We examined these combinations in SK N AS and two more neuroblastoma cell lines NB 1691 and SH SY5Y to make certain that the synergistic effect was not particular to any cell line. We initially measured the IC50 of topotecan and NSC676914 in these cells at 24 hrs, and they had been 2 uM and seven uM for SK N AS. 550 www.selleckchem.com/products/at13387.html nM and 4 uM for NB 1691. 200 nM and 2 uM for SH SY5Y respectively. Sub IC50 doses of topotecan and NSC 676914 have been then used in mixture in all three cell lines along with the com bination index was calculated for every dose combination. Every one of the blend treatment information points in nor malized isobolograms had been far from the diagonal additive line and in direction of the origin indicating that very low doses of NSC676914 and topotecan act synergistically in all three cell lines.
To facilitate long term trans lation of our findings towards the clinic, we examined bortezo mib, an FDA approved drug acknowledged to inhibit NF B as one particular of its mechanisms, within the subsequent in vitro and in vivo research. In addition, this drug has become reported to inhibit each of the top rated 4 pathways recognized in our synthetic lethal screen. Consequently, we predicted bortezomib would have synergistic effects in combination with topotecan in neuroblastoma cells. To check this hypothesis, we initial measured the IC50 of bor tezomib for these 3 cell lines at 24 hrs as seven nM, three. 5 nM and three. 5 nM for SK N AS, NB 1691 and SH SY5Y respectively. As anticipated, we observed from standard ized isobolograms that sub IC50 doses of topotecan and bortezomib acted synergistically to inhibit cell development since the information factors in normalized isobolograms were also far from the diagonal additive line.
This synergistic inhibitory result among topotecan and bor tezomib was confirmed in an independent experiment utilizing electronic cell sensing to watch cell growth. In addition, we have been in a position to detect a rise in apoptosis using the mixture of topote can and bortezomib as evidenced by elevated caspase three exercise. To verify that bortezomib enhances topotecan induced development inhibition as a result of NF B pathway, Western blotting was carried out to display that SK N AS cells taken care of with both bortezomib and topotecan inhibited the degradation of total I B a protein, suggesting that the NF B was certainly inacti vated. Taken with each other, these information demon strated that topotecan and NF B inhibitors could synergistically inhibit growth via inhibition in the NF B pathway and induction of apoptosis in neuro blastoma cells.
Delayed tumor progression in human neuroblastoma xenograft handled with topotecan and bortezomib In an effort to investigate the synergistic effect of topotecan and bortezomib in vivo, we tested the drug blend in SCID beige mice bearing human neuroblastoma xeno grafts. SK N AS cells were intravenously injected in to the mice and monitored by Xenogen.