The sesquiterpene lactone dehydroleucodine triggers senescence and apoptosis in association with accumulation of DNA damage markers.

Sesquiterpene lactones (SLs) are plant-derived compounds that display anti-cancer effects. Some SLs derivatives have a marked killing effect on cancer cells and have therefore reached clinical trials. Little is known regarding the mechanism of action of SLs. We studied the responses of human cancer cells exposed to various concentrations of dehydroleucodine (DhL), a SL of the guaianolide group isolated and purified from Artemisia douglasiana (Besser), a medicinal herb that is commonly used in Argentina. We demonstrate for the first time that treatment of cancer cells with DhL, promotes the accumulation of DNA damage markers such as phosphorylation of ATM and focal organization of γH2AX and 53BP1. This accumulation triggers cell senescence or apoptosis depending on the concentration of the DhL delivered to cells. Transient DhL treatment also induces marked accumulation of senescent cells. Our findings help elucidate the mechanism whereby DhL triggers cell cycle arrest and cell death and provide a basis for further exploration of the effects of DhL in in vivo cancer treatment models

Exploratory study on the effects of biodegradable nanoparticles with drugs on malignant B cells and on a human/mouse model of Burkitt lymphoma.

The aim of this study was to determine if Rituximab coated Biodegradable Nanoparticles (BNPs) loaded with Chlorambucil and Hydroxychloroquine could induce apoptosis of B-Chronic Lymphocytic Leukemia (B-CLL), MEC-1 and BJAB cells in vitro and evaluate their toxic and therapeutic effects on a Human/Mouse Model of Burkitt Lymphoma at an exploratory, proof of concept scale. We found that Rituximab-Chlorambucil-Hydroxychloroquine BNPs induce a decrease in cell viability of malignant B cells in a dose-dependent manner. The mediated cytotoxicity resulted from apoptosis, and was confirmed by monitoring the B-CLL cells after Annexin V/propidium iodide staining. Additional data revealed that these BNPs were non toxic for healthy animals, and had prolonged survival in this mice model of human lymphoma

DhL treatment inhibits cell proliferation in a dose-dependent manner.

<p>Unsynchronized HeLa (A) and MCF-7 cells (B) and synchronized HeLa (C and D) cells were treated with 0, 5, 10, 20, or 30 µM DhL for 72 or 96 h and counted every 24 h. The total number of cells counted each 24 h (Total cells) were compared with the number of viable cells (Viable cells) (D). Data are expressed as the mean ± SEM of 3 independent experiments. (A), (B) and (C) * p≤0.05, ** p≤0.01, *** p≤0.001 vs. control group (0 µM DhL). (D) * p≤0.05, ** p≤0.01, *** p≤0.001 total cells vs. viable cells.</p

DhL induces cellular senescence in HeLa cells.

<p>Synchronized HeLa cells treated with 0–20 µM DhL for 48 h were analyzed for: (A) protein concentration and (B) SA-β-Gal activity (at pH 6) in cell extracts. Equal volumes of supernatants were assayed. C) SA-β-Gal activity at pH 6 <i>in situ</i>. Representative panels for control and treated cells stained for SA-β-Gal and examined by bright field microscopy are shown. Bar: 50 µm. Right: percentages of SA-β-Gal-positive cells. (D) Senescence-associated heterochromatin foci (SAHF) in control and treated cells stained with DAPI and examined by bright field and fluorescence microscopy (merge: bright field/DAPI) and fluorescence microscopy (DAPI). The insets are magnifications of the boxed areas in the DAPI column. Arrowheads indicate the SAHF. Bar: 10 µm. Right: percentages of SAHF-positive cells. (E) Synchronized HeLa cells treated with 20 µM DhL and then with fresh medium plus DMSO (“Fresh medium”) for the time indicated in the upper panel (treatment 1–4) or with fresh medium for 24 h (treatment 5) were analyzed for protein concentration and for SA-β-Gal activity at pH 6. The protein concentration (lower left) and SA-β-Gal activity (lower right) in cell extracts from each treatment are shown. Equal volumes of extract were assayed. Data represents the mean ± SEM of 2 experiments. * p≤0.05, ** p≤0.01, *** p≤0.001 vs. control group (0 µM).</p

Treatment with 30 µM DhL induces apoptosis in HeLa cells.

<p>Unsynchronized (A) and synchronized (B) HeLa cells were treated with 0, 20, or 30 µM DhL for 24 or 48 h. DNA content was assessed by flow cytometry (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053168#pone.0053168.s002" target="_blank">Fig. S2</a>, DNA distributions). The percentages of unsynchronized and synchronized cells in the sub-G1 phase are shown. (C) Synchronized HeLa cells were treated with 0, 20, or 30 µM DhL for 24 or 48 h. Apoptotic cells were assessed by TUNEL assay. Left: representative panels with apoptotic cells indicated by arrowheads. Bar: 50 µm. Right: percentages of apoptotic cells at 24 and 48 h. (D) Unsynchronized HeLa cells were treated as in (C) and subjected to Annexin V assay. Left: representative panels with apoptotic cells stained with Annexin V (bright cells) at 24 h. Representative fields of Annexin V positive cells for 48 and 72 h treatment are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053168#pone.0053168.s002" target="_blank">Fig. S2</a> C. Bar: 50 µm. Right: percentages of apoptotic cells at 24, 48 and 72 h. Data represent mean ± SEM of 3 independent experiments. * p≤0.05, ** p≤0.01, *** p≤0.001 vs. control group (0 µM DhL).</p

20 µM DhL inhibits cell growth by inducing transient arrest in the G2/M phase and permanent accumulation in the G1 phase.

<p>(A) Synchronized HeLa cells were treated with 0–20 µM DhL for 72 h and subjected to DNA flow cytometry at indicated times. Representative DNA distributions from one experiment are shown. (B) Percentage of cells in G1 (left panel), S (middle panel), and G2/M (right panel) phases were determined using the WinMDI 2.9 program. (C) Left: unsynchronized HeLa cells were treated with 0–20 µM DhL and stained with DAPI (to visualize nuclei) and antibodies specific to phospho-H3 at the indicated times. Right: percentage of phospho-H3 positive cells. Bar: 10 µm. (D) Cells were treated with the indicated concentrations of DhL for 48 h, counted, and replated after treatment. Cells that had the ability to form colonies were scored based on clonogenic survival assay 10 days post-treatment. Left: fixed and stained colonies from each treatment representative of 3 independent experiments. Right: number of colonies counted expressed as a percentage of the control (defined as 100%). Data represent mean ± SEM of 3 independent experiments. * p≤0.05, ** p≤0.01, *** p≤0.001 vs. control group (0 µM DhL).</p

DhL delays mitosis entry extends the permanence of mitosis, and downregulates cyclin B1.

<p>Synchronized HeLa cells were treated with 0, 10, or 20 µM DhL and analyzed by phase contrast in live time-lapse microscopy from 0 to 32 h treatment; images were acquired every 15 min using a Nikon Eclipse TE 2000-U microscope. (A) Representative images of cells at the beginning of treatment (T = 0), prophase, metaphase, anaphase, telophase, and cytokinesis. Upper right of each panel: average time (h:min) to reach each phase. Bar: 20 µm. The average times of mitosis and cytokinesis (B) and of the phases of mitosis (C) were calculated by analyzing 4 movies following 200 cells for each treatment. (D) Immunoblot analysis of cyclin B1. Left: immunoblot representative of 3 independent experiments. β-actin was employed as a loading control. Right: mean intensity ± SEM obtained from densitometric analysis of 3 independent experiments. * p≤0.05, ** p≤0.01 vs. control group (0 µM DhL).</p