A Bis-benzopyrroloisoquinoline Alkaloid Incorporating a Cyclobutane Core and a Chlorophenanthroindolizidine Alkaloid with Cytotoxic Activity from <i>Ficus fistulosa</i> var. <i>tengerensis</i>

Tengerensine (<b>1</b>), isolated as a racemate and constituted from a pair of bis-benzopyrroloisoquinoline enantiomers, and tengechlorenine (<b>2</b>), purified as a scalemic mixture and constituted from a pair of chlorinated phenanthroindolizidine enantiomers, were isolated from the leaves of <i>Ficus fistulosa</i> var. <i>tengerensis</i>, along with three other known alkaloids. The structures of <b>1</b> and <b>2</b> were determined by spectroscopic data interpretation and X-ray diffraction analysis. The enantiomers of <b>1</b> were separated by chiral-phase HPLC, and the absolute configurations of (+)-<b>1</b> and (−)-<b>1</b> were established via experimental and calculated ECD data. Compound <b>1</b> is notable in being a rare unsymmetrical cyclobutane adduct and is the first example of a dimeric benzopyrroloisoquinoline alkaloid, while compound <b>2</b> represents the first naturally occurring halogenated phenanthroindolizidine alkaloid. Compound (+)-<b>1</b> displayed a selective in vitro cytotoxic effect against MDA-MB-468 cells (IC₅₀ 7.4 μM), while compound <b>2</b> showed pronounced in vitro cytotoxic activity against all three breast cancer cell lines tested (MDA-MB-468, MDA-MB-231, and MCF7; IC₅₀ values of 0.038–0.91 μM)

Cudraflavone C inhibits PI3K activity.

<p>The effect of negative control (1%DMSO), Cud C or LY-294002 (100 μM) on p110α/p85α, p110β/p85α, p110δ/p85α, and p120γ PI3K activity were quantified using the PI3K-Glo^™ Class I Profiling Kit. All data represents the mean ± s.d. from at least three independent experiments. Symbol “*” presents the statistical significance concluded from Student’s independent <i>t</i>-test with p-value ≤0.05.</p

Cudraflavone C induces tumor-specific cell death in colorectal cancer cells.

<p>(A) Chemical structure of Cud C. (B) KM12, HT29, Caco-2, HCC2998, HCT116 and SW48 colorectal cancer cells were exposed to various concentrations of Cud C for 72 hours. Cell viability was recorded using CellTitre Glo^® luminescence assay. (C) KM12, Caco-2 and CCD 841 CoN were treated with 0.1% DMSO (control) or 10μM Cud C (Cud C) for 72 hours followed by microscopy analysis (×100 magnification). (D) Apoptotic cell death in KM12, Caco-2 and CCD 841 CoN cells was quantified using Annexin V/7-AAD flow cytometry at 72 hours following treatment. (E) Caspase activities in KM12 and Caco-2 cells were assessed by Caspase Glo^® assay at 72 hours following treatment. (F) 10μM Cud C induced mitochondrial membrane depolarization. Caco-2 and KM12 cells stained with JC-1 at 72 hours after treatment with Cud C. The green dye represents JC-1 monomers in cytoplasm while the red dye represents JC-1 aggregates in nucleus. Cells were observed under fluorescence microscope (×100 magnification). All data represent the mean ± s.d. from at least three independent experiments. Symbol “*” presents the statistical significance concluded from Student’s independent <i>t</i>-test with p-value ≤0.05.</p

Top 20 pharmaceutical perturbagens exhibiting positive correlation to the gene signature induced by Cud C treatment.

<p>Top 20 pharmaceutical perturbagens exhibiting positive correlation to the gene signature induced by Cud C treatment.</p

IC₅₀ of Cud C and 5-fluorouracil in colorectal cancer and non-transformed colon epithelial cells.

<p>IC₅₀ of Cud C and 5-fluorouracil in colorectal cancer and non-transformed colon epithelial cells.</p

Differential gene expression regulated by cudraflavone C in Caco-2 cells.

<p>(A) Heatmaps generated based on the genes regulated by Cud C. Caco-2 cells were exposed to 10 μM Cud C for 48 hours. GeneChip^® Human Transcriptome Array 2.0 (Affymetrix, USA) was applied. Gene expression changes that ≥2-fold were considered significant. Control 1 and Control 2 represent gene expression from cells treated with vehicle control (1% DMSO); Cud C 1 and Cud C 2 were gene expression from cells treated with Cud C (10 μM). (B) qPCR was used to validate the microarray data. Caco-2 cells were exposed to 10 μM Cud C for 12, 24, 48 or 72 hours The left and right panels present genes that are up and down-regulated respectively. All data represent the mean ± s.d. from at least three independent experiments. Symbol “*” indicates the statistical significance concluded from Student’s independent <i>t</i>-test with p-value ≤0.05.</p

Additional file 10 of Buffy coat signatures of breast cancer risk in a prospective cohort study

Additional file 10: Fig. S3. ROC curves for the tested classifiers. Individual ROC curves are shown for each cross-validation fold. SVM: support vector machines; PLR: penalized logistic regression; NNET: neural network; RF: random forests; LogitBoost: boosted logistic regressison; KNN: k-nearest neighbours; PAM: Prediction Analysis for Microarrays; RPART: classification and regression tre

Additional file 6 of Buffy coat signatures of breast cancer risk in a prospective cohort study

Additional file 6. Supplementary Table 6. Top GO Biological Processes, GO Cellular Components, GO Molecular Functions, Reactome, and KEGG Pathways enriched from significantly differentially methylated regions (FDR 0.075) identified when overlapping DMRs identified in the main analysis with DMRs identified when samples were limited to participants aged 50 and above at recruitment

Additional file 7 of Buffy coat signatures of breast cancer risk in a prospective cohort study

Additional file 7: Fig. S2. Relative proportions of hypermethylated, hypomethylated and all regions of the dataset when annotated by Enhancer status as annotated in the FANTOM5 enhancer atlas for the GM12878 human lymphoblastoid cell line; andgenic annotations

Additional file 1 of Buffy coat signatures of breast cancer risk in a prospective cohort study

Additional file 1. Supplementary Methods, Supplementary Tables and References