# HMT exerts an Anticancer Effect by Targeting PAK-1
## 3 Results
### HMT Induces G1 and Sub-G1 Arrest  
To evaluate whether HMT can modulate cell cycle progression, cell cycle analysis was performed by treating PC-3 cells with high concentrations of HMT (10, 200, and 400 ug/mL) for 24 h. As shown in Figure 3A, HMT gradually increased G1 and sub-G1 durations in a dose-dependent manner compared to those in the nontreated cells. Particularly, the increase in sub-G1 arrested cells treated with 400 ug/mL HMT was 76% (Figure 3A). Figure 2: \(Cont.\)  
To confirm the presence of HMT-mediated apoptotic cells, we stained PC-3 cells treated with 200 ug/mL HMT for 24 h with the CellEvent Caspase-3/7 detection dye. As shown in Figure 3C, we detected no cas3/7 fluorescent cells in the control group. On the other hand, HMT-treated cells showed green fluorescence in almost all cells. We next assessed whether HMT-mediated G1 and sub-G1 arrest (apoptosis) were dependent on changes in G1-regulating and apoptotic-related proteins. HMT treatment decreased the levels of G1-regulating proteins (CDK6) and increased apoptosis through the inhibition of BCL-2 and PARP and the increase of cleaved caspase-3 (Figure 3D). The apoptosis pathways activate the programmed cell death through the mitochondria, which are involved in the regulation of apoptosis [20]. The chemical-induced apoptotic pathway involving mitochondria is regulated by apoptosis-related key proteins such as BC1-2/Bax and cytochrome C, with subsequent activation of caspase-3 and PARP [20; 21; 22; 23]; BCI-2, caspase-3, and PARP are representative markers of apoptosis. Figure 2: Inhibitory effect of HMT on cell migration and invasion in PC-3 cells. Cells were treated with HMT (50 ug/mL) for 24 h. (**A**) The cells invading into the Matrigel-coated membrane were photographed (X100), and the number of invasive cells was calculated as a percentage of invasion. (**B**) The cells migrating into the scratched area were photographed (X100), and the number of migratory cells was calculated as a percentage of migration. (**C**) HMT-treated PC-3 cell lysates were prepared and subjected to Western blotting for cell motility-related proteins in the PAK-1 signaling pathway (PAK-1, LIMK1/2, p-LIMK1/2, cofilin, p-cofilin, AKT, p-AKT, and \(\beta\)-actin). The bar graph indicates the ratio of the protein of interest to \(\beta\)-actin by densitometric analysis presented as fold changes of the control. Data represent mean \(\pm\) SD. \({}^{*}\)\(p\) < 0.05, \({}^{**}\)\(p\) < 0.01, and \({}^{**}\)\(p\) < 0.001 compared with control. Figure 3: Effect of HMT on cell cycle arrest and apoptosis in PC-3 cells. PC-3 cells were treated with HMT (100, 200, and 400 \(\upmu\)g/mL) for 24 h. (**A**) The treated cells were stained with propidium iodide (PI) and analyzed by flow cytometry. (**B**) Bar graphs show the quantification of the cell cycle population (%). Data represent mean \(\pm\) SD. * \(p\) \(<\) 0.05, **_p_ \(<\) 0.01, and ***_p_ \(<\) 0.001 compared with control. (**C**) PC-3 cells were treated with HMT (200 \(\upmu\)g/mL) for 24 h and stained with 2 \(\upmu\)M CellEvent for apoptosis analysis and imaged using a fluorescence microscope (200\(\times\)) (Nikon, Tokyo, Japan). (**D**) HMT (100 and 200 \(\upmu\)g/mL)-treated PC-3 cell lysates were prepared and subjected to Western blotting for cell cycle- and apoptosis-related proteins (PAK-1, CDK6, BCL-2, cleaved caspase-3, PARP, and \(\upbeta\)-actin). Band density of proteins was quantified using the Gelpro analyzer (Media Cybernetic, Bethesda, MD, USA). The bar graph indicates the ratio of the protein of interest to \(\upbeta\)-actin by densitometric analysis presented as fold changes of the control. Data represent mean \(\pm\) SD. *_p_ \(<\) 0.05, **_p_ \(<\) 0.01, and ***_p_ \(<\) 0.001 compared with control. PAK-1 Mediates HMT-Induced G1 Arrest and Apoptosis and Suppresses Cell Proliferation and Cell Motility  
Next, we assessed whether the anticancer effect of HMT is dependent on PAK-1.