c-Myc/NOXA pathway participates in ixazomib induced proliferation inhibition in ESCC cells.
Based on these previous studies, the proteasome inhibitors increased the accumulation of polyubiquitinated proteins and trigger the endoplasmic reticulum stress-induce apoptosis in several cancer cells(Fribley et al. , 2004; Mozos et al. , 2011; Niewerth et al. , 2015). To assess the role of the ixazomib in apoptosis by endoplasmic reticulum stress in ESCC cells, we determined by RT-PCR the expression levels of endoplasmic reticulum stress related genes in Kyse150 cell treated with ixazomib. Results of these experiments showed significant increase in the mRNA expression levels of endoplasmic reticulum stress related genes in response to ixazomib. The increased in NOXA expression was the most prominent compared with other ERS related gene (Figure 4A). As NOXA has been demonstrated to play a role in ixazomib-induced proliferation inhibition in ESCC cell, we knocked down the NOXA by siRNA, the results show that silencing NOXA rescued ixazomib-induced proliferation inhibition in ESCC cells (Figure 4B, C).
Several studies have evidenced that c-Myc was a prime transcription factor to transactivate NOXA (Nikiforov et al., 2007; Knorr et al., 2015). We determined the protein levels of NOXA and c-Myc in both Kyse150 and Kyse510 cell lines in response to ixazomib. As shown in Fig. 5A, ESCC cells express undetectable NOXA protein levels, which were clearly upregulated following ixazomib treatment. Furthermore, the c-Myc protein expression followed a trend similar to that observed for NOXA protein expression. We knocked down the c-Myc by siRNA, same phenomenon can also be found that reduced c-Myc function rescued ixazomib-induced proliferation inhibition in ESCC cells (Figure 5B, C). These observations indicated that c-Myc/NOXA signaling pathways play a major role in ixazomib induced proliferation inhibition in ESCC cells.