Journal: Cancer Drug Resistance

Article Title: Daunorubicin can eliminate iPS-derived cancer stem cells via ICAD/CAD-independent DNA fragmentation

doi: 10.20517/cdr.2019.01

Figure Lengend Snippet: Comparison of apoptotic features among L1210, miPS-LLCcm, and Hela cells. A: Mouse leukemia L1210 cells were treated with 1 μmol/L daunorubicin for the indicated periods. Protein levels of activated caspase-3 and its substrate ICAD were determined by western blotting; B: DNA fragmentation in L1210 cells was analyzed. Cells were treated with daunorubicin for 24 or 36 h; C: DNA fragmentation in miPS-LLCcm and L1210 cells treated with staurosporine. Cells were treated with various concentrations of staurosporine for 3 h (miPS-LLCcm) or 12 h (L1210), after which DNA fragmentation was observed; D: Western blot analysis of PARP-1 and ICAD levels in staurosporine-treated cells (3 h for miPS-LLCcm and 12 h for L1210); E: ICAD levels in Hela cells treated with 1 μmol/L daunorubicin for the indicated periods were analyzed by western blotting; F: DNA fragmentation in Hela cells treated with 1 μmol/L daunorubicin

Article Snippet: The mouse leukemia cell line L1210 (JCRB cell bank, Osaka, Japan) was maintained in RPMI1640 containing 10% FBS, 2 mmol/L L-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin.

Techniques: Comparison, Western Blot

Journal: Marine Drugs

Article Title: Another Facet to the Anticancer Response to Lamellarin D: Induction of Cellular Senescence through Inhibition of Topoisomerase I and Intracellular Ros Production

doi: 10.3390/md12020779

Figure Lengend Snippet: Senescence-like growth arrest in cancer cells exposed to LamD. ( A ) May-Grunwald Giemsa staining of P388 cells exposed to 5 µM or 0.2 µM LamD for the indicated times. Arrows show apoptotic phenotype and * show senescence-like phenotype; ( B ) examination of DAPI-stained nuclei in P388 cells exposed to 0.2 µM LamD for 24 h. Original magnification ×400. Numbers indicate the percentage of cells displaying DNA damage foci; ( C ) cell cycle distribution in P388 cells exposed to 0.2 µM LamD for the indicated times; ( D ) kinetics of the effects of LamD (0.2 µM or 5 µM) on ATP levels in P388 cells; * p < 0.05 between two groups.

Article Snippet: The mouse leukemia cell line P388, its topoisomerase I-mutated subclone (P388CPT5) resistant to camptothecin ([ ], a gift from JF Riou, Rhône-Poulenc Rorer, France) and the HBL cutaneous melanoma cell [ , , ] line (kindly provided by Pr.

Techniques: Staining

Journal: Marine Drugs

Article Title: Another Facet to the Anticancer Response to Lamellarin D: Induction of Cellular Senescence through Inhibition of Topoisomerase I and Intracellular Ros Production

doi: 10.3390/md12020779

Figure Lengend Snippet: ( A ) ( left ) Representative flow cytometric profiles of senescent P388 cells treated with 0.2 µM LamD for 24 h (or kept untreated) then stained with C12FDG, a fluorogenic substrate for SA-β-galactosidase before analysis; ( right ) Kinetics of the effects of 0.2 µM LamD on senescence of P388 cells assessed by flow cytometry after C12FDG staining, * p < 0.05 between two groups; ( B ) senescence was assessed in human HBL melanoma cells and SAOS2 osteosarcoma cells upon LamD exposure (20 nM for 72 h) detecting SA-β-galactosidase by cytochemistry; Numbers indicate the percentage of positive cells; ( C ) representative histological sections from tumors of HBL-injected SCID mice treated or not with the indicated doses of LamD for 2 weeks then stained for detection of SA-β-gal as in F and tumor volumes were measured (3 mice per group).

Article Snippet: The mouse leukemia cell line P388, its topoisomerase I-mutated subclone (P388CPT5) resistant to camptothecin ([ ], a gift from JF Riou, Rhône-Poulenc Rorer, France) and the HBL cutaneous melanoma cell [ , , ] line (kindly provided by Pr.

Techniques: Staining, Flow Cytometry, Injection

Journal: Marine Drugs

Article Title: Another Facet to the Anticancer Response to Lamellarin D: Induction of Cellular Senescence through Inhibition of Topoisomerase I and Intracellular Ros Production

doi: 10.3390/md12020779

Figure Lengend Snippet: Senescence-like growth arrest depends on the effects of LamD on topoisomerase I ( A ) P388 cells and P388CPT5 cells were incubated in the presence of 0.2 µM LamD, 0.2 µM camptothecin (CPT) or kept untreated, then cells were counted every day. Data are means ± SD of three independent experiments. * p < 0.05 between two groups; ( B ) cell cycle analysis of P388 and P388CPT5 cells exposed to 0.2 µM LamD or CPT for 24 h. Data are representative of at least four independent experiments; ( C ) immunoblot analysis of cdc25c protein expression in P388 and P388CPT5 cells exposed to 0.2 µM LamD or CPT for 18 h. G3PDH was used as loading control. Data are from one representative of two independent experiments.

Article Snippet: The mouse leukemia cell line P388, its topoisomerase I-mutated subclone (P388CPT5) resistant to camptothecin ([ ], a gift from JF Riou, Rhône-Poulenc Rorer, France) and the HBL cutaneous melanoma cell [ , , ] line (kindly provided by Pr.

Techniques: Incubation, Cell Cycle Assay, Western Blot, Expressing, Control

Journal: Marine Drugs

Article Title: Another Facet to the Anticancer Response to Lamellarin D: Induction of Cellular Senescence through Inhibition of Topoisomerase I and Intracellular Ros Production

doi: 10.3390/md12020779

Figure Lengend Snippet: ( A ) Analysis of senescence by flow cytometry after C12FDG staining of P388 and P388CPT5 cells treated with 0.2 µM LamD or CPT. Data are means ± SD of four independent experiments. * p < 0.05 compared to untreated group; ( B ) LamD, like CPT, induced the expression of P21 in P388 cells. ( left ) RT-PCR analysis of P21 and Bax mRNA expression in P388 and P388CPT5 cells treated with 0.2 µM LamD or CPT for 18 h. β-tubulin was used as control. Data are representative of two independent experiments; ( upper right ) immunoblot analysis of P21 protein expression in P388 cells treated as above. G3PDH served as control; ( lower right ) immunofluorescence analysis of P21 protein expression in P388 cells treated as above then counterstained with DAPI before analysis under the microscope. Data are representative of two independent experiments; ( C ) P388 cells were exposed to 0.2 µM LamD or CPT then at indicated times assayed for telomerase activity using the TRAP assay. IC refers to the 36-bp internal control band.

Article Snippet: The mouse leukemia cell line P388, its topoisomerase I-mutated subclone (P388CPT5) resistant to camptothecin ([ ], a gift from JF Riou, Rhône-Poulenc Rorer, France) and the HBL cutaneous melanoma cell [ , , ] line (kindly provided by Pr.

Techniques: Flow Cytometry, Staining, Expressing, Reverse Transcription Polymerase Chain Reaction, Control, Western Blot, Immunofluorescence, Microscopy, Activity Assay, TRAP Assay

Journal: Marine Drugs

Article Title: Another Facet to the Anticancer Response to Lamellarin D: Induction of Cellular Senescence through Inhibition of Topoisomerase I and Intracellular Ros Production

doi: 10.3390/md12020779

Figure Lengend Snippet: Intracellular generation of ROS in cancer cells treated with LamD ( A ) P388 cells were incubated with 0.2 µM LamD alone or in association with 100 µM Vitamin C (VitC) and at indicated times, cells were stained with H2DCFHDA before flow cytometric analysis; ( B ) P388 cells were either kept untreated or exposed to LamD at indicated doses for 24 h then GSH content was determined by flow cytometry. As control, cells were treated with 10 mM BSO for 12 h to deplete intracellular GSH. Results are expressed as fluorescence as a percentage of untreated cells (mean ± SD of three independent experiments in triplicates); ( C ) representative flow cytometric profiles of P388 cells treated with 0.2 µM LamD for 36 h ( lower panels ) or kept untreated ( upper panels ) then stained with either H2DCFHDA ( right panels ) or MitoSox ( left panels ) before analysis.

Article Snippet: The mouse leukemia cell line P388, its topoisomerase I-mutated subclone (P388CPT5) resistant to camptothecin ([ ], a gift from JF Riou, Rhône-Poulenc Rorer, France) and the HBL cutaneous melanoma cell [ , , ] line (kindly provided by Pr.

Techniques: Incubation, Staining, Flow Cytometry, Control, Fluorescence

Journal: Marine Drugs

Article Title: Another Facet to the Anticancer Response to Lamellarin D: Induction of Cellular Senescence through Inhibition of Topoisomerase I and Intracellular Ros Production

doi: 10.3390/md12020779

Figure Lengend Snippet: ( A ) HBL and HBL ρ0 cells were incubated with increasing doses of LamD (0.05 µM, 0.2 µM, 0.5 µM) for 24 h. As control of the absence of functional mitochondria in HBL ρ0, cells were incubated with the mitochondrial oxidative agent, eleclomol (300 nM for 18 h). ROS generation was detected by flow cytometrica analyses of H2DCFHDA fuorescence. Data are means ± SD of two experiments in triplicates; ( B ) P388 cells were kept untreated or treated with 0.2 µM LamD for 24 h in the presence or absence of one of the following ROS inhibitors; 1 µM Rotenone, 1 µM Antimycin A, 10 µM DPI, 250 µM Apocynin, 1mM Allopurinol, 10 µM Ketoconazol, 10mM L-NAME. Then, cells were stained with H2DCFHDA before analysis. Data are means ± SD of three independent experiments, * p < 0.05; ( C ) flow cytometric profiles of H2DCFHDA fluorescence in HBL cells exposed to 0.2 µM LamD for 18 h in the presence or absence of 100 µM DPI. Profiles are representative of two independent experiments in duplicates.

Article Snippet: The mouse leukemia cell line P388, its topoisomerase I-mutated subclone (P388CPT5) resistant to camptothecin ([ ], a gift from JF Riou, Rhône-Poulenc Rorer, France) and the HBL cutaneous melanoma cell [ , , ] line (kindly provided by Pr.

Techniques: Incubation, Control, Functional Assay, Staining, Fluorescence

Journal: Marine Drugs

Article Title: Another Facet to the Anticancer Response to Lamellarin D: Induction of Cellular Senescence through Inhibition of Topoisomerase I and Intracellular Ros Production

doi: 10.3390/md12020779

Figure Lengend Snippet: LamD-induced ROS generation participates in the occurrence of the senescent phenotype. P388 cells ( A ) or HBL and HBL ρ0 cells; ( B ) were incubated with 0.2 µM LamD alone or in association with 10 µM DPI for 24 h then stained with C12FDG before flow cytometric analysis. Data are means ± SD of three independent experiments. * p < 0.05 compared to control.

Article Snippet: The mouse leukemia cell line P388, its topoisomerase I-mutated subclone (P388CPT5) resistant to camptothecin ([ ], a gift from JF Riou, Rhône-Poulenc Rorer, France) and the HBL cutaneous melanoma cell [ , , ] line (kindly provided by Pr.

Techniques: Incubation, Staining, Control

Journal: Marine Drugs

Article Title: Another Facet to the Anticancer Response to Lamellarin D: Induction of Cellular Senescence through Inhibition of Topoisomerase I and Intracellular Ros Production

doi: 10.3390/md12020779

Figure Lengend Snippet: ( A ) Phase contrast microscopy analysis of HBL ρ0 cells exposed to 0.2 µM LamD for 36 h. (original magnification ×400). Dashed lines delineate representative cells. Note the presence of enlarged cells after LamD exposure; ( B ) P388 and P388 CPT5 cells were incubated with 0.2 µM LamD for 24 h then stained with H2DCFHDA before flow cytometric analysis. Data are means ± SD of two independent experiments in duplicates; * p < 0.05 compared to control.

Article Snippet: The mouse leukemia cell line P388, its topoisomerase I-mutated subclone (P388CPT5) resistant to camptothecin ([ ], a gift from JF Riou, Rhône-Poulenc Rorer, France) and the HBL cutaneous melanoma cell [ , , ] line (kindly provided by Pr.

Techniques: Microscopy, Incubation, Staining, Control

Journal: Marine Drugs

Article Title: Another Facet to the Anticancer Response to Lamellarin D: Induction of Cellular Senescence through Inhibition of Topoisomerase I and Intracellular Ros Production

doi: 10.3390/md12020779

Figure Lengend Snippet: Links between ROS generation and DNA damage induced by LamD ( A ) right Morphological examination of DAPI-stained nuclei in P388 cells exposed to 0.2 µM LamD alone or in the presence of 10 µM DPI for 24 h (original magnification ×400); left P388 cells were treated as above then H2AXgamma positive cells were detected by flow cytometry as described in materials and methods * p < 0.05 compared to control; ( B ) cell cycle distribution in P388 cells exposed to LamD and/or DPI as above.

Article Snippet: The mouse leukemia cell line P388, its topoisomerase I-mutated subclone (P388CPT5) resistant to camptothecin ([ ], a gift from JF Riou, Rhône-Poulenc Rorer, France) and the HBL cutaneous melanoma cell [ , , ] line (kindly provided by Pr.

Techniques: Staining, Flow Cytometry, Control