*2.3. Salternamide A Suppresses the Hypoxia-Induced Accumulation of HIF-1α via the Regulation of Signal Transduction Pathways*

Recent studies have reported that the PI3K/Akt/mTOR and p42/p44 MAPK pathways are associated with the regulation of HIF-1α protein synthesis at the translational level [10,20]. The p42/p44 MAPK also enhances the transcriptional activity of HIF-1α [11]. To address the potential involvement of these pathways in the SA-mediated suppression of HIF-1α accumulation, the expression of the proteins in the signal transduction pathway was determined by Western blot analysis. As shown in Figure 3A, activated (phosphorylated form) PI3K, Akt, mTOR, and RPS6 expression under hypoxic conditions was downregulated by the treatment of SA (10 μM) in a time-dependent manner. A subsequent study also revealed that SA effectively suppressed the expressions of these signaling proteins in a concentration-dependent manner (Figure 3B). In addition, SA also downregulated the activation of p70S6K1 (Thr389), 4E-BP1 (Thr37/46), eIF4E (Ser209), and RPS6 (Ser235/236), which are downstream target molecules of mTOR complex 1 (mTORC1) signaling pathways. Therefore, these data suggest that the suppression of HIF-1α protein expression by SA might be partly associated with the downregulation of mTORC1 signaling pathways under hypoxic conditions.

**Figure 3.** Effect of SA on hypoxia-response protein expressions. (**A**) HCT116 cells were treated at the indicated time points under normoxic or hypoxic conditions in the presence or absence of SA (10 μM) before immunoblotting. (**B** and **C**) HCT116 cells were treated for 8 h under normoxic or hypoxic conditions in the presence or absence of increasing SA concentrations.

Recent reports have also revealed that the transcription factor STAT3 is involved in the transcriptional regulation of HIF-1α and that the activation of STAT3 activity is enhanced by hypoxia [21,22]. In the present study, we also found that hypoxia enhanced STAT3 activation (phosphorylated at the Tyr<sup>705</sup> residue), and this effect was abrogated by the treatment of SA in a concentration-dependent manner (Figure 3C).

Overall, these findings suggest that the suppression of the PI3K/Akt/mTOR, p42/p44 MAPK, and STAT3 signaling pathways by SA might be associated, in part, with the downregulation of HIF-1α protein synthesis.
