The Notch inhibitor, FLI-06, increases the chemosensitivity of head and neck Squamous cell carcinoma cells to taxanes-based treatment

Description

In this study, we show that the use of novel, small-molecule inhibitors of Notch signaling, such as FLI-06, can have a beneficial effect on increasing the chemosensitivity of HNSCC to taxane-based chemotherapy. Inhibition of Notch signaling by FLI-06 alone virtually blocks the proliferation and growth of HNSCC cells in 2D cultures, which is accompanied by down-regulation of key Notch target genes and proteins. Mechanistically, FLI-06 treatment causes cell cycle arrest in the G1-phase and induction of apoptosis in HNSCC. Combining FLI-06 with Docetaxel shows a synergistic effect and partially blocks the cell growth of aggressive HNSCC cells via enhanced apoptosis and modification of c-JunS243 phosphorylation via GSK-3β inhibition. In conclusion, inhibition of Notch signaling in HNSCC cells that retain active Notch signaling significantly supports taxane-based anticancer activities via modulation of both the GSK-3β and the c-Jun. The dataset contains: - cell cycle raw data - PI/annexin raw data - qPCR raw data - Calculation and Visualization of synergy scores for Drug Combinations (SynergyFinder 3.0 software)

Steps to reproduce

The total RNA was extracted using RNeasy kit (QIAGEN) according to the manufacturers protocol. The cDNA synthesis was performed through High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). The qPCR analysis was performed with the LightCycler® 480 II instrument (Roche) using PowerUp SYBR Green Master Mix (Applied Biosystems) protocol and 25 ng of cDNA on each reaction. The following primer sequences were used: GAPDH (forward 5’-GTGGAGTCTACTGGTGTCTTC-3’, reverse 3’-GTGCAGGAGGCATTGCTTACA-5’), HES1 (forward 5′-TCAACACGACACCGGATAAAC-3′ and reverse 3′-GCCGCGAGCTATCTTTCTTCA-5′), HEY1 (forward 5′-CGGCTCTAGGTTCCATGTCC-3′ and reverse 3′-GCTTAGCAGATCCCTGCTTCT-5′), NOTCH1 (forward 5’-CAACTGCCAGAACCTTGTGC-3’ and reverse 3′-GGCAACGTCAACACCTTGTC-5′), NOTCH2 (forward 5′-GGCACGTCAGGGGTTAATTG-3′ and reverse 3′- GCGGAAACCATTCACACCGTTGAT-5′), NOTCH3 (forward 5′-GCAGATGGCTCAACGGCACTG-3′ and reverse 3′-GGGGTCTCCTCCTTGCTATCCTG-5′), JAG1 (forward 5′-GCCGAGGTCCTATACGTTGC-3′ and reverse 3′-CCGAGTGAGAAGCCTTTTCAA-5′), DLL1 (forward 5′-TGCCTGGATGTGATGAGCAGCA-3′ and reverse 3′-ACAGCCTGGATAGCGGATACAC-5′), CCNA2 (forward 5′-CTCTACACAGTCACGGGACAAAG-3′ and reverse 3′-CTGTGGTGCTTTGAGGTAGGTC-5′), CCNB1 (forward 5′-GACCTGTGTCAGGCTTTCTCTG-3′ and reverse 3′-GGTATTTTGGTCTGACTGCTTGC-5′), CCNE1 (forward 5′-TGTGTCCTGGATGTTGACTGCC-3′ and reverse 3′-CTCTATGTCGCACCACTGATACC-5′), CCND1 (forward 5′-TCTACACCGACAACTCCATCCG-3′ and reverse 3′-TCTGGCATTTTGGAGAGGAAGTG-5′). CDKN1A (forward 5′-AGGTGGACCTGGAGACTCTCAG-3′ and reverse 3′-TCCTCTTGGAGAAGATCAGCCG-5′), CDKN1B (forward 5′-ATAAGGAAGCGACCTGCAACCG-3′ and reverse 3′-TTCTTGGGCGTCTGCTCCACAG-5′) The HNSCC cells were seeded into 6-well microplates at a density of 5 × 105 cells/ml. After 24 h, the cells were exposed to a range of drugs concentrations and incubated for 48 h. After harvest, cell were centrifuged and fixed with 70 % ice-cold ethanol and stored at − 20 °C. PI/RNase (PI/RNase Staining Buffer, BD Pharmingen, Catalogue Number 550825, Franklin Lakes, New Jersey, U.S.) staining was performed directly before the flow cytometric analysis (BD FACSCalibur, CellQuest Pro Version 6.0. software for the Macintosh operating system.). The PI fluorescence intensity of individual nuclei was determined and at least 10000 events an acquisition rate of 100–300 events/s. For Annexin V/Propidium iodide (PI), cells were prepared as described above. The number of apoptotic and necrotic cells was measured with the FITC Annexin V Apoptosis Detection Kit I (BD Pharmingen). The antiproliferative effect of each drug combination was analyzed using SynergyFinder 3.0 software. Inhibition of proliferation by appropriate combinations of the tested drugs was assessed by MTT assay. Next, results of combined FLI-06 and DTX treatment were evaluated by comparing differential responses to drug combinations with a reference mathematical model (the high single agent (HSA) model, according to the software recommendation).

Institutions

Categories

Funding

Related Links

Licence