6-O-angeloylplenolin inhibits osteoclastogenesis in vitro via suppressing c-Src/NF-κB/NFATc1 pathways and ameliorates bone resorption in collagen-induced arthritis mouse model.

One of the effective therapeutic strategies to treat rheumatoid arthritis (RA)-related bone resorption is to target excessive activation of osteoclasts. We discovered that 6-O-angeloylplenolin (6-OAP), a pseudoguaianolide from Euphorbia thymifolia Linn widely used for the treatment of RA in traditional Chinese medicine, could inhibit RANKL-induced osteoclastogenesis and bone resorption in both RAW264.7 cells and BMMs from 1 µM and protect a collagen-induced arthritis (CIA) mouse model from bone destruction in vivo. The severity of arthritis and bone erosion observed in paw joints and the femurs of the CIA model were attenuated by 6-OAP administered at both dosages (1 or 5 mg/kg, i.g.). BMD, Tb.N and BV/TV were also improved by 6-OAP treatment. Histological analysis and TRAP staining of femurs further confirmed the protective effects of 6-OAP on bone erosion, which is mainly due to reduced osteoclasts. Molecular docking indicated that c-Src might be a target of 6-OAP and phosphorylation of c-Src was suppressed by 6-OAP treatment. CETSA and SPR assay further confirmed the potential interaction between 6-OAP and c-Src. Three signaling molecules downstream of c-Src that are vital to the differentiation and function of osteoclasts, NF-κB, c-Fos and NFATc1, were also suppressed by 6-OAP in vitro. In summary, the results demonstrated that the function of c-Src was disrupted by 6-OAP, which led to the suppression of downstream signaling vital to osteoclast differentiation and function. In conclusion, 6-OAP has the potential to be further developed for the treatment of RA-related bone erosion.

Pentacyclic triterpene-amino acid derivatives induced apoptosis and autophagy in tumor cells, affected the JNK and PI3K/AKT/mTOR pathway.

A series of pentacyclic triterpene-amino acid derivatives were synthesized and tested for anti-proliferative activity. The results showed that most of the target compounds had good anti-proliferative activity. 2c did not contain protecting groups and hydrochloride, had excellent cytotoxicity, so it had been selected for further study in the mechanism of action in T24 cells. The data from transcriptome sequencing indicated that 2c was found to be closely related to apoptosis and autophagy. Observation of fluorescence staining and analysis from flow cytometry demonstrated that 2c induced apoptosis and cause cell cycle arrest in S/G2 phase in T24 cells. Molecular mechanism studies exhibited that 2c induced apoptosis in the intrinsic and extrinsic pathways. 2c also induced cellular autophagy in T24 cells. Results from Western Blotting showed that 2c could activate JNK pathway and inhibit PI3K/AKT/mTOR pathway. In conclusion, 2c was deserved further investigation in the field of anti-tumor.

Spectrum-Effect Relationship Analysis of Bioactive Compounds in Zanthoxylum nitidum (Roxb.) DC. by Ultra-High Performance Liquid Chromatography Mass Spectrometry Coupled With Comprehensive Filtering Approaches.

Zanthoxylum nitidum (Roxb.) DC. (ZN), with strong effects of anti-inflammation and antioxidant activities is treated as a core herb in traditional Chinese medicine (TCM) preparation for treating stomachache, toothache, and rheumatoid arthritis. However, the active ingredients of ZN are not fully clarified due to its chemical complexity. In the present study, a double spectrum-effect analysis strategy was developed and applied to explore the bioactive components in herbs, and ZN was used as an example. Here, the chemical components in ZN were rapidly and comprehensively profiled based on the mass defect filtering-based structure classification (MDFSC) and diagnostic fragment-ion-based extension approaches. Furthermore, the fingerprints of 20 batches of ZN samples were analyzed by high-performance liquid chromatography, and the anti-inflammatory and antioxidant activities of the 20 batches of ZN samples were studied. Finally, the partial least squares regression (PLSR), gray relational analysis models, and Spearman's rank correlation coefficient (SRCC) were applied to discover the bioactive compounds in ZN. As a result, a total of 48 compounds were identified or tentatively characterized in ZN, including 35 alkaloids, seven coumarins, three phenolic acids, two flavonoids, and one lignan. The results achieved by three prediction models indicated that peaks 4, 12, and 17 were the potential anti-inflammatory compounds in ZN, whereas peaks 3, 5, 7, 12, and 13 were involved in the antioxidant activity. Among them, peaks 4, 5, 7, and 12 were identified as nitidine, chelerythrine, hesperidin, and oxynitidine by comparison with the standards and other references. The data in the current study achieved by double spectrum-effect analysis strategy had great importance to improve the quality standardization of ZN, and the method might be an efficiency tool for the discovery of active components in a complex system, such as TCMs.

Design, Synthesis, and Evaluation of Novel 2-Methoxyestradiol Derivatives as Apoptotic Inducers Through an Intrinsic Apoptosis Pathway.

In order to discover novel derivatives in the anti-tumor field, reported anti-tumor pharmacophores (uridine, uracil, and thymine) were combined with 2-methoxyestradiol, which has been characterized as having excellent biological properties in terms of anti-tumor activity. Thus, 20 hybrids were synthesized through etherification at the 17ß-OH or 3-phenolic hydroxyl group of 2-methoxyestradiol, and evaluated for their biological activities against the human breast adenocarcinoma MCF-7 cell lines, human breast cancer MDA-MB-231 cell lines, and the normal human liver L-O2 cell lines. As a result, all the uridine derivatives and single-access derivatives of uracil/thymine possessed good anti-proliferative activity against tested tumor cells (half maximal inhibitory concentration values from 3.89 to 19.32 µM), while only one dual-access derivative (21b) of thymine possessed good anti-proliferative activity (half maximal inhibitory concentration ≈ 25 µM). Among them, the uridine derivative 11 and the single-access derivative of uracil 12a possessed good anti-proliferative selectivity against tested tumor cells. Furthermore, basic mechanism studies revealed that hybrids 11 and 12a could induce apoptosis in MCF-7 cells through mitochondrial pathway. These hybrids induced morphological changes in MCF-7 cells, causing mitochondrial depolarization. These two hybrids also had the following effects: arrest of the cell cycle at the G2 phase; upregulation of Apaf-1, Bax, and cytochrome c; downregulation of Bcl-2 and Bcl-xL for both mRNA and protein; and increase of the expression for caspase-8 and -9. Finally, apoptotic effector caspase-3 was increased, which eventually caused nuclear apoptosis at least through an intrinsic pathway in the mitochondria. Additionally, hybrids 11 and 12a could specifically bind to estradiol receptor alpha in a dose-dependent manner.

Synthesis and antitumor activity of fluorouracil - oleanolic acid/ursolic acid/glycyrrhetinic acid conjugates.

Due to the obvious adverse effects of 5-fluorouracil that limit its clinical usefulness and considering the diverse biological activities of pentacyclic triterpenes, twelve pentacyclic triterpene-5-fluorouracil conjugates were synthesized and their antitumor activities were evaluated. The results indicated that all the single substitution targeted hybrids (7a-12a) possessed much better antiproliferative activities than the double substitution targeted hybrids (7b-12b). Hybrid 12a exhibited good antiproliferative activities against all the tested MDR cell lines. Furthermore, it was revealed that 12a could induce intracellular calcium influx, the generation of ROS, arrest the cell proliferation at the G1 phase, and activate the apoptotic signaling caspase-8, which eventually activates the apoptotic effector caspase-3 and causes the later nuclear apoptosis.

In Silico Prediction of Chemical Toxicity Profile Using Local Lazy Learning.

BACKGROUND: Chemical toxicity is an important reason for late-stage failure in drug R&D. However, it is time-consuming and expensive to identify the multiple toxicities of compounds using the traditional experiments. Thus, it is attractive to build an accurate prediction model for the toxicity profile of compounds. MATERIALS AND METHODS: In this study, we carried out a research on six types of toxicities: (I) Acute Toxicity; (II) Mutagenicity; (III) Tumorigenicity; (IV) Skin and Eye Irritation; (V) Reproductive Effects; (VI) Multiple Dose Effects, using local lazy learning (LLL) method for multi-label learning. 17,120 compounds were split into the training set and the test set as a ratio of 4:1 by using the Kennard-Stone algorithm. Four types of properties, including molecular fingerprints (ECFP_4 and FCFP_4), descriptors, and chemical-chemical-interactions, were adopted for model building. RESULTS: The model 'ECFP_4+LLL' yielded the best performance for the test set, while balanced accuracy (BACC) reached 0.692, 0.691, 0.666, 0.680, 0.631, 0.599 for six types of toxicities, respectively. Furthermore, some essential toxicophores for six types of toxicities were identified by using the Laplacian-modified Bayesian model. CONCLUSION: The accurate prediction model and the chemical toxicophores can provide some guidance for designing drugs with low toxicity.

Synthesis of acyl oleanolic acid-uracil conjugates and their anti-tumor activity.

BACKGROUND: Oleanolic acid, which can be isolated from many foods and medicinal plants, has been reported to possess diverse biological activities. It has been found that the acylation of the hydroxyl groups of the A-ring in the triterpene skeleton of oleanolic acid could be favorable for biological activities. The pyrimidinyl group has been constructed in many new compounds in various anti-tumor studies. RESULTS: Five acyl oleanolic acid-uracil conjugates were synthesized. Most of the IC50 values of these conjugates were lower than 10.0 µM, and some of them were even under 0.1 µM. Cytotoxicity selectivity detection revealed that conjugate 4c exhibited low cytotoxicity towards the normal human liver cell line HL-7702. Further studies revealed that 4c clearly possessed apoptosis inducing effects, could arrest the Hep-G2 cell line in the G1 phase, induce late-stage apoptosis, and activate effector caspase-3/9 to trigger apoptosis. CONCLUSIONS: Conjugates of five different acyl OA derivatives with uracil were synthesized and identified as possessing high selectivity toward tumor cell lines. These conjugates could induce apoptosis in Hep-G2 cells by triggering caspase-3/9 activity.Graphical abstractFive acyl oleanolic aicd-uracil conjugates were synthesized. These conjugates exhibited selective cytotoxicity toward tumor cells achieved via inducing apoptosis by activation of caspase-3/9.

Antiproliferative activity and apoptosis inducing effects of nitric oxide donating derivatives of evodiamine.

The first series of nitric oxide donating derivatives of evodiamine were designed and prepared. NO releasing ability of all target derivatives was evaluated in BGC-823, Bel-7402 and L-02 cells. The cytotoxicity was evaluated against three human tumor cell lines (Bel-7402, A549 and BGC-823) and normal human liver cells L-02. The nitrate derivatives 11a and 11b only exhibited moderate activity and furoxan-based derivatives 13a-c, 14a and 14b showed promising activity. 13c showed good cytotoxic selectivity between tumor and normal liver cells and was further investigated for its apoptotic properties on human hepatocarcinoma Bel-7402 cells. The molecular mode of action revealed that 13c caused cell-cycle arrest at S phase and induced apoptosis in Bel-7402 cells through mitochondria-related caspase-dependent pathways.

Conjugation of Uridine with Oleanolic Acid Derivatives as Potential Antitumor Agents.

According to fused two bioactive moieties together by bonds covalently and available as a new single hybrid entity known as pharmacophore hybridization, a total of 10 targeted uridine-oleanolic acid hybrids were synthesized. Most of these hybrids showed excellent proliferation inhibition against tested Hep-G2, A549, BGC-823, MCF-7, and PC-3 tumor cell lines (IC50 < 8 µm), even with some IC50 values under 0.1 µm. The detection of cytotoxicity selectivity revealed that hybrids 5 and 18 exhibited low cytotoxicity toward normal human liver cell HL-7702. Further studies revealed that selected hybrid 5 could induce apoptosis in Hep-G2 cells through the investigation of acridine orange/ethidium bromide, Hoechst 33258 fluorescence stainings, and annexin V/propidium iodide assay. It was also found that hybrid 5 could induce mitochondrial membrane potential disruption, arrest Hep-G2 cell line at G1 phase, and activate effector caspase-3/9 to trigger cell apoptosis.

Synthesis of oleanolic acid dimers linked at C-28 and evaluation of anti-tumor activity.

Five dimeric oleanolic acids linked at C-28 by 1,6-hexanediamine, or built around the carbon chains of varying lengths between two carboxyl groups were synthesized, to investigate the effect of internal spacer length and species upon the stereochemical features and anti-tumor activity of the resultant bis-oleanolic acids. The IC50 values of these dimeric compounds for cytotoxicity evaluation in vitro against Hep-G2, A549, BGC-823, MCF-7 and PC-3 tumor cell lines, were mainly under 10.0 µM. This result was much better than the inhibition of proliferation against tested tumor cell lines of the monomer oleanolic acid and the commercial anticancer drug 5-fluorouracil. The cytotoxicity selectivity detection revealed that dimer 11c exhibited low cytotoxicity towards normal human liver cell HL-7702. A combination of fluorescence staining observation and flow cytometric analysis indicated that 11c could induce Hep-G2 cell apoptosis. Molecular mechanism studies suggested that 11c induced apoptosis is mediated through the intrinsic apoptotic pathway with changes in mitochondrial membrane potential by finally activating effector caspase-3/9 to trigger cell apoptosis. Further studies revealed that 11c caused cell cycle arrest at G1 phase in Hep-G2 cells. Taken together, these results suggest that 11c may be a potential candidate for further cancer research.