MMTV-PyMT;Apc+/+ and MMTV-PyMT;ApcMin/+ cells were isolated from primary tumors from the mouse mammary gland [11 (link)] and grown in RPMI 1640 media supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin, and 1:5000 plasmocin (Invivogen, San Diego, CA). All cells were routinely passaged using 0.25% trypsin/EDTA and maintained at 37°C with 5% CO2. Experiments were performed with cells between passage 10–20. Cells were treated for 24 hours with each chemotherapeutic agent or solvent control: doxorubicin (500 nM MP Biomedicals, LLC, Santa Ana, CA) or cisplatin (16 μM; cis-Diammineplatinum (III) dichloride, Sigma-Aldrich) as described previously [12 (link)]. STAT3 DNA binding was blocked by 24-hour treatment with the small molecule inhibitor A69 [25 (link), 26 (link)].
Doxorubicin
Manufactured by MP Biomedicals
Sourced in United States, France
Doxorubicin is a chemotherapeutic agent used in the treatment of various types of cancer. It is a cytotoxic anthracycline antibiotic that works by interfering with the DNA replication process in cancer cells, ultimately leading to their death. The core function of Doxorubicin is to inhibit the growth and proliferation of cancer cells.
Automatically generated - may contain errors
Lab products found in correlation
Cisplatin, by Merck Group (3 mentions)
Plasmocin, by InvivoGen (2 mentions)
Cis diammineplatinum 3 dichloride, by Merck Group (2 mentions)
Paclitaxel, by MP Biomedicals (2 mentions)
Paclitaxel, by Merck Group (2 mentions)
Rpmi 1640 media, by InvivoGen (1 mentions)
Verapamil, by MP Biomedicals (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by MP Biomedicals (1 mentions)
Doxorubicin, by Merck Group (1 mentions)
5 bromo 2 deoxyuridine brdu, by BD (1 mentions)
Sp600125, by Merck Group (1 mentions)
Melphalan, by Merck Group (1 mentions)
Carboplatin, by Merck Group (1 mentions)
Ku 55955, by Selleck Chemicals (1 mentions)
Ve 821, by Selleck Chemicals (1 mentions)
Nu7441, by Bio-Techne (1 mentions)
Etoposide, by Merck Group (1 mentions)
Annexin 5 fitc apoptosis detection kit, by Keygen Biotech (1 mentions)
N acetyl l cysteine nac, by Beyotime (1 mentions)
Mg132, by MedChemExpress (1 mentions)
13 protocols using doxorubicin
1
Breast Cancer Cell Culture Protocol
2
Cytotoxicity Assay for Drug Resistance
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Paclitaxel, doxorubicin, verapamil, and MTT 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyl tetrazolium bromide (MTT) were purchased from MP Biomedicals (Santa Ana, CA). P-gp inhibitors identified though in silico screening were purchased in small quantities through SIA MolPort (Riga, Latvia). Stock solutions (10–100 mmol/L) of all drugs and experimental compounds were prepared in DMSO and stored as aliquots at −20°C. On the day of the experiment, working solutions of compounds prepared in DMSO were further diluted in culture media such that the final DMSO concentration was ≤1% (v/v). MTT solution was prepared as 5 mg/mL in phosphate buffered saline (PBS; 137 mmol/L NaCl, 2.7 mmol/L KCl, 10 mmol/L Na2HPO4, 1.8 mmol/L KH2PO4) and sterile filtered through 0.22 μm nylon filter before use. Cell culture materials were purchased from Corning Inc. (Corning, NY) unless otherwise stated. GraphPad Prism∼ version 6.05 for Windows was used for plotting data and IC50 values were calculated from nonlinear, four-parameter logistic curve fitting (GraphPad Software, La Jolla, CA).
3
Chemotherapeutic agents and inhibitor treatments
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Cells were treated for 24 h with each chemotherapeutic agent or solvent control: doxorubicin (MP Biomedicals, LLC, Santa Ana, CA), paclitaxel (Sigma-Aldrich) or cisplatin (cis-Diammineplatinum (III) dichloride, Sigma-Aldrich). Drug concentrations for MMTV-PyMT-derived cells were 2.5 μM paclitaxel, 16 μM cisplatin, or 500 nM doxorubicin. MDA-MB-157 cells were treated with 0.078 μM paclitaxel, 4 μM cisplatin or 12.5 nM doxorubicin. These drug doses were selected after treatment of the MMTV-PyMT;Apc+/+ cells from 24–72 h showed approximately a 50 % reduction in cell population (data not shown). For the combination treatments, chemical inhibitors were added to the media 18 h after chemotherapeutic agents, resulting in a 6 h treatment with a combination of cisplatin or doxorubicin and 50 μM PP2 (Src inhibitor, Sigma-Aldrich) or 50 μM SP600125 (JNK inhibitor, Sigma-Aldrich). For BrdU incorporation assays, treatment was the same as above with the addition of 5-bromo-2’-deoxyuridine (BrdU, 10 μM, BD Pharmigen, Franklin Lakes, NJ) 8 h after chemotherapeutic agents.
4
LY294002 Inhibition of PI3K Activity
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LY294002 (Bio-Techne Corporation, Danvers MA, USA), a compound derived from the naturally occurring biflavonoid quercetin, which inhibits PI3K activity via competitive inhibition of an ATP binding site [67 (link),68 (link)] was freshly prepared in dimethyl sulfoxide (DMSO) and used at the final concentrations indicated. Doxorubicin (MP Biomedicals, Santa Ana, CA, USA) was prepared in DMSO and used as pro-oxidant control.
5
Chemotherapeutic Agents Preparation Protocol
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Vincristine, vinblastine, etoposide, paclitaxel (Taxol), cisplatin, carboplatin and melphalan were purchased from Sigma-Aldrich (St Louis, MO, USA), and doxorubicin from MP Biomedicals (Santa Ana, CA, USA). Cell culture chemicals were obtained from Gibco (Grand Island, NY, USA), and other reagents from Merck (Darmstadt, Germany). BO-1922 was synthesized according to our published procedure [42 (link)]. cisplatin was freshly prepared by dissolving in dimethyl sulfoxide, immediately diluted with medium, and administrated into the culture dishes or wells.
6
Comparative Analysis of MMTV-PyMT Breast Cancer Cells
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MMTV-PyMT;Apc+/+ and MMTV-PyMT;ApcMin/+ cells were isolated as previously described from primary mouse mammary tumors [11] and grown in RPMI 1640 media supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin, and 1:5000 plasmocin (Invivogen). MMTV-PyMT;ApcMin/+ cells, expressing one truncated and one WT allele of APC, resemble the triple negative subtype of breast cancer cells (Supplemental Figure 1 ) [11] . Cells were passaged using 0.25% trypsin/EDTA and maintained at 37 °C with 5% CO2. Cells were plated and treated 24 hours later with each chemotherapeutic agent or solvent control: doxorubicin (500 nM, MP Biomedicals, LLC), etoposide (10 μM, Sigma), paclitaxel (2.5 μM, Sigma), cisplatin (16 μM, Sigma), VE-821 (10 μM, Selleckchem), KU-55955 (10 μM, Selleckchem), or NU-7441 (5 μM, Tocris). Drug treatments were performed for 24 hours unless otherwise indicated.
7
HMEC-1 Conditioned Media Preparation
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Media in which non-senescent and senescent HMEC-1 cells were cultured (conditioned media) were collected for functional analyses. Briefly, 2 × 104 and 1 × 105 HMEC-1 cells were cultured for 72 h in the presence of vehicle (0.01% DMSO) or doxorubicin (0.05 µM; MP Biomedicals, LLC, Santa Ana, CA, USA), respectively. Following this incubation time, media were replaced with minimum volumes of serum- and doxorubicin-free media, and cells were cultured for an additional 24 or 48 h. Conditioned media were collected and centrifuged for 5 min at 5000× g (D3024R microcentrifuge, SCILOGEX, EEUU) before use. Finally, protein concentrations were estimated by Bradford assays using a BSA-based calibration curve.
8
Fbw7 Expression and Doxorubicin-Induced Apoptosis
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Cells were first transfected with an Fbw7 expression plasmid or vector control, and then Doxorubicin (MP Biomedicals, Illkirch-Graffenstaden France) was used to induce apoptosis. We used the Annexin V-FITC Apoptosis Detection Kit (KeyGEN BioTECH, Nanjing, China), according to the manufacturer’s instructions, and the percentage of apoptotic cells was detected by flow cytometry analysis.
9
Preparation and Storage of Anticancer Agents
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The compound APR-246 and the proteasome inhibitor MG132 were purchased from MedChemExpress (China). Cycloheximide (CHX), carboplatin, doxorubicin, paclitaxel, and fluorouracil (5-FU) were purchased from MP Biomedicals (France). N-acetyl-L-cysteine (NAC) was bought from Beyotime Biotechnology (Beijing, China). For in vitro experiments, 100 mM APR-246 and 750 mM NAC were dissolved in DMSO, and ultrasonic was used for improving its solubility. MG132, CHX, and paclitaxel were dissolved in DMSO to 50 mg/mL, 50 mg/mL, and 100 mg/mL, respectively. Carboplatin (10 mg/mL), doxorubicin (100 mg/mL), and 5-FU (10 mg/mL) were dissolved in water. For in vivo experiments, APR-246 was rinsed with sterilized PBS and ultrasonized into clear solutions at concentration 100 mg/mL, and NAC was directly dissolved at 5 mg/mL in the drinking water. All the reagents were stored at -20°C for long-term preservation.
10
Murine Cell Chemotherapeutic Treatment
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Murine cells were plated and treated 24 h later with each chemotherapeutic agent or a solvent control: doxorubicin (500 nM, MP Biomedicals, LLC), Valspodar (10 μM, Sigma, St. Louis, MO, USA), Reversan (5 μM, Sigma), or A69 (10 μM [33 (link),34 (link)]). Drug treatments were performed for 24 h unless otherwise indicated.
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