Lenvatinib

Manufactured by Selleck Chemicals

Sourced in United States, Italy

Lenvatinib is a chemical compound used in laboratory research settings. It is a tyrosine kinase inhibitor that targets multiple receptor tyrosine kinases involved in angiogenesis and tumor growth. The core function of Lenvatinib is to serve as a research tool for investigating cellular signaling pathways and their potential therapeutic implications.

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47 protocols using lenvatinib

Leptin and Lenvatinib Effects on PTC Cells

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For in vitro experiments, two human PTC cell lines, K1 and TPC-1, were used. These cell lines contained the BRAF V600E and RET/PTC1 mutation, respectively [11 (link)]. Cells were grown in a DMEM medium (Thermo Fisher Scientific Inc., Waltham, MA, USA), supplemented with a 10% foetal bovine serum (FBS) (Thermo Fisher Scientific), penicillin (100 IU/ml), streptomycin (100 mg/ml), and amphotericin B (2.5 mg/ml) (Sigma-Aldrich, Milan, Italy), and maintained at 37°C in a humidified atmosphere containing 5% CO₂. Short tandem repeat profiling was used to authenticate these cell lines. Cultured cells were treated with 200 or 500 ng/ml of leptin (Sigma-Aldrich) for 96 h (leptin), 50 μM of lenvatinib (Selleckchem, Aurogene Srl, Rome, Italy) (lenvatinib) for 24 h, or with 500 ng/ml of leptin for 96 h plus 50 μM of lenvatinib for an additional 24 h (leptin+lenvatinib). Untreated cells were used as a control (ctrl).

Celano M., Maggisano V., Lepore S.M., Sponziello M., Pecce V., Verrienti A., Durante C., Maranghi M., Lucia P., Bulotta S., Damante G, & Russo D. (2019). Expression of Leptin Receptor and Effects of Leptin on Papillary Thyroid Carcinoma Cells. International Journal of Endocrinology, 2019, 5031696.

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Evaluating Cell Viability with CCK-8

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The Cell Counting Kit-8 (Bimake) assay was used to estimate the viability of the cells. Huh7 and HepG2 cells were seeded in 96-well plates (5,000 cells/well). Next, different concentrations of lenvatinib (Selleck) or BGJ (Selleck) were added into each well separately and incubated for 48 h. After that, the culture medium containing lenvatinib or BGJ was sucked out. The cells were washed with phosphate buffer solution (PBS). Then, the mixture of 10 μl CCK-8 solvent and 90 μl of fresh medium was added to each well and incubated for 60 min. Finally, the absorbance at 450 nm was measured using a microplate reader (EON, BioTek Instruments, Inc.). The cell survival rate was calculated. The experiment was repeated four times.

Guo J., Zhu P., Ye Z., Wang M., Yang H., Huang S., Shu Y., Zhang W., Zhou H, & Li Q. (2021). YRDC Mediates the Resistance of Lenvatinib in Hepatocarcinoma Cells via Modulating the Translation of KRAS. Frontiers in Pharmacology, 12, 744578.

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Establishing Lenvatinib-Resistant Liver Cancer Cell Lines

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Human liver cancer cell lines (HuH7 and PLC/PRF/5), HEK293T cells, and the mouse liver cancer cell line Hep1–6 were purchased in 2018 from the Shanghai Cell Bank of the Chinese Academy of Sciences (Shanghai, China). All human cancer cells and mouse liver cancer cells were cultured in DMEM with 10% FBS, glutamine and 1% penicillin–streptomycin (Gibco) at 37°C and 5% CO₂. To establish lenvatinib-resistant (LR) cell lines, HuH7 and PLC/PRF/5 cells were cultured with increasing doses of lenvatinib (Selleck) starting from 3 μmol/L and 20 μmol/L, respectively. The cell culture media was replaced every 48 hours until the cells spread across 90% of the culture dish and then passaged. After the passaged cells were replated, the concentration was increased by 0.5 μmol/L until HuH7 cells proliferated quickly at a concentration of 30 μmol/L lenvatinib and PLC/PRF/5 cells proliferated quickly at 60 μmol/L lenvatinib. This process took at least 6 months. The resistant cell strains were named HuH7 LR and PLC/PRF/5 LR. The identities of all cell lines were confirmed via short tandem repeat profiling and the Mycoplasma test results were negative. Cell stocks were conducted within five passages, and all experiments were completed within eight passages.

Hu B., Zou T., Qin W., Shen X., Su Y., Li J., Chen Y., Zhang Z., Sun H., Zheng Y., Wang C.Q., Wang Z., Li T.E., Wang S., Zhu L., Wang X., Fu Y., Ren X., Dong Q, & Qin L.X. (2022). Inhibition of EGFR Overcomes Acquired Lenvatinib Resistance Driven by STAT3–ABCB1 Signaling in Hepatocellular Carcinoma. Cancer Research, 82(20), 3845-3857.

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Establishment of Lenvatinib-resistant Cell Lines

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To determine the IC₅₀ of SMMC-7721 and Huh7 cells to Lenvatinib treatment, the cells were incubated with different concentrations of Lenvatinib in 96-well plates, followed by cell viability measurements 3 days later. In order to establish Lenvatinib-resistant cell lines, SMMC-7721 and Huh7 cells were incubated with Lenvatinib (Selleck, S1164) at a concentration just below their IC₅₀, and increased by 0.2 μM/L per week for 6 to 7 months. Two Lenvatinib-resistant cell lines were obtained, termed 7721-LR and Huh7-LR. These two Lenvatinib-resistant cell lines were maintained by continued culture in the presence of Lenvatinib.

Yu T., Yu J., Lu L., Zhang Y., Zhou Y., Zhou Y., Huang F., Sun L., Guo Z., Hou G., Dong Z, & Wang B. (2021). MT1JP-mediated miR-24-3p/BCL2L2 axis promotes Lenvatinib resistance in hepatocellular carcinoma cells by inhibiting apoptosis. Cellular Oncology (Dordrecht), 44(4), 821-834.

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Comprehensive Compound Acquisition Protocol

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Sorafenib (#T0093L) was purchased from TargetMol (Shanghai, China); lenvatinib (#S1164), brivanib (#S1084) and bafilomycin A1 (#S1413) were purchased from Selleck (Shanghai, China); cisplatin (#P4394), doxorubicin (#D1515), N-acetyl-l-cysteine (NAC, #A7250), and Hoechst (#94403) were purchased from Sigma‒Aldrich (Shanghai, China); canagliflozin (#A11100) was purchased from AdooQ Bioscience (Nanjing, China); and oligomycin (#HY-N6782), antimycin A (#HY-105755), phloretin (#HY-N0142), z-VAD-FMK (#HY-16658B), necrostatin-1 (#HY-15760), and ferrostatin-1 (#HY-100579) were purchased from MedChemExpress (Shanghai, China).

Zhou J., Feng J., Wu Y., Dai H.Q., Zhu G.Z., Chen P.H., Wang L.M., Lu G., Liao X.W., Lu P.Z., Su W.J., Hooi S.C., Ye X.P., Shen H.M., Peng T, & Lu G.D. (2022). Simultaneous treatment with sorafenib and glucose restriction inhibits hepatocellular carcinoma in vitro and in vivo by impairing SIAH1-mediated mitophagy. Experimental & Molecular Medicine, 54(11), 2007-2021.

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Targeted Cancer Signaling Inhibitors

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Sorafenib (VEGFR and PDGFRβ), Lenvatinib (RET, VEGFR, PDGFRβ, and FGFR), Erlotinib (EGFR), Afatinib (EGFR), Crizotinib (MET), and AZ628 (Ras/MAPK) were purchased from Selleck (TX, USA) and dissolved in DMSO. Recombined EGF, HGF, FGF2, PDGF-BB, and VEGF were purchased from PeproTech (NJ, USA), and recombined insulin was bought from Selleck (TX, USA).

Song S., Yu Z., You Y., Liu C., Xie X., Lv H., Xiao F., Zhu Q, & Qin C. (2022). EGFR/MET promotes hepatocellular carcinoma metastasis by stabilizing tumor cells and resisting to RTKs inhibitors in circulating tumor microemboli. Cell Death & Disease, 13(4), 351.

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RET Fusion Cancer Cell Line Maintenance

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The mesothelioma cell line EHMES-10 [17 (link)–19 (link)] containing a NCOA4-RET fusion, and human lung adenocarcinoma cell line LC-2/ad [20 (link)] and thyroid papillary carcinoma cell line TPC-1 [21 (link)] containing a CCDC6-RET fusion were used in this study. All cells were maintained in RPMI-1640 medium supplemented with 10% FBS, penicillin (100 U/mL), and streptomycin (10 μg/mL) in a humidified CO₂ incubator at 37°C. All cells were passaged for less than 3 months before renewal from frozen early-passage stocks. Cells were regularly screened for mycoplasma using a MycoAlert Mycoplasma Detection Kit (Lonza, Rockland, ME, USA). Alectinib, vandetanib, and lenvatinib were obtained from Selleck Chemicals (Houston, TX, USA).

Arai S., Kita K., Tanimoto A., Takeuchi S., Fukuda K., Sato H, & Yano S. (2017). In vitro and in vivo anti-tumor activity of alectinib in tumor cells with NCOA4-RET. Oncotarget, 8(43), 73766-73773.

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Lenvatinib Inhibits Cell Invasion and Anchorage-Independent Growth

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The RET inhibitor lenvatinib was obtained from Selleckchem (Cat#. S1164). Cell invasion was assessed as above using 0.7 ml of 10% FBS-medium below the well in the absence or presence of lenvatinib (100 nM or 500 nM). After 24 hours, the invasion cells on the bottom surface of the membrane were counted as described above. To evaluate anchorage-independent growth, 2.5X10³ cells/well were seeded in 0.4% agar on top of a base layer containing 0.6% agar in the 6-well plate. Cells were treated 2 times per week with 2 ml of cell culture media in the absence or presence of lenvatinib (100 nM or 500 nM) for 21 days. At the end of the experiment, colonies were fixed with methanol, stained with 0.5% crystal violet, and counted.

Ban K., Feng S., Shao L, & Ittmann M. (2017). RET signaling in prostate cancer. Clinical cancer research : an official journal of the American Association for Cancer Research, 23(16), 4885-4896.

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Lenvatinib treatment of HCC organoids

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Human HCC organoids were constructed as previously described.²¹ A total of 2000 organoid cells were seeded in a 96‐well plate with 7 μl Matrigel matrix droplets (Corning Life Sciences, Tewksbury, MA). The organoids were then treated with the medium containing 35 μM lenvatinib (Selleck) for 6 days. Organoids were visualized using a bright field microscope at 10× magnification. Five randomly chosen visual fields were accessed per well and each experiment group was repeated with 3 wells. The diameters of organoids were quantified using ImageJ software.

Liu D., Liu W., Chen X., Yin J., Ma L., Liu M., Zhou X., Xian L., Li P., Tan X., Zhao J., Liao Y, & Cao G. (2022). circKCNN2 suppresses the recurrence of hepatocellular carcinoma at least partially via regulating miR‐520c‐3p/methyl‐DNA‐binding domain protein 2 axis. Clinical and Translational Medicine, 12(1), e662.

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Lenvatinib and Inhibitor Compounds

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Lenvatinib was purchased from Selleck and applied as indicated concentrations. Giemsa staining solution was obtained from Sigma. MK-2206 (10 μM) and SC-79 (5 μM) were purchased from Selleck. 10058-F4 (5μM) was purchased from MCE.

Ma X.L., Hu B., Tang W.G., Xie S.H., Ren N., Guo L, & Lu R.Q. (2020). CD73 sustained cancer-stem-cell traits by promoting SOX9 expression and stability in hepatocellular carcinoma. Journal of Hematology & Oncology, 13, 11.

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