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Trametinib

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Trametinib is a laboratory reagent used for research purposes. It is a selective inhibitor of the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase (MEK) enzymes, which are involved in cell signaling pathways. Trametinib can be used to study the role of the MAPK/ERK pathway in various biological processes.

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Lab products found in correlation

Vemurafenib, by Apexbio (2 mentions) Trastuzumab, by Apexbio (2 mentions) Bt474, by Apexbio (2 mentions) A8218, by Genentech (2 mentions) Mda mb 361, by Merck Group (2 mentions) Penicillin streptomycin, by Merck Group (2 mentions) Trastuzumab, by Genentech (2 mentions) Mda mb 361, by American Type Culture Collection (2 mentions) Mda mb 453, by American Type Culture Collection (2 mentions) Skbr3, by American Type Culture Collection (2 mentions) Dabrafenib, by Apexbio (1 mentions) Doxorubicin, by Apexbio (1 mentions) Dasatinib, by Merck Group (1 mentions) Ly294002, by Cell Signaling Technology (1 mentions) Azd8330, by Apexbio (1 mentions) Accuri c6 flow cytometer, by BD (1 mentions) Shp099, by Chemietek (1 mentions) Selumetinib, by Selleck Chemicals (1 mentions) Cetuximab, by Merck Group (1 mentions) Anti erk, by Cell Signaling Technology (1 mentions)

15 protocols using trametinib

1

Trametinib Effects on MiaPaCa-2 Cells

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To determine the effects of MEK inhibitor drug Trametinib (ApexBio, Houston, TX) on MiaPaCa-2 cells, a concentrated stock solution of Trametinib was first weighed and dissolved at 100 mM in DMSO. The stock solution was diluted in DMEM media and added to cells at 10 or 50 nM for 24 h, before any analysis was made.
Yong L.K., Lai S., Liang Z., Poteet E., Chen F., van Buren G., Fisher W., Mo Q., Chen C, & Yao Q. (2016). Overexpression of Semaphorin-3E enhances pancreatic cancer cell growth and associates with poor patient survival. Oncotarget, 7(52), 87431-87448.
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2

Cell Viability Assay for Drug Screening

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Cells were seeded in triplicate in 96-well culture plates at 2000 cells per well and grown overnight in the absence of any drug treatment. The following day, the indicated concentration of drug or an equivalent volume of DMSO vehicle control was added and the cells were grown for an additional three days. After three days of treatment, cell viability was assessed using CellTiter-Glo luminescent cell viability agent (Promega) according to manufacturer instructions. Luminescence was measured with a SpectraMax i3x multi-mode plate reader. The cell survival rate of drugged cells was normalized to the DMSO control group. The dose-response curves and IC50 values were generated using Prism (GraphPad). The BRAF inhibitors dabrafenib and vemurafenib, the MEK inhibitors trametinib and binimetinib, and the non-specific chemotherapeutic doxorubicin were obtained from ApexBio and Selleckchem.
Turner E., Chen L., Foulke J.G., Gu Z, & Tian F. (2022). CRISPR/Cas9 Edited RAS & MEK Mutant Cells Acquire BRAF and MEK Inhibitor Resistance with MEK1 Q56P Restoring Sensitivity to MEK/BRAF Inhibitor Combo and KRAS G13D Gaining Sensitivity to Immunotherapy. Cancers, 14(21), 5449.
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3

Apoptosis and Cell Cycle Analysis in HCT116 PTPRS KO Cells

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HCT116 PTPRS KO Cells and control cells were treated with the ERK inhibitors SCH772984 (942183-80-4 Santa Cruz Biotechnology), VX11-E (A3931 ApexBio), VRT752271 (B1106 ApexBio); MEK inhibitors Trametinib (A3018 ApexBio) and AZD8330 (A8374 ApexBio); PI3K inhibitor LY294002 (Cell Signaling 9901), Dasatinib (CDS023389 Sigma) and the AKT inhibitor VIII (A6730 Sigma). All treatments were for 48 hours and the noted concentrations, unless specified otherwise. Following treatments, Annexin 5 apoptosis assay or a cell cycle analysis using Brdu and propidium iodide staining were performed as described previously [42 (link)]. The analyses were performed with a BD Accuri-C6 Flow Cytometer.
Davis T.B., Yang M., Wang H., Lee C., Yeatman T.J, & Pledger W.J. (2019). PTPRS drives adaptive resistance to MEK/ERK inhibitors through SRC. Oncotarget, 10(63), 6768-6780.
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4

Generating Liver Tumors and Evaluating Treatments

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For primary liver cancer, oncogene-expressing DNA constructs were delivered by hydrodynamic tail vein injection into mice at 6–8 weeks of age to generate liver tumors, as described.[21 (link)] For metastasized liver tumors, mice were put under anesthesia and shaved at the left subcostal area. A left subcostal incision in line with the left ear was made through the skin and peritoneum. Then, the inferior half of the spleen was exposed through the incision, and MC38 tumor cells in PBS solution (100 μl) were injected through the inferior end of the spleen over a time period of 1–2 min. In the pharmaceutical treatment, SHP099 (Chemietek, Indianapolis, IN), trametinib (GSK1120212; APExBIO, Houston, TX), and Maraviroc (UK-427857; AdooQ Bioscience, Irvine, CA, USA) were dissolved in DMSO to make stock solutions at 100, 10, and 90 mg/ml. SHP099 was further diluted (1:24) in Ringer’s solution, trametinib was diluted (1:29) in Ringer’s solution, and Maraviroc was diluted in olive oil (1:8) as delivery vehicle. All three molecules were delivered by i.p. injection. Detailed experimental procedures are provided in the Supporting Information. We followed standard protocols to perform immunoblotting, immunostaining, RNA extraction, real-time quantitative PCR analysis, ELISA, and flow cytometry as well as cell culture.
Liu J.J., Xin B., Du L., Chen L., Long Y, & Feng G.S. (2023). Pharmaceutical SH2 domain–containing protein tyrosine phosphatase 2 inhibition suppresses primary and metastasized liver tumors by provoking hepatic innate immunity. Hepatology (Baltimore, Md.), 77(5), 1512-1526.
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5

Elucidating Inflammatory Signaling Pathways

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Erlotinib was purchased from MedChem Express. Cetuximab was from MERCK Serono. Trametinib and vemurafenib were from ApexBio. Selumetinib and MG132 were from Selleckchem. Recombinant human IL-36γ (catalog 6835), mouse IL-36γ (catalog 6996), and human IL-36Ra (catalog 1275) were from R&D Systems. Pam3CSK4 was from InvivoGen. The goat anti–IL-36γ (catalog AF2320) and anti-mouse KLF4 (catalog AF3158) antibodies were from R&D Systems. The rabbit anti–IL-36γ (catalog LS‑C201142) and its blocking peptide (catalog LS-E45854) were from LifeSpan BioSciences. The anti–human KLF4 antibody (catalog AM09057PU-N) was from Acris. The anti–β-actin (catalog A5441), anti-FLAG (catalog F1804), and anti-myc (catalog C3956) antibodies were from Sigma-Aldrich. The anti–human KLF4 (catalog 12173), anti-ERK (catalog 9107), anti–phospho-ERK (catalog 4370), and anti–phospho-threonine/proline (catalog 9391) antibodies were from Cell Signaling Technology. The anti-T7 antibody was from Abcam (catalog ab9138). The anti-HA antibody was from Santa Cruz Biotechnology (catalog sc-805). The secondary antibodies used were alkaline phosphatase–conjugated mouse IgG (catalog S372B), rabbit IgG (catalog S373B), and goat IgG (V115A) from Promega. Live C. acnes was prepared as previously described (63 (link)).
Satoh T.K., Mellett M., Meier-Schiesser B., Fenini G., Otsuka A., Beer H.D., Rordorf T., Maul J.T., Hafner J., Navarini A.A., Contassot E, & French L.E. (2020). IL-36γ drives skin toxicity induced by EGFR/MEK inhibition and commensal Cutibacterium acnes. The Journal of Clinical Investigation, 130(3), 1417-1430.
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6

Trametinib-Loaded HDL Nanoparticle Synthesis

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In brief, trametinib (GSK1120212, ApexBio), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), and 1-palmitoyl-2-hexadecyl-sn-glycero-3-phosphocholine (PHPC; Avanti Polar Lipids) were mixed in a ratio of 1:3:1 and dissolved in chloroform/methanol (4:1 by volume) solvent and dried to form a thin film. Human apolipoprotein A1 (APOA1) proteins, separated from human plasma, were added to the film, and the solution was incubated at 37°C until the film was hydrated and a homogeneous solution was formed. The solution was sonicated, and aggregates were removed by centrifugation to yield small trametinib-loaded nanoparticles (Tra-HDL). For fluorescence detection purposes, DiO (Invitrogen) was incorporated when flow cytometry techniques were applied. The nanoparticle solution was washed extensively with sterilized PBS by using a 100-kD filter (Vivaspin, Vivaproducts) and filtered through a 0.22-µm nylon filter before being administered to animals. trametinib incorporation efficiency was determined by HPLC (Shimadzu), and the final yield of the compound in the nanoparticle was ∼50%.
Hogstad B., Berres M.L., Chakraborty R., Tang J., Bigenwald C., Serasinghe M., Lim K.P., Lin H., Man T.K., Remark R., Baxter S., Kana V., Jordan S., Karoulia Z., Kwan W.H., Leboeuf M., Brandt E., Salmon H., McClain K., Poulikakos P., Chipuk J., Mulder W.J., Allen C.E, & Merad M. (2018). RAF/MEK/extracellular signal–related kinase pathway suppresses dendritic cell migration and traps dendritic cells in Langerhans cell histiocytosis lesions. The Journal of Experimental Medicine, 215(1), 319-336.
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7

Pharmacological Inhibition of Signaling Pathways

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For in vitro and in vivo studies, JNK inhibitor SP600125, MEK inhibitor Trametinib (GSK1120212), MLK inhibitor URMC-099, and CK2 inhibitor CX-4945 (Silmitasertib) were purchased from APExBIO (A4604, A3018, B4877, A833010, respectively). p38 inhibitor SB203580 was purchased from Selleckchem (Cat No S1076) for the in vitro experiments. For in vivo studies, water soluble SB203580 hydrochloride was purchased from APExBIO (B1285). SW-538 (SW034538) was provided by Elizabeth J. Goldsmith et al.
Yesilkanal A.E., Yang D., Valdespino A., Tiwari P., Sabino A.U., Nguyen L.C., Lee J., Xie X.H., Sun S., Dann C., Robinson-Mailman L., Steinberg E., Stuhlmiller T., Frankenberger C., Goldsmith E., Johnson G.L., Ramos A.F, & Rosner M.R. (2021). Limited inhibition of multiple nodes in a driver network blocks metastasis. eLife, 10, e59696.
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8

Breast Cancer Cell Line Authentication and Resistance

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Human breast cancer cell lines BT474, SK-BR-3, MDA-MB-361, and MDA-MB-453 were purchased from the ATCC at the beginning of the project in August 2018. Cell lines were cultured in DMEM (Sigma #D6429) supplemented with 10% FBS (Sigma #10500–064) and 1% penicillin/streptomycin (Sigma #P4333), except MDA-MB-361 cells which were grown in DMEM supplemented with 10% FBS, 1% penicillin/streptomycin, 2 mmol/L l-glutamine (Sigma #G7513), and 1× MEM nonessential amino acid solution (Sigma #M7145). lapatinib-resistant (LR) and trastuzumab-resistant (TR) BT474 cell lines were developed by incubation with gradually increased doses of lapatinib (APExBIO #A8218) or trastuzumab (Genentech) over a period of 4 to 5 months. Resistant cells were subsequently maintained with 0.1 μmol/L lapatinib or 50 μg/mL trastuzumab. The compounds were removed from the culture medium three to four days prior to experiments. Cell authentication was not routinely conducted given that cell lines were utilized for a maximum of 20 passages before thawing another aliquot of the same stock to maintain the original phenotype. Mycoplasma testing was not routinely performed on the cells. Trametinib was purchased from APExBIO (#A3887). For in vivo studies, lapatinib was dissolved in a buffer containing 1% Tween-80 and 5% hydroxypropyl methylcellulose just before use.
Zhang J., Pearson A.J., Sabherwal N., Telfer B.A., Ali N., Kan K., Xu Q., Zhang W., Chen F., Li S., Wang J., Gray N.S., Risa-Ebrí B., Finegan K.G., Cross M.J., Giurisato E., Whitmarsh A.J, & Tournier C. (2022). Inhibiting ERK5 Overcomes Breast Cancer Resistance to Anti-HER2 Therapy By Targeting the G1–S Cell-Cycle Transition. Cancer Research Communications, 2(3), 131-145.
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9

PDX Tumor Models for Drug Efficacy

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Tumors were established from dissociated PDX and tumor harboring mice were randomized into treatment and control cohorts of 6 animals when the tumor average size reached about 100 mm3 (single Afuresertib experiment) or individually distributed into different treatment groups when tumors reached about 100 mm3 (combination experiment). For Afuresertib treatment, drug (100 or 20 mg/kg) and vehicle were administered by oral gavage 5 times a week. Afuresertib (Selleck, S7521) was dissolved in 20% PEG-400 (Lipoid), 1% DMSO and 79% H2O. For trametinib (ApexBio, A3018) treatment, drug (0.5 mg/kg) was dissolved in 4% DMSO/ corn oil and administered by i.p. injection five times a week. Tumor size was measured three times a week using a caliper and mouse weight was measured twice a week. No mice needed to be euthanized due to severe body weight loss (>20% than baseline).
Manzella G., Schreck L.D., Breunis W.B., Molenaar J., Merks H., Barr F.G., Sun W., Römmele M., Zhang L., Tchinda J., Ngo Q.A., Bode P., Delattre O., Surdez D., Rekhi B., Niggli F.K., Schäfer B.W, & Wachtel M. (2020). Phenotypic profiling with a living biobank of primary rhabdomyosarcoma unravels disease heterogeneity and AKT sensitivity. Nature Communications, 11, 4629.
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10

Combination Inhibitor Treatment Protocols

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Trametinib (GSK1120212, MEK inhibitor, APExBio Technology, Shanghai, China), JQ1 (BET bromodomain inhibitor, APExBio Technology, Shanghai, China), and hydroxychloroquine (HCQ, autophagy inhibitor, APExBio Technology, Shanghai, China) were dissolved in DMSO.
Zhang X., Mao T., Xu H., Li S., Yue M., Ma J., Yao J., Wang Y., Zhang X., Ge W., Wang Y., Shentu D, & Wang L. (2022). Synergistic blocking of RAS downstream signaling and epigenetic pathway in KRAS mutant pancreatic cancer. Aging (Albany NY), 14(8), 3597-3606.
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