Two parental and their subtypes highly chemotherapy refractory cell lines LAN-1WT, LAN-1RETO neuroblastoma and 79HF6WT, 79HF6RETO glioblastoma multiforme derived from human tumors were used in this investigation. The LAN-1 cells were isolated from a bone marrow metastasis of a 2-year-old boy with neuroblastoma (clinical Stage IV), and the 79HF6 cells were isolated from a female adult patient. The etoposide-resistant sublines usyed in this work exhibit CSC features among a set of CSC markers, broad spectrum of cross-resistance to several cytostatics, and radioresistance. The phenotype characteristics and the CSC features were published previously (5 (link)). Cells were cultured in Dulbecco’s modified Eagle medium (DMEM), supplemented with 10% fetal calf serum (FCS) and 1% penicillin/streptomycin (all purchased from Biochrom AG, Berlin, Germany), and kept in a humidified atmosphere of 5% CO2 at 37°C. Resistant to etoposide (RETO) cells were constantly cultured in the medium containing 4 μg/ml etoposide (Teva, Germany). The cell doubling time (tD) was determined in the exponential phase of the growth with the GSI in house program gd (©M. Krämer, 2003).
Etoposide
Manufactured by Teva Pharmaceuticals
Sourced in Germany, United Kingdom
Etoposide is a laboratory reagent used for various research and experimental applications. It is a topoisomerase II inhibitor, which plays a role in DNA replication and cell division processes. Etoposide is commonly utilized in scientific investigations, but a detailed description of its core function would require more specific information about the intended use, which is not provided.
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Lab products found in correlation
Cisplatin, by Teva Pharmaceuticals (3 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
L glutamine, by Merck Group (2 mentions)
Doxorubicin, by Merck Group (2 mentions)
Dulbecco s modified eagle medium dmem, by Harvard Bioscience (1 mentions)
Penicillin streptomycin, by Harvard Bioscience (1 mentions)
Fetal calf serum (fcs), by Harvard Bioscience (1 mentions)
Abiplatin, by Teva Pharmaceuticals (1 mentions)
Melphalan, by Merck Group (1 mentions)
Azacitidine, by Merck Group (1 mentions)
Cytarabine, by Merck Group (1 mentions)
Ponatinib, by Bio-Techne (1 mentions)
Vincristine, by Pfizer (1 mentions)
Paclitaxel, by Pfizer (1 mentions)
Trichloroacetic acid tca, by Thermo Fisher Scientific (1 mentions)
Insulin, by Merck Group (1 mentions)
Sorafenib, by LC Laboratories (1 mentions)
Nilotinib, by LC Laboratories (1 mentions)
Ab9722, by Abcam (1 mentions)
Anakinra, by Amgen (1 mentions)
14 protocols using etoposide
1
Characterization of Chemotherapy-Resistant Neuroblastoma and Glioblastoma Cell Lines
2
DNA Damage Agents Treatment Protocol
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DNA-damage agents used in this study were; DOX (2 µM) (Sigma-Aldrich) dissolved in 100% DMSO (Sigma-Aldrich); Etoposide (30 µM) (TEVA); Abiplatin (60 µM) (TEVA). Cells were subjected to this treatment for 3 h (or with the appropriate vehicle control). After reagent removal, cells were washed once with PBSx1 and released to fresh medium for 24 h and then analyzed.
3
3D Tumor Model Chemotherapy Evaluation
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Chemotherapy treatments were performed by incubating 3D cultures in growth medium with the indicated dosage of carboplatin (NDC: 61703-360-22; NOVAPLUS, Irving, TX, USA) diluted to 5–2560 μM, paclitaxel (NDC: 61703-342-22; Hospira Worldwide, Inc. Lake Forest, IL, USA), etoposide (NDC: 0703-5653-01; Teva Pharmaceuticals, Irvine, CA, USA) diluted to 1–100 μM, or gemcitabine (NDC: 0781-3282-75; Eli Lily and Company, Indianapolis, IN, USA) diluted to 0.01 to 1000 μM. Chemotherapy agents were allowed to incubate for 72 hours with the exception of caroboplatin incubations which were conducted for a total of 96 hours with a refresh at 48 hours. The incubation times chosen for this study are consistent with previous reports and reflect the observation that 3D tumor models typically require a longer period of drug exposure relative to traditional monolayer cultures and these treatment schedules more closely resemble in vivo therapeutic assessment scenarios19 (link)40 (link)42 (link). All chemotherapy drugs were clinical grade formulations purchased from the central pharmacy at the Massachusetts General Hospital. Treatment response was assessed immediately after removal of the chemotherapeutic agent.
4
Cytotoxicity Assay for Anticancer Drugs
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Azacitidine (Sigma), cisplatin (Teva), cytarabine (Sigma), etoposide (Teva), fludarabine (Sagent), melphalan (Sigma), methotrexate (Hospira), paclitaxel (Hospira), vincristine (Hospira), 5-FU (Teva), doxorubicin (APP Pharmaceuticals), and anakinra (Amgen) were stored according to manufacturer’s recommendation. Lipopolysaccharide (LPS) from E. coli serotype 0111:B4 was purchased from Enzo Life Sciences. Nilotinib and sorafenib (LC Laboratories) and ponatinib (Tocris Bioscience) were dissolved in DMSO and stored at −80 °C. Insulin was purchased from Sigma. Trichloroacetic acid (TCA) was purchased from Fisher Scientific. Antibody against IL-1β (ab9722) was purchased from Abcam, phospho-JNK (9251) and phospho-p38 MAPK (9211) from Cell Signaling Technology, and p38 MAPK (sc-535) from Santa Cruz Biotechnology.
5
Generating Chemoresistant RB Cell Lines
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All chemoresistant RB cell lines characterized were generously provided by Dr H. Stephan. To generate these cell lines, established Y-79, WERI-Rb1 and RB-355 cells (see above) were continuously treated with consecutively increasing concentrations of etoposide or cisplatin (both from Teva, Berlin, Germany) until the chemoresistant sublines exhibited a at least 10-fold higher IC50 value in WST-1 viability assays than the respective parental controls (11 (link)). The chemoresistant cell lines were subsequently cultivated as described above for RB cell lines with additional treatment of the appropriate cytostatic drug twice a week (every 3–4 days). For details on final concentrations of the drugs used, see Table I .
6
Murine Lymphoma Cell Death Assay
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Murine lymphoma (EL4)
cells (ATCC, Piscataway, NJ) were propagated
in RPMI 1640 media (Sigma) supplemented with 10% fetal calf serum
(FCS) and 2 mMl -glutamine (Sigma). Cell number and viability
were monitored using the trypan blue dye exclusion assay on a Vi-cell
system (Beckman Coulter, Brea, CA). EL4 cell death was induced by
addition of 10 μM etoposide (Teva, Leeds, UK) for 18 h at 37
°C. In preparation for flow cytometry, cells (10 million) were
then pelleted (600 g, 4 °C, 4 min), washed in ice-cold HBS+ buffer
(HBS, 2 mM CaCl2, 1% FCS), and resuspended in 100 μL of the
same buffer containing either C2Am-Cys95 or C2Am-NHBn95 (2 μM),
in combination with 50 nM of the necrosis probe Sytox green (Life
Technologies, Grand Island, NY) for 15 min at 37 °C, with orbital
shaking (300 r.p.m.). The resulting cell suspension was washed twice
with cold HBS+ buffer, kept briefly on ice, and analyzed in a LSRII
cytometer (BD Biosciences, Rockville, MD), equipped with 488 and 630
nm lasers and counting 20 000 cells per event.
cells (ATCC, Piscataway, NJ) were propagated
in RPMI 1640 media (Sigma) supplemented with 10% fetal calf serum
(FCS) and 2 mM
were monitored using the trypan blue dye exclusion assay on a Vi-cell
system (Beckman Coulter, Brea, CA). EL4 cell death was induced by
addition of 10 μM etoposide (Teva, Leeds, UK) for 18 h at 37
°C. In preparation for flow cytometry, cells (10 million) were
then pelleted (600 g, 4 °C, 4 min), washed in ice-cold HBS+ buffer
(HBS, 2 mM CaCl2, 1% FCS), and resuspended in 100 μL of the
same buffer containing either C2Am-Cys95 or C2Am-NHBn95 (2 μM),
in combination with 50 nM of the necrosis probe Sytox green (Life
Technologies, Grand Island, NY) for 15 min at 37 °C, with orbital
shaking (300 r.p.m.). The resulting cell suspension was washed twice
with cold HBS+ buffer, kept briefly on ice, and analyzed in a LSRII
cytometer (BD Biosciences, Rockville, MD), equipped with 488 and 630
nm lasers and counting 20 000 cells per event.
7
DNA Damage Response in Myogenic Differentiation
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All treatments and differentiation were performed in GM. Idoxuridine (IdU) and bromo-deoxyuridine (BrdU) were purchased from Sigma-Aldrich (#I7125, #B5002) and dissolved in DMSO at final concentration of 25 mM. Thymidine (Sigma-Aldrich, #T5018) and uridine (Sigma-Aldrich, #U3750) were dissolved in PBS at final concentration of 25 mM. Recombinant BMP-2 were purchased from Pepro-Tech (#120–02) and prepared according to manufacturer’s instructions to final concentration of 200 μg/ml. Experimental concentrations are indicated in Figure legends and Results.
Spontaneous myogenic differentiation was obtained by incubating the cells for additional 5 days after reaching confluence.
The induction of DNA damage was performed by treating cells with 0.5 μM Etoposide (Teva Pharmaceuticals) or 0.5 μM Doxorubicin (Sigma Aldrich, #D1515) for 24 hours. Cisplatin was purchased from Hospira (Lot #Y091881AB; Brussel, Belgium) and used at final concentration of 0.5 μM.
ATM and p38 kinase inhibitors, KU 55933 (Tocris Bioscience, #3544) or SB 203580 (Sigma Aldrich, #S8307) respectively, were used at 10 μM in pre-treatment for 3 hours followed by co-treatment with IdU for the indicated time.
Spontaneous myogenic differentiation was obtained by incubating the cells for additional 5 days after reaching confluence.
The induction of DNA damage was performed by treating cells with 0.5 μM Etoposide (Teva Pharmaceuticals) or 0.5 μM Doxorubicin (Sigma Aldrich, #D1515) for 24 hours. Cisplatin was purchased from Hospira (Lot #Y091881AB; Brussel, Belgium) and used at final concentration of 0.5 μM.
ATM and p38 kinase inhibitors, KU 55933 (Tocris Bioscience, #3544) or SB 203580 (Sigma Aldrich, #S8307) respectively, were used at 10 μM in pre-treatment for 3 hours followed by co-treatment with IdU for the indicated time.
8
Cytotoxic Compound Combination Protocol
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SAHA (SAHA, Merck, Kenilworth, NJ, USA) and Entinostat (MS275, Alexis Biochemicals, Roma, Italy) were dissolved in DMSO (Sigma-Aldrich, Milano, Italy) and used at 5 μm . Etoposide (Teva, Castleford, UK) was used at 34 μm ; all-trans retinoic acid was used at 1 μm ; valproic acid (Sigma-Aldrich) was used at 1 mm .
9
Evaluating Compound-Induced Cell Death
10
Synergistic Antiviral-Cytotoxic Drug Interactions
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Amantadine, ribavirin, pleconaril, lamivudine, and doxorubicin were purchased from Sigma-Aldrich, acyclovir and ganciclovir from HEXAL AG, Holzkirchen, Germany. Retrovir was obtained from ViiV Healthcare, London, UK, Foscavir from Clinigen Healthcare, Staffordshire, UK and brivudine from Berlin Chemie, Germany. Etoposide and cisplatin were purchased from TEVA GmbH and 5FU from Medac, both Hamburg, Germany.
The simultaneous effect of antiviral drugs and cytostatics was analyzed by the isobologram method (50 % isodose) as described previously [30 (link)]. Briefly, the IC50 for both substances were first determined using the MTT proliferation assay. Applying fixed percentages of the IC50 for the first drug (20, 40, 60, 80 and 100 %) and varying the concentration of the second drug from 0.1 to 50 μM, the variation in the resulting IC50 was determined for every percentage. The same procedure was carried out inversely for the second drug. Dose-response curves were then plotted and evaluated.
The simultaneous effect of antiviral drugs and cytostatics was analyzed by the isobologram method (50 % isodose) as described previously [30 (link)]. Briefly, the IC50 for both substances were first determined using the MTT proliferation assay. Applying fixed percentages of the IC50 for the first drug (20, 40, 60, 80 and 100 %) and varying the concentration of the second drug from 0.1 to 50 μM, the variation in the resulting IC50 was determined for every percentage. The same procedure was carried out inversely for the second drug. Dose-response curves were then plotted and evaluated.
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