Paclitaxel (Tocris, Bristol, United Kingdom) stock (6 mg/mL) was prepared by dissolving Paclitaxel in a solution comprised 50% Cremophor EL and 50% absolute ethanol and stored at −20°C for a maximum of 14 days. On the day of the experiment, Paclitaxel stock was diluted to 0.2 mg/mL in normal saline (NaCl 0.9%) just before injection. The vehicle for Paclitaxel which comprised a mixture of Cremophor EL and ethanol was diluted at the time of injection with normal saline in the same proportion as the Paclitaxel solution. Paclitaxel (2 mg/kg) or its vehicle was intraperitoneally (i.p.) injected into mice at a dose of 10 μL/g body mass, once per day for 5 consecutive days.
Paclitaxel
Manufactured by Bio-Techne
Sourced in United Kingdom
Paclitaxel is a purified natural product derived from the bark of the Pacific yew tree. It is a mitotic inhibitor that functions by disrupting the normal breakdown of microtubules during cell division. Paclitaxel is commonly used as a reference standard in various research and development applications.
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Lab products found in correlation
Cisplatin, by Bio-Techne (5 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (4 mentions)
Docetaxel, by Bio-Techne (4 mentions)
Vincristine, by Bio-Techne (4 mentions)
Verapamil, by Merck Group (3 mentions)
Vinblastine, by Bio-Techne (3 mentions)
Cremophor el, by Merck Group (3 mentions)
3h paclitaxel, by Moravek Biochemicals (2 mentions)
Anti ctla4 antibody clone 9h10, by BioXCell (2 mentions)
Colchicine, by Bio-Techne (2 mentions)
Mitoxantrone, by Bio-Techne (2 mentions)
3 4 5 dimethylthiazol yl 2 5 diphenyltetrazolium bromide mtt, by Merck Group (2 mentions)
Monoclonal antibody against gapdh, by Cell Signaling Technology (2 mentions)
Cisplatin, by Merck Group (2 mentions)
Vinblastine sulfate, by Merck Group (1 mentions)
Demecolcine, by Merck Group (1 mentions)
Nocodazole, by Bio-Techne (1 mentions)
Bovine brain tubulin, by Cytoskeleton (1 mentions)
Peroxidase conjugated affinipure f ab 2 fragment goat anti mouse igg h l, by Jackson ImmunoResearch (1 mentions)
Hydroxypropyl methylcellulose, by Spectrum Chemical (1 mentions)
37 protocols using paclitaxel
1
Paclitaxel Administration Protocol
2
Synthesis and Characterization of Maytansinoid Compounds
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S-methyl DM1 and other maytansinoids were synthesized as described previously [6 (link)]. 3[H]-S-methyl DM1 was synthesized at American Radiolabeled Chemicals. 1,2-dioleoyl-sn-glycero-3-phosphocholine in chloroform (Avanti Polar Lipids) was stored under argon at-20o C. 3[H]-Paclitaxel was from Moravek Biochemicals. MCF7 cell tubulin and bovine brain tubulin (>99%; isolated by the method described in [10 (link)]) were purchased from Cytoskeleton, Inc. Paclitaxel was from Tocris; nocodazole, vinblastine sulfate, demecolcine, monoclonal anti-α-tubulin clone B-5–1–2 murine IgG1 T6074, RIPA buffer from Sigma; Halt Protease & Phospatase Inhibitor Cocktail (no EDTA or other chelators) was from Thermo Scientific; DilC18(3) and MagicMark XP protein size marker were purchased from Invitrogen; peroxidase-conjugated AffiniPure F(ab’)2 fragment goat anti mouse IgG (H+L) 115–036–062 was purchased from Jackson Labs; ECL Advanced Blocking Agent CPK1075 was purchased from GE Healthcare.
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S-methyl DM1 and other maytansinoids were synthesized as described previously [6 (link)]. 3[H]-S-methyl DM1 was synthesized at American Radiolabeled Chemicals. 1,2-dioleoyl-sn-glycero-3-phosphocholine in chloroform (Avanti Polar Lipids) was stored under argon at-20o C. 3[H]-Paclitaxel was from Moravek Biochemicals. MCF7 cell tubulin and bovine brain tubulin (>99%; isolated by the method described in [10 (link)]) were purchased from Cytoskeleton, Inc. Paclitaxel was from Tocris; nocodazole, vinblastine sulfate, demecolcine, monoclonal anti-α-tubulin clone B-5–1–2 murine IgG1 T6074, RIPA buffer from Sigma; Halt Protease & Phospatase Inhibitor Cocktail (no EDTA or other chelators) was from Thermo Scientific; DilC18(3) and MagicMark XP protein size marker were purchased from Invitrogen; peroxidase-conjugated AffiniPure F(ab’)2 fragment goat anti mouse IgG (H+L) 115–036–062 was purchased from Jackson Labs; ECL Advanced Blocking Agent CPK1075 was purchased from GE Healthcare.
3
Preclinical Evaluation of Chemotherapeutics
4
Paclitaxel-Induced Neuropathic Pain Model
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Paclitaxel (Tocris, Bristol, UK) was dissolved in a solution made up of 50% Cremophor EL and 50% absolute ethanol to a concentration of 6 mg/ml and stored at −20°C, for a maximum of 14 days, and then diluted in normal saline (NaCl 0.9%), to a final concentration of 0.2 mg/ml just before administration. The vehicle for Paclitaxel was diluted at the time of injection with normal saline in the same proportion as the Paclitaxel solution. Paclitaxel 2 mg/kg or its vehicle were administered to mice intraperitoneally (i.p.), in a volume of 10 μl/g body mass, once per day for 5 consecutive days. Other groups and ours have observed that this treatment regimen produce thermal hyperalgesia in mice20 (link)21 (link)22 (link)23 (link)24 (link).
Minocycline (Sigma-Aldrich, St Louis, MO, USA) was dissolved in phosphate buffered saline and administered to mice i.p. in a volume of in a volume of 10 μl/g body mass. Minocycline (50 mg/kg) was administered alone or coadministered with Paclitaxel daily for 5 days. This dose of Minocycline has been shown to prevent the development of Paclitaxel-induced neuropathic pain18 (link).
Minocycline (Sigma-Aldrich, St Louis, MO, USA) was dissolved in phosphate buffered saline and administered to mice i.p. in a volume of in a volume of 10 μl/g body mass. Minocycline (50 mg/kg) was administered alone or coadministered with Paclitaxel daily for 5 days. This dose of Minocycline has been shown to prevent the development of Paclitaxel-induced neuropathic pain18 (link).
5
Salinomycin Analogue 20-Ethyl Carbonate-Na: Anti-Cancer Stem Cell Potential
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The salinomycin analogue 20-ethyl carbonate-Na (SAEC) was kindly provided by Björn Borgström and Daniel Strand at the Centre for Analysis and Synthesis, Department of Chemistry, Lund University [18 (link),19 (link)]. SAEC was used because of its higher toxicity and anti-cancer stem cell specificity compared to the mother compound salinomycin [18 (link),19 (link)]. A 10 mM stock solution was made in 100 % dimethyl sulfoxide (DMSO), which was stored at −20 °C. A serial dilution using PBS with 5000, 2500, 1000, 500, 10, 50, and 10 nM concentrations was prepared and used. The conventional chemotherapeutic drug, paclitaxel was obtained from Tocris Bioscience (Abingdon, United Kingdom). A 100 mM stock solution was made in 100 % DMSO which was stored at −20 °C. A serial dilution using PBS with 8000, 2000, 500, 125, 31.2, 0.78, and 0.19 nM concentrations was prepared and used.
6
Crizotinib, Paclitaxel, and Doxorubicin Cytotoxicity Assay
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Crizotinib, paclitaxel, and doxorubicin were purchased from Tocris Bioscience Company (Bristol, UK). 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) was obtained from Sigma Aldrich (St Louis, MO, USA). Primary antibodies for Ki-67, MET, and phospho-MET as well as goat anti-rabbit Alexa Fluor®594 secondary antibody, and Fluoroshield mounting medium with DAPI were purchased from Abcam (Cambridge, MA, USA).
7
Pharmacological Modulation of Capsaicin-Induced Ablation
8
Establishment and Characterization of Cell Lines
9
Cal51 Cell Line Characterization
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Cal51 cells expressing stably integrated RFP-tagged histone H2B and GFP-tagged a-tubulin were generated as previously described (Zasadil et al., 2014 (link)). Cells were maintained at 37 ºC and 5% CO2 in a humidified, water-jacketed incubator and propagated in Dulbecco’s Modified Eagle’s Medium (DMEM) – High Glucose formulation (Cat #: 11965118) supplemented with 10% fetal bovine serum and 100 units/mL penicillin-streptomycin. Paclitaxel (Tocris Bioscience, Cat #: 1097/10) used for cell culture experiments was dissolved in DMSO. The Cal51 cells were obtained from the DSMZ-German Collection of Microorganisms and Cell Cultures and were free from mycoplasma contamination prior to study. Karyotype analysis confirms the near-diploid characteristic of the cell line and the presence of both fluorescent markers suggests they are free of other contaminating cell lines.
10
Epigenetic Modulation and Drug Resistance in R-Cancer Cells
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To induce epigenetic modulation in the R-cancer cells, the cells were differentiated by the withdrawal of the stem cell medium, mTeSR1, and its replacement with complete RPMI medium.
For the colony-formation assay, the R-cancer cells were induced to differentiate for 3, 7, 13, and 40 days. On the next day post-cell counting, after the transfer of the cells in complete RPMI medium to freshly prepared Matrigel-coated 10 mm dishes, cisplatin (5 μM, Tocris) or paclitaxel (5 nM, Tocris) was added to the differentiated R-cancer (dR-cancer) cells for 3 days. After drug treatment, the cells were maintained in complete RPMI media for 30 days. The colonies were stained with 0.5% crystal violet staining solution, and images were obtained as described previously14 .
For the stability testing of drug resistance or expression profiling, cisplatin-resistant (CR) colonies were identified and selected under microscopy on a clean bench and maintained in complete RPMI medium.
For the colony-formation assay, the R-cancer cells were induced to differentiate for 3, 7, 13, and 40 days. On the next day post-cell counting, after the transfer of the cells in complete RPMI medium to freshly prepared Matrigel-coated 10 mm dishes, cisplatin (5 μM, Tocris) or paclitaxel (5 nM, Tocris) was added to the differentiated R-cancer (dR-cancer) cells for 3 days. After drug treatment, the cells were maintained in complete RPMI media for 30 days. The colonies were stained with 0.5% crystal violet staining solution, and images were obtained as described previously14 .
For the stability testing of drug resistance or expression profiling, cisplatin-resistant (CR) colonies were identified and selected under microscopy on a clean bench and maintained in complete RPMI medium.
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