We therefore evaluated the effect of 519-23-3 miR-200c on apoptosis induced by the proteasome inhibitor bortezomib. This clinically used drug was chosen since it has been shown that Noxa 917879-39-1 cost induction is important for bortezomib-induced cell death. Treatment of HCT116 cells with clinically relevant doses of bortezomib led to a time- and dose-dependent induction of Noxa protein. As can be seen in Figure 5A, overexpression of miR-200c in HCT116 cells treated with bortezomib led to a downregulation of Noxa at all doses. Surprisingly, at the same time miR-200c overexpression resulted in increased bortezomib-induced apoptosis as assessed by immunoblotting for cleaved caspase 3 and cleaved PARP. In order to directly test how apoptosis induction is affected by miR-200c overexpression, Annexin V/PI staining was performed on HCT116 left untreated or treated with bortezomib. Again, in both cases miR-200c overexpression led to increased cell death, as compared to a scrambled pre-miR control oligonucleotide. A similar result was obtained in the HEK293 cell line. Also, this effect was not restricted to proteasome inhibition, as cells treated with the DNAdamaging drug doxorubicin showed increased apoptosis induction upon miR-200c overexpression as well. Since the effects of miR-200c on Noxa and cell death induced by bortezomib apparently contradict one another, we went on to examine the effect of miR-200c on apoptosis in a setting without Noxa expression. Therefore, we knocked down Noxa expression in bortezomib-treated HCT116 cells using siRNA oligos. Knockdown of Noxa led to an expected decrease in both Noxa protein levels and proteasome inhibitor-induced apoptosis as measured by Annexin V/PI staining. Interestingly, when Noxa was knocked down, miR-200c overexpression had an even more pronounced effect on apoptosis induction. Indeed, in cells transfected with control siRNA oligos, miR-200c overexpression led to a increase in apoptosis, as compared to cells transfected with scrambled pre-miRs. In contrast, in cells with Noxa knocked down the increase in apoptosis. To further investigate the relationship between miR-200c, Noxa and bortezomib-induced cell death, we went on to ectopically express a Noxa construct lacking the miR-200c
