Resistant Hep3B cells were developed in clinically relevant models [116 (link)], as previously reported [117 (link)]. In T-75 flasks, Hep3B cells were seeded overnight and incubated at 37 °C. The cells were then treated with sorafenib at the 10% inhibitory concentration (IC10) (0.4 µM) of sorafenib (Biovision, Milpitas, CA, USA #BAY 43-9006) to mimic the clinically consumed low dose of chemotherapeutic drugs by a cancer patient who is then exposed to escalating doses over time [116 (link)]. To develop resistance, the survived cells were transplanted to a new flask and then treated with sorafenib at escalating concentrations over six months. Then, sorafenib at concentration IC10 (0.4 µM) was persistently retained in the culture media to ensure and maintain resistance. MTT cell viability assay was performed each month to validate the resistance behavior of the cells.
Sorafenib
Manufactured by Abcam
Sourced in United States
Sorafenib is a small-molecule inhibitor of multiple kinases, including BRAF, VEGFR, and PDGFR. It is commonly used as a research tool in the study of cell signaling pathways and cancer biology.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Caspase 8, by Cell Signaling Technology (2 mentions)
Phospho erk, by Cell Signaling Technology (2 mentions)
Z vad fmk, by Merck Group (2 mentions)
Caspase 9, by Cell Signaling Technology (2 mentions)
Pd98059, by Merck Group (2 mentions)
Dulbecco s modified eagle medium dmem, by Thermo Fisher Scientific (2 mentions)
Pd173074, by Merck Group (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (2 mentions)
Propidium iodide, by Merck Group (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Ac devd cho, by Merck Group (1 mentions)
Caspase 3, by Novus Biologicals (1 mentions)
Phenylmethylsulfonyl fluoride pmsf, by Merck Group (1 mentions)
Ethylene glycol tetraacetic acid (egta), by Merck Group (1 mentions)
Histone h3, by Cell Signaling Technology (1 mentions)
Ribonuclease a, by Merck Group (1 mentions)
9 protocols using sorafenib
1
Development of Sorafenib-Resistant Hep3B Cells
2
Sorafenib and HDAC Inhibitor Combination Therapy
3
Pancreatic and Prostate Cell Lines Comparison
4
Anticancer Agent Evaluation Protocol
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Antibodies were obtained from the following commercial sources: PARP, caspase-3, caspase-8, caspase-9, phospho-Erk, and Erk (Cell Signaling Technology); p-c-Met, c-Met, and FGFR3 (Santa Cruz Biotechnology); pan-actin (Millipore); phospho-stat3-Ser727 and Stat3 (BD Biosciences). propidium iodide, MTT, z-VAD–FMK, PD98059, PD173074, and all of the other chemical reagents were obtained from Sigma. RPMI-1640 medium, Dulbecco's Modified Eagle Medium (DMEM), FBS, penicillin, streptomycin, and all other tissue culture regents were obtained from GIBCO/BRL Life Technologies. Sorafenib (purity ≥ 99%) was purchased from Biovision. Tivantinib (ARQ 197) was purchased from ChemieTek. For in vitro administration, Sorafenib, tivantinib or DE605 (structure and scheme of DE605 synthesis shown in supplemental Fig. S1 ) were dissolved in DMSO (Sigma) to a concentration of 10 mM and further diluted to appropriate final concentration in RPMI 1640 with 10% fetal bovine serum. DMSO in the final solution did not exceed 0.1% (v/v). For in vivo testing, Sorafenib or DE605 were dissolved in Cremophor EL/ethanol (50:50; Sigma Cremophor EL, 95% ethanol) at 4 × concentration. This 4 × solution was prepared fresh every 4 days. Final dosing concentration was prepared by diluting the 4 × solution to 1× with sterile water. The 1× solution was prepared just before it was given to the mice.
5
Anticancer Effects of Copper Compounds
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The HCC cell lines Huh7 and PLC/PRF/5 were purchased from ATCC (Middlesex, UK). DS, 5-fluorouracil (5-FU), copper chloride (CuCl2), copper gluconate (CuGlu), poly lactic-co-glycolic acid (PLGA), poly(vinyl acetate), cyanomethyl diphenylcarbamodithioate (PVA), dichloromethane, poly-2-hydroxyethyl methacrylate (poly-HEMA) and bovine serum albumin (BSA) were purchased from Sigma (Dorset, UK). DMEM and fetal calf serum (FCS) were supplied by Lonza (Wokingham, UK). Ki-67 and BAX antibodies were purchased from Cell Signaling (Danvers, MA, USA). NFkBp65 and ALDH1 antibodies were from Abcam. Sorafenib was from BioVision (CA, USA). Cell culture inserts were purchased from Fisher (Loughborough, UK). Lipo-DS was provided by Prof. Xing Tang (Shenyang Pharmaceutical University, China).
6
Sorafenib Mass Spectrometry Analysis
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Methanol (≥99.9%), acetonitrile (ACN) and deionized water, as well as LC-MS CHROMASOLV, were purchased from Honeywell (Wunstorfer, Strasse, Seelze, Germany). Trifluoroacetic acid (TFA) and formic acid (FA) were purchased from Fisher Scientific (Loughborough, UK). Hydrochloric acid (HCl) (37%) was purchased from VWR chemicals (France). C18 columns, lysis buffer, Pierce trypsin protease, lysyl-endopeptidase LysC and Pierce protease inhibitor tablets were obtained from Thermo Scientific (Rockford, IL, USA). Bradford’s reagent and bovine serum albumin were bought from Sigma-Aldrich (St. Louis, MO, USA). Sorafenib was obtained from BioVision (Milpitas, CA, USA #BAY 43-9006).
7
Huh7 HCC Cell Sorafenib and Avicularin Treatments
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The Huh7 HCC cells were obtained from the Cell Bank of the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). The cells were cultured in Dulbecco's Modified Eagle Medium supplemented with 10% fetal bovine serum (FBS; both Thermo Fisher Scientific, Inc., Waltham, MA, USA), 1% antibiotics (100 U/ml penicillin and 100 mg/ml streptomycin) and 2 mM glutamine at 37°C in a humidified atmosphere of 5% CO2. The cells were treated with sorafenib (5 µmol/l; BioVision, Inc.) as the positive control, and treated with different concentrations (100, 50 and 25 µg/ml) of avicularin (Aladdin Biochemical Technology, Co., Ltd., Shanghai, China) as the treatment groups.
8
Cell Line and Primary Hepatocyte Cultivation
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The HCC cell lines HepG2 (ATCC HB-8065), PLC (ATCC CRL-8024), Hep3B (ATCC HB-8064) and Huh7 were cultured as described [53 (link)]. Primary human hepatocytes were isolated by the Biobank of the Department of General, Visceral and Transplant Surgery in Ludwig-Maximilians University using a two-step collagenase perfusion technique with modifications [54 (link)].
The murine Hepa129 cell line originates from a C3H/HeN mouse and was obtained from the NCI-Frederick Cancer Research and Development Center (DCT Tumor Repository). The murine Hepa1-6 cell line (ATCC CRL-1830) was also used. Isolation and culture of primary murine hepatocytes (PMH) were performed as described [55 (link)].
For stimulation experiments, cells were treated with trichostatin A (TSA) (Cayman Chemical, Ann Arbor, MI, USA), suberoylanilide hydroxamic acid (SAHA) (Cayman Chemical) and sorafenib (Biovision, Milpitas, CA, USA) at the concentrations and for duration as indicated.
The murine Hepa129 cell line originates from a C3H/HeN mouse and was obtained from the NCI-Frederick Cancer Research and Development Center (DCT Tumor Repository). The murine Hepa1-6 cell line (ATCC CRL-1830) was also used. Isolation and culture of primary murine hepatocytes (PMH) were performed as described [55 (link)].
For stimulation experiments, cells were treated with trichostatin A (TSA) (Cayman Chemical, Ann Arbor, MI, USA), suberoylanilide hydroxamic acid (SAHA) (Cayman Chemical) and sorafenib (Biovision, Milpitas, CA, USA) at the concentrations and for duration as indicated.
9
Sorafenib Cardiac Perfusion Protocol
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Sorafenib (BAY 43-9006) was purchased from BioVision (Milpitas, CA), dissolved in DMSO, and freeze-stored in stock aliquots. The stock solution was added to the perfusate to achieve a final concentration of 1–30 µM with a constant concentration of 0.1% DMSO. Isoproterenol and caffeine were administered at a final concentration of 10 nM and 30 mM, respectively. If not otherwise stated, all chemicals were obtained from Sigma-Aldrich (Vienna, Austria).
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