*2.2. Suppression of HIF-1α Accumulation by Salternamide A in HCT116 Cells Is Independent of Proteasomal Degradation*

In general, the accumulation of HIF-1α depends on the balance between its degradation and synthesis (translation) [18]. To determine whether SA is able to suppress HIF-1α protein accumulation by promoting its degradation, the cells were pretreated with the proteasome inhibitor MG132, followed by SA treatment in HCT116 cells. As shown in Figure 2A, pretreatment with MG132 resulted in the accumulation of HIF-1α, but SA efficiently abrogated the accumulation of HIF-1α despite proteasome suppression, indicating that SA decreases HIF-1α protein accumulation through a pathway independent of proteasomal degradation.

**Figure 2.** Effect of SA on the degradation of HIF-1α. (**A**) HCT116 cells were treated with a proteasome inhibitor (10 μM MG132) and 10 μM SA under normoxic or hypoxic conditions before immunoblotting; (**B**) for VHL and Hsp90 immunoblotting, HCT116 cells were treated with SA and cultured for 8 h under normoxic or hypoxic conditions, respectively.

The von Hippel-Lindau (VHL) tumor suppressor protein recruits an E3-ubiquitin ligase that targets HIF-1α for proteasomal degradation [4]. In addition, heat-shock protein 90 (Hsp90) binds to HIF-1α and promotes its stability [19]. To determine whether the suppression of HIF-1α protein expression by SA is associated with these adaptor proteins, Western blot analysis was performed under hypoxic conditions with the treatment of SA in HCT116 cells. As a result, SA did not significantly enhance the VHL or abrogate Hsp90 expression in the HCT116 cells (Figure 2B). These data suggest that the suppression of HIF-1α accumulation by SA under hypoxic conditions might not be associated with the enhancement of the degradation of HIF-1α under these conditions. Further study revealed that SA did not affect HIF-1α gene transcription or HIF-1α mRNA stability (data not shown). Overall, the suppressive effect of SA on the accumulation of HIF-1α protein expression under hypoxic conditions might be due in part to the downregulation of the translation of HIF-1α mRNA. These translational regulations should be further clarified in detail.
