Total coumarins of Pileostegia tomentella induces cell death in SCLC by reprogramming metabolic patterns, possibly through attenuating ß-catenin/AMPK/SIRT1.

BACKGROUND: Small-cell lung cancer (SCLC) is a high malignant and high energy-consuming type of lung cancer. Total coumarins of Pileostegia tomentella (TCPT) from a traditional folk medicine of Yao minority, is a potential anti-cancer mixture against SCLC, but the pharmacological and molecular mechanism of TCPT remains largely unknown. METHODS: Screening of viability inhibition of TCPT among 7 cell lines were conducted by using CCK-8 assays. Anti-proliferative activities of TCPT in SCLC were observed by using colony formation and flow cytometry assays. Morphological changes were observed by transmission electron microscope and Mito-Tracker staining. High Throughput RNA-seq analysis and bio-informatics analysis were applied to find potential targeted biological and signaling pathways affected by TCPT. The mRNA expression of DEGs and protein expression of signalling proteins and metabolic enzymes were verified by qPCR and Western blot assays. Activity of rate-limiting enzymes and metabolite level were detected by corresponding enzyme activity and metabolites kits. Xenograft nude mice model of SCLC was established to observe the in vivo inhibition, metabolism reprogramming and mechanism of TCPT. RESULTS: TCPT treatment shows the best inhibition in SCLC cell line H1688 rather than other 5 lung cancer cell lines. Ultrastructural investigation indicates TCPT induces mitochondria damage such as cytoplasm shrinkage, ridges concentration and early sight of autolysosome, as well as decrease of membrane potential. Results of RNA-seq combined bio-informatics analysis find out changes of metabolism progression affected the most by TCPT in SCLC cells, and these changes might be regulated by ß-catenin/AMPK/SIRT1 axis. TCPT might mainly decline the activity and expression of rate-limiting enzymes, OGDH, PDHE1, and LDHA/B to reprogram aerobic oxidation pattern, resulting in reduction of ATP production in SCLC cells. Xenograft nude mice model demonstrates TCPT could induce cell death and inhibit growth in vivo. Assimilate to the results of in vitro model, TCPT reprograms metabolism by decreasing the activity and expression of rate-limiting enzymes (OGDH, PDHE1, and LDHA/B), and attenuates the expression of ß-catenin, p-ß-catenin, AMPK and SIRT1 accordance with in vitro data. CONCLUSION: Our results demonstrated TCPT induces cell death of SCLC by reprograming metabolic patterns, possibly through attenuating master metabolic pathway axis ß-catenin/AMPK/SIRT1.

Total coumarins in Pileostegia tomentella inhibits proliferation of small cell lung cancer H1688 cells by inducing ferroptosis / 中国药理学通报

Aim To explore the mechanism by which total coumarins in Pileostegia tomentella (TCPT) inhibits the proliferation of small cell lung cancer (SCLC) H1688 cells via inducing ferroptosis. Methods The gradient concentrations of TCPT were used to treat H1688 cells. CCK-8 assay was applied for detection of proliferative inhibition of H1688 cells. Transmission electron microscopy was used to approach the morphological changes of H1688 cells under the treatment of TCPT. Additionally, dichlorofluorescein (DCFH-DA) probe was used to detect the intracellular reactive oxygen species (ROS) level. BODIPY 581/ 589 Cll probe was applied to examine the intracellular lipid peroxide formation. Western blotting was employed to detect the expression levels of glutathione peroxidase 4 (GPX4), kelch-like ECH-associated protein (KEAP1), nuclear factor E2 related factor 2 (NRF2), ferritin heavy chain 1 (FTH1) proteins in HI688 cells. Results The proliferation of small cell lung cancer cell H1688 was dramatically inhibited after TCPT intervention (P < 0. 05, P < 0. 01). The morphological characteristics of ferroptosis induced by TCPT were observed by transmission electron microscope. TCPT could also effectively elevate intracellular level of ROS and lipid peroxide. In HI688 cells the expression of ferroptosis markers GPX4, NRF2, and FTH1 was down-regulated, while the expression of KEAP1 was up-regulated, and there were statistically significant differences among the markers mentioned a-bove (P<0. 01). Conclusions Total coumarins in TCPT can significantly inhibit the proliferation of H1688 cells, possibly through increasing ROS and intracellular lipid peroxide levels and eventually inducing ferroptosis.

Effects of three aqueous extraction preprocessing methods on total coumarins extract of Angelicae dahuricae Radix / 中成药

AIM To investigate the effects of aqueous extraction at normal temperature,40 ℃,and water boiling on total coumarins extract of Angelicae dahuricae Radix.METHODS For the extracts Ⅰ-Ⅳ obtained by ethanol reflux extraction and three aqueous extraction preprocessing methods,respectively,UV and HPLC were adopted in the content determination of total coumarins and imperatorin,then the powder properties and dissolution rates were compared.RESULTS The contents of total coumarins in four extracts were 3.98％,6.03％,6.81％ and 4.46％,while those of imperatorin were 0.633％,0.540％,0.465％ and 0.155％,respectively.The angles of repose were 48.455°,42.587°,42.689° and 42.024° with the bulk densities of 0.214,0.324,0.316 and 0.354 g/cm3,respectively.Within 100 min,extract Ⅰ demonstrated higher moisture adsorption rate and equilibrium moisture absorption content than the other three extracts.The accumulative dissolution rates of various extracts were much higher than that of medicinal material fine powder within 120 min.CONCLUSION All the three aqueous extraction preprocessing methods can obviously improve the powder properties of total coumarins extract of A.dahuricae Radix and increase the dissolution rate of medicinal material fine powder.

Total coumarins of Hedyotis diffusa induces apoptosis of myelodysplastic syndrome SKM-1 cells by activation of caspases and inhibition of PI3K/Akt pathway proteins.

ETHNOPHARMACOLOGICAL RELEVANCE: Hedyotis diffusa is an ethno-medicine used for anti-cancer treatment in the clinic of traditional Chinese medicine (TCM). The total coumarins of Hedyotis diffusa (TCHD) was a selected extract with observed antiproliferative activity, which has not been tested in treatment of myelodysplastic syndromes (MDS) or acute myeloid leukemia (AML). AIM OF THE STUDY: This study aimed to evaluate the apoptosis-inducing effect of TCHD on human MDS cell line (SKM-1) and explore its action mechanism in association with caspase family and PI3K/Akt signaling pathway. MATERIALS AND METHODS: The chemical constituents and total coumarins content of TCHD were determined by High Performance Liquid Chromatography-tandem mass spectrometry (HPLC-MS/MS) and UV-vis spectrophotometry, respectively. MTT assay, Hoechst 33258 staining, and Annexin V-FITC/PI double labeling were applied to evaluate TCHD's efficacy on SKM-1 cells. Western blot analysis was also used to clarify the action mechanism of TCHD on protein expression level. RESULTS: Two compounds, p-coumaric acid and E-6-O-p-coumaroyl scandoside methyl ester, were identified in TCHD, and its total coumarins content reached 87.4%. By MTT assay, apoptosis-inducing effect of TCHD on SKM-1 cells was found in a dose-dependent manner after 24-48h treatment, with IC50 values of 104.48µg/ml and 100.66µg/ml, respectively. Morphological and flow cytometry observation also confirmed such effect of TCHD. Western blot analysis clarified its action mechanism associating with the activation of caspases and inhibition of PI3K/Akt pathway proteins. CONCLUSIONS: This is the first report regarding the apoptosis-inducing efficacy and mechanism of TCHD on SKM-1 cells, providing a promising candidate of TCM for MDS and AML therapy with fewer side effects.

Quality standard for Weigela japonica var.sinica / 中成药

AIM To establish the quality standard for Weigela japonica Thunb.var.sinica (Rehd.) Bailey (W.j.).METHODS TLC was adopted in this medicinal material's qualitative identification after morphological identification and microscopic identification.The contents of water,total ash,acid-insoluble ash and extracts were detected according to Chinese Pharmacopoeia methods.Then the contents of scopoletin and total coumarins were determined by HPLC and UV,respectively.RESULTS The morphologies and microscopic characters of W.j.could be distinguished from other same generic plants.The clear TLC spot displayed good resolution.The contents of water,total ash,acid-insoluble ash,water-soluble extract and acid-soluble extract were no more than 12.0％,no more than 2.0％,no more than 0.5％,no less than 5.0％ and no less than 4.5％,respectively.Scopoletin and total coumarins showed good linear relationships within the ranges of 1.25-40.0 μg/mL(r =0.999 7)and 2.0-64.0 μg/mL (r =0.999 9),whose average recoveries were 98.19％ (RSD =0.90％) and 99.21％ (RSD =2.5％),respectively.CONCLUSION This accurate and reliable method can be used for the quality control of W.j..

Proliferation Inhibition and Its Mechanism of Total Coumarins on Leukemia Cells / 中国药学杂志

OBJECTIVE: To explore the effect of total coumarins isolated from Hedyotis diffusa (total coumarins from Hedyotis diffusa, TCHD) on proliferation inhibition of leukemia cells, and to explore its related mechanism. METHODS: The purity of TCHD prepared by ethanol reflux extraction was tested by ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) system. The cells (KG-1 Kasumi-1, THP 1 cells, U937 cells and K562 cells) were treated with TCHD(0.02, 0.04, 0.06, 0.08, 0.10 mg·mL-1) for 24 or 48 h, the inhibitive effect of TCHD on cells growth were determined by MTT method. After Kasumi-1 cells were incubated with TCHD for 24 h, the apoptosis of cells were analyzed by flow cytometry stained with Annexin V/PI. The expression levels of caspase-3, caspase-8, caspase-9, PARP and Bcl-2 family protein were assayed by Western blot. RESULTS: TCHD in certain concentration range could markedly inhibit the proliferation of AML cells, their IC50 on Kasumi-1, THP-1 KG-1, U937 and K562 cells were 0.077, 0.083, 0.096, 0.087, 0.096 mg·mL-1 for 24 h, and 0.059, 0.067, 0.072, 0.064, 0.068 mg·mL-1 for 48 h. TCHD has significant inhibitory effect on Kasumi-1, which was stronger than those on other cell lines, and showed a dose- and time-dependent manner(r=0.357, P<0.05). The apoptotic proportion of Kasumi-1cells in 0, 0.02, 0.04, 0.06, 0.08, 0.10 mg·mL-1 TCHD treatment groups for 24 h were (5.33±0.41)%, (7.99±0.45)%, (10.22±0.32)%, (20.10±1.99)%, (28.66±0.67)% and (33.24±2.12)%, respectively. After treated with TCHD(0.02-0.06 mg·mL-1) for 24 h, G0/G1 phase ratio of Kasumi-1 detected by flow cytometry were (51.43±3.21)%, (62.91±2.35)% and (76.42±4.14)%, respectively, which were significantly higher than that of the control group (35.8±5.25)% (P<0.05).Western blot results showed that different concentrations of TCHD could activate caspase-8, caspase-9, caspase-3 and PARP, promote the expression of cyto-C, down-regulate the cyclin E and CDK6, CDK2, p-CDK2 and cyclin D1 protein, and up-regulate the expression of p21 proteinin concentration- dependent manner(P<0.01). CONCLUSION: TCHD can obviously inhibit the proliferation of Kasumi-1 in a dose- and time-dependent manner, which may relate to the apoptosis of Kasumi 1 induced by activating caspase-3, 9, PARP protein through the mitochondrial pathways and Kasumi-1 cell block in G0/G1 phase through the influence of CDK2, p-CDK2, CDK4/6, cyclin E, cyclin D1 and p21.