2.4.3. New Compound 3 Inhibited Proliferation of A549 Cells

The known compounds, epiremisporine B (**4**) [16] and penicitrinone A (**5**) [28], were reported to exhibit anticancer activities in previous studies. Epiremisporine E (**3**) was selectively tested for clonogenic assay as it is a new compound and possesses cytotoxic activity against A549. The effect of compound **3** on colony formation of A549 cells was examined using the clonogenic assay (Figure 8). The A549 cell colonies were visualized as blue discs, through crystal violet staining. It was clearly observed that compound **3** (25 μM) significantly reduced the colony formation of A549 cells. Moreover, compound **3** almost completely inhibited the colony formation at 50 μM.

**Figure 8.** Effect of epiremisporine E (**3**) on the colony formation of A549 cells. (**A**) The effect of **3** against A549 cell colony formation. Clonogenicity was evaluated by the monolayer colony formation assay. Representative images show the blue colonies of A549 cells stained with crystal violet. (**B**) Histogram presentation of A549 cell colony quantification. *\* p* < 0.05; *\*\*\* p* < 0.001 compared with the control. *<sup>a</sup>* 5-Fluorouracil (5-FU) was used as a positive control.

2.4.4. Effects of Epiremisporine E (**3**) and Epiremisporine B (**4**) on Protein Expressions of Pro-caspase 3 and Cleaved-caspase 3 in A549 Cells

Caspase 3 activation is a hallmark of apoptosis. Caspase 3 activation involves the cleavage of pro-caspase 3 (the inactive precursor form of caspase 3), leading to the formation of cleaved-caspase 3 (which is the active caspase 3). Upon apoptosis, the pro-caspase 3 would decrease and the cleaved-caspase 3 would increase accordingly. We further investigated whether epiremisporine E (**3**) and epiremisporine B (**4**) were able to influence these enzymatic activities of caspase 3. The results showed that compounds **3** and **4** suppressed pro-caspase 3 and increased the cleaved-caspase 3 (Figures 9 and 10). Furthermore, compounds **3** and **4** markedly induced apoptosis of A549 cells through caspase 3-dependent pathways.

**Figure 9.** Western blot analysis for Bcl-2 (**a**), Bax (**b**), pro-caspase 3 (**c**), and cleaved-caspase 3 (**d**) in each group. Treatment with epiremisporine C (**3**) significantly reduced the expression levels of Bcl-2 and pro-caspase 3, and increased the expression levels of Bax and cleaved-caspase 3. As-terisks indicate significant differences (\* *p* < 0.05, \*\* *p* < 0.01, and \*\*\* *p* < 0.001) compared with the control group.
