*2.1. Salternamide A Suppresses Hypoxia-Induced HIF-1α Protein Accumulation in Various Cancer Cells*

To investigate whether SA (Figure 1A) affects HIF-1α induced by hypoxia, HCT116 cells were exposed to normoxic or hypoxic (CoCl2 treatment) conditions for 2, 4, 8, 12, or 24 h in the presence of 10 μM SA. As shown in Figure 1B, HIF-1α expression was significantly induced by hypoxia-mimetic CoCl2 treatment, starting from as early as 4 h. However, SA effectively suppressed hypoxia-induced HIF-1α protein expression at 8 h along with marked suppression at 12 and 24 h (Figure 1B). In addition, when treated with SA for 8 h under hypoxic conditions, SA suppressed the accumulation of hypoxia-induced HIF-1α protein in a concentration-dependent manner (Figure 1C).

**Figure 1.** Effect of SA on hypoxia-induced HIF-1α protein accumulation in various cancer cells. (**A**) Chemical structure of SA; (**B**) HCT116 cells were treated at the indicated time points under normoxic or hypoxic conditions (CoCl2 treatment) in the presence or absence of SA (10 μM); (**C**) HCT116 cells were treated for 8 h under normoxic or hypoxic conditions in the presence or absence of increasing SA concentrations; (**D**) MDA-MB-231, SK-HEP-1, and SNU-638 cells were treated with 10 μM SA for 8 h under normoxic or hypoxic conditions. Immunoblotting analysis was performed to determine HIF-1α and β-actin protein levels.

To further examine whether the suppressive effect of SA on HIF-1α expression is applicable to a variety of cancer cell lines with different genetic backgrounds (wild-type or mutated p53), given that HIF-1α is destabilized by p53 [17] in different organs, SK-HEP-1 (liver), SNU-638 (gastric), and MDA-MB-231 (breast) cancer cells were treated with 10 μM SA for 8 h. SA effectively suppressed the expression of HIF-1α in the tested cancer cells, similar to the results shown in HCT116 cells (Figure 1D). These findings suggest that SA suppresses HIF-1α expression in various cancer cell types by blocking HIF-1α protein accumulation in response to hypoxic conditions.
