The Effects Of Bortezomib To The T-ALL Cell Line CCRF-CEM In The Notch1Signal Pathway

Objective: Investigate the effect of bortezomib and with the combination ofMTX on the T-cell acute lymphoblastic leukemia (T-ALL) proliferation. Discuss theexpress of Notch1pathway signal in the T-ALL cell line (CCRF-CEM) by usingbortezomib, in order to search a new way in the T-ALL treatment.Methods:1. Cell culture. Human T-ALL cell line CCRF-CEM was cultured inRPMI medium1640.2. Cell growth inhibition was tested by MTT assay. CCRF-CEMwere incubated at a density of5000cells/well in96-well plates, and were addedbortezomib and MTX by the different concentration (bortezomib0.25ng/ml，0.5ng/ml，1ng/ml，10ng/ml，20ng/ml，40ng/ml; MTX0.25ng/ml,1ng/ml,5ng/ml,10ng/ml,20ng/ml). The combination of the two different drugs, bortezomib and MTX(bortezomib0.25ng/ml+MTX1ng/ml, bortezomib0.25ng/ml+MTX10ng/ml,bortezomib0.25+MTX20ng/ml, bortezomib0.5ng/ml+MTX1.0ng/ml,bortezomib0.5ng/ml+MTX20ng/ml, bortezomib0.5ng/ml+MTX40ng/ml) wasalso used in the MTT test. Compare different drug concentrations and time toCCRF-CEM cell growth inhibition.3. Choose different concentration of bortezomibused in the CCRF-CEM with24h,48h and72h, then harvest the cell. Total RNA wasextracted by Trizol according to the manufacture’’s instructions. Synthesis offirst-strand cDNA was carried out with Prime Script RT reagent kit with gDNAEraser. RT-PCR was used to detect the expression of Notch1, Hes, Myc and NF-KB1mRNA.Results:1. We found there were significant inhibition in CCRF-CEM cell lineby used the bortezomib and MTX (p<0.05). And the more inhibition happened bytime and the concentration increased of the two drugs.2. The inhibition by using bothbortezomib and MTX were more obvious than the total effects of two single drugused.3. The express of Notch1and NF-ΚB1in CCRF-CEM were reduced by theeffect of bortezomib（P<0.05）, but the Myc and Hes gene expression were notdetected the significant difference（P>0.05）. Conclusion:1. Both bortezomib and MTX can significantly inhibit theproliferation of CCRF-CEM;2. Bortezomib can enhance the inhibition of MTX toCCRF-CEM cell line;3. The lower express of Myc and Hes may contribute to theinhibition of CCRF-CEM proliferation by adding bortezomib...