*2.4. Salternamide A Induces Apoptotic Cell Death in Human Colorectal Cancer Cells*

A previous study revealed the anti-proliferative activity of SA in a panel of cancer cell lines [16]. To further elucidate the mechanism of action of the anti-proliferative activity of SA, the effect of SA

on the regulation of the cell cycle was determined in HCT116 cells. When treated with SA for 72 h, SA primarily inhibited the growth of HCT116 cells in a concentration-dependent manner (Figure 4A). SA (10 μM) increased the cell population in the sub-G1 phase at 48 h (Figure 4B, right panel). To further elucidate whether cell cycle arrest is associated with the regulation of cell cycle checkpoint proteins, the expression of G2/M cell cycle regulatory proteins was analyzed by Western blotting. As shown in Figure 4C, the expression of p-CDC25C (Ser216), CDC25C, p-CDC2 (Thr161), CDC2, cyclin B1, and cyclin A were significantly suppressed, but the levels of p-chk1 (Ser345) and p-chk2 (Thr168) were upregulated by the treatment of SA. These findings suggest that the anti-proliferative activity of SA is associated, in part, with the induction of the G2/M phase cell cycle arrest by modulating cell cycle regulators in HCT116 cells.

**Figure 4.** Effect of SA on cell cycle distribution in HCT116 human colorectal cancer cells. (**A**) Anti-proliferative effect of SA on HCT116 cells. HCT116 cells were treated with various concentrations of SA for 72 h, and cell proliferation was determined with the SRB assay. The data represent the mean percentage ± SD compared to the DMSO-treated control group. Each experiment was performed in triplicate (*n =* 3). \* *p* < 0.05 compared with the control group. (**B**) HCT116 cells were treated with SA for 24 or 48 h. The cell cycle distribution was analyzed by flow cytometry. (**C**) Effects of SA on the expression of cell cycle-related proteins in HCT116 colorectal cancer cells. HCT116 cells (2 <sup>×</sup> 105 cells/mL) were treated with SA for 24 h. Subsequently, the protein expression levels of cell cycle-related proteins were analyzed by Western blotting.

To further confirm whether the induction of the sub-G1 peak by SA (10 μM) at 48 h is related to apoptotic cell death, the cells were treated with SA (10 μM) for 48 h, and the quantification of Annexin-V/PI staining, a marker for apoptosis, was determined by flow cytometry. The number of cells that were positive for the double staining of Annexin-V/PI was significantly increased by SA treatment, suggesting that SA is able to induce apoptotic cell death in cancer cells (Figure 5A). To further unveil the mechanism of SA-induced apoptosis in HCT116 cells, the expression of apoptosis-associated proteins was analyzed by Western blotting. As shown in Figure 5B,C, the expression of cleaved

caspase-8, cleaved caspase-3, and cleaved PARP was upregulated, but the expression of pro-caspase-8, caspase-3, pro-PARP, Bcl-2 and Bcl-xL was downregulated in a time- and concentration-dependent manner. In addition, LC3-II is an autophagy-specific marker, and LC3-II formation (LC3 lipidation) is also a key step in autophagy-associated cell death [23]. To evaluate whether the cytotoxic activity of SA is related, in part, to the induction of autophagy, the expression level of LC3-II was determined by 48 h of SA (10 μM) treatment. SA also significantly induced the expression of LC3-II in a timeand concentration-dependent manner. Taken together, these results suggest that the anti-proliferative activity of SA might be due, in part, to the G2/M phase cell cycle arrest and the induction of apoptosis and autophagic cell death in HCT116 cells.

**Figure 5.** Effects of SA on apoptosis and autophagy in HCT116 human colorectal cancer cells. (**A**) HCT116 cells (1 <sup>×</sup> 105 cells/mL) were treated with SA for 48 h. Following treatment, HCT116 cells were harvested and stained with Annexin V-FITC and PI and analyzed using flow cytometry as described in the experimental section. (**B**) HCT116 cells were treated at the indicated time points in the presence or absence of SA (10 μM) prior to Western blotting. (**C**) Effects of SA on the expression of apoptosis- and autophagy-related proteins in HCT116 colorectal cancer cells. HCT116 cells (2 <sup>×</sup> 105 cells/mL) were treated with SA for 48 h; subsequently, the protein expression levels of apoptosis- and autophagy-related proteins were analyzed by Western blotting.
