cyclopeptide Astin C analogues and investigation of their immunosuppressive activity.

To further investigate on the structure-activity relationships of immunosuppressive Astin C, seventeen analogues 1-17 were designed and synthetized via amino acid substitution strategy by the solid-phase peptide synthesis method for the first time. In comparison with Astin C (IC50 = 12.6 ±â€¯3.3 µM), only compounds 2 (IC50 = 38.4 ±â€¯16.2 µM), 4 (IC50 = 51.8 ±â€¯12.7 µM), 5 (IC50 = 65.2 ±â€¯15.6 µM), and 8 (IC50 = 61.8 ±â€¯12.4 µM) exhibited immunosuppressive activity in the Lymph node cells of mice. These results showed that the Astin C analogues containing D-amino acid residues, hydrophobic long-chain alkyl substituents, and aryl substituents performed better than those carrying hydrophilic amino acid residues and short-chain alkyl substituents. Moreover compounds 15, 16, and 17 had no immunosuppressive activity, which suggested that cis-3,4-dichlorinated proline played an important role in the immunosuppressive activity of Astin C.

Natural cyclopeptide RA-V inhibits the NF-κB signaling pathway by targeting TAK1.

Rubiaceae-type cyclopeptides (RAs) are a type of plant cyclopeptides from the Rubia that have garnered significant attention owing to their unique bicyclic structures and amazing antitumour activities. Our recent work has shown that RAs suppress inflammation and angiogenesis and induce apoptosis. However, the underlying mechanism and targets remained unknown. Nuclear factor κB (NF-κB) signaling pathway plays a critical role in these biological processes, prompting us to investigate whether and how RAs affect this pathway. By screening compound libraries using NF-κB-dependent luciferase reporter, we observed that RA-V is the best NF-κB inhibitor. Further experiments demonstrated that RA-V interrupted the TAK1-TAB2 interaction and targeted TAK1 in this pathway. Moreover, RA-V prevented endotoxin shock and inhibited NF-κB activation and tumor growth in vivo. These findings clarify the mechanism of RA-V on NF-κB pathway and might account for the majority of known bioactivities of RA-V, which will help RA-V develop as new antiinflammatory and antitumour therapies.

Enhancement of immune response of piglets to PCV-2 vaccine by porcine IL-2 and fusion IL-4/6 gene entrapped in chitosan nanoparticles.

In order to develop a novel effective immunomodulator to enhance pig resistance against post-weaning multi systemic wasting syndrome (PMWS), a recombinant plasmid co-expressing pig interleukin-2 (IL-2) and fusion interleukin-4/6 (IL-4/6) genes, designated VRIL4/6-2, was constructed and encapsulated in chitosan (CS) nanoparticles prepared by the ionotropic gelation method. Then 21-day old piglets were divided into two groups and intramuscularly injected respectively with VPIL4/6-2-CS and saline along with the porcine circovirus-2 (PCV-2) vaccine. The blood was collected from each piglet on days 0, 7, 14, 28, 56 and 84 after vaccination to assay the immunological changes. Content of IgG2a, CD4+, CD8+ T cells increased significantly in the sera or blood of piglets treated with VPIL4/6-2-CS (P<0.05). Furthermore, the expression level of IL-2, IL-4, IL-6, IL-15, TLR-2, TLR-7, Bcl-2, TNF-α, CD45 and STATs (STAT1, STAT2, STAT3, STAT4) genes were significantly elevated in the treated piglets respectively in different days after inoculation (P<0.05). The growth weight gain of the treated piglets was markedly improved in comparison with the controls (P<0.05). These indicate that VPIL-4/6-2 entrapped with chitosan nanoparticles is a safe and promising effective adjuvant to promote the immune response of pig to PCV-2 vaccination.

NF-κB and JNK mediated apoptosis and G0/G1 arrest of HeLa cells induced by rubiarbonol G, an arborinane-type triterpenoid from Rubia yunnanensis.

ETHNOPHARMACOLOGICAL RELEVANCE: Rubia yunnanensis is a medicinal plant mainly grown in Yunnan province in Southwest China, and its root named "Xiaohongshen" has been used as a herb in Yunnan for the treatment of cancers. Three major types of chemical components, Rubiaceae-type cyclopeptides, quinones, and triterpenoids, were identified from R. yunnanensis, in which some of compounds including rubiarbonol G (RG), a unique arboriane-type triterpenoid, showed cytotoxicity on cancer cells. But the cytotoxic mechanism of RG has not been reported. AIM OF THE STUDY: To investigate the cytotoxic mechanism of RG on cancer cells. MATERIALS AND METHODS: RG was evaluated its cytotoxicity on 7 cancer cell lines by the SRB assay, and detected the effect on apoptosis and cell cycle arrest by Annexin V-FITC/PI apoptosis assay and DNA contents analysis. The expression and activity of apoptosis and cell cycle related proteins were also investigated by western blot and caspase activity assay. Furthermore, the effect of RG on NF-κB signaling was also tested by luciferase assay, western blot, and immunofluorescence staining. RESULTS: RG showed potent cytotoxicity on 7 human cancer cell lines, whose activity was attributed to apoptosis induction and G0/G1 arrest in HeLa cells. Results from the mechanism study showed that RG promoted the activation of ERK1/2 and JNK pathway in MAPK family, which in turn increased the expression of p53, thereby triggering the G0/G1 arrest through p53/p21/cyclin D1 signaling. Moreover, RG-mediated JNK activation down-regulated the expression of the anti-apoptotic protein Bcl-2, which caused the release of cytochrome c to the cytosol and activated the cleavage of caspase cascade and poly(ADP-ribose) polymerase, thereby inducing apoptosis in HeLa cells. In addition, RG was also found to inhibit the activation of NF-κB signaling by down-regulating the expression and attenuating the translocation to nucleus of NF-κB p65, by which the down-stream p53, cyclin D1, Bcl-2, and caspases were regulated, thereby triggering apoptosis and G0/G1 arrest in HeLa cells. CONCLUSION: These results indicated that RG induces mitochondria-mediated apoptosis and G0/G1 cell cycle arrest by activation of JNK signaling as well as inactivation of NF-κB pathway in HeLa cells, which suggests that RG is one of the key active ingredients accounting for the anti-tumor effect of R. yunnanensis.