# HMT exerts an Anticancer Effect by Targeting PAK-1
## 4 Discussion  
Since PAKs were discovered in 1994, several studies demonstrated their crucial roles in numerous cellular processes, such as cell cycle progression and cell survival [24]. PAK-1 belongs to the Group I of the PAK family and was identified for the first time in a screening for proteins that interacted with GTP-bound Rac [25]. PAK-1 is overexpressed in a variety of cancers, including prostate cancer [26]. PAK-1 was recently shown to be a valid therapeutic target for cancer treatment [27]. Since then, several PAK-1 inhibitors have been developed for use as biological markers and therapeutic agents [28]. Recent studies investigating the antitumor effect of natural products suggested that they may lead to promising alternative therapy for the treatment of cancer. Traditional Chinese or Korean medicine is widely accepted as an alternative treatment for cancer [29; 30; 31]. Figure 6: HMT contains decursin that inhibits PAK-1. (**A**) TLC analyses of HMT. For the detection of decursin, the TLC plate was dipped in 10% sulfuric acid to char carbon-containing compounds. (**B**) HPLC profile. Upper section: standard of decursin, lower section: HMT. (**C**) Changes in PAK-1 expression in response to decursin in PC-3 cells. Cells were treated with decursin (at 10 \(\upmu\)M) for 24 h. Cell lysates were prepared and subjected to Western blotting using PAK-1 antibodies. 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_ < 0.01 compared with control. Furthermore, research on traditional Chinese and Korean medicinal and herbal formulations has been recognized internationally by medical researchers [32], and several studies elucidated the molecular and cellular mechanisms of herbal medicine-derived phytochemicals in the context of cancer research [33; 34; 35]. In this study, we investigated the effect of oriental medicinal herbs on PAK-1 signaling. Previously, we reported the effect of PAK-1 inhibitor EOPK (essential oil of _Pinus koraiensis_) in HCT 116 cells [36]. Now, we have identified a second PAK-1 inhibitor isolated from HMT. Recent studies have shown that there are several possible links between PAK and PI3K and Raf-MAPK pathways [5; 36; 37; 38]. To confirm the involvement of PAK-1 in the HMT-mediated anticancer effect, we evaluated expression changes of proteins involved in the PAK-1 pathway by knocking down PAK-1 with siRNA. As shown in Figure 4, knockdown of PAK-1 by siRNA reduced the expression of the G1-regulatory protein cyclin D1, LIMK1/2, and cofilin and induced expression of the apoptotic protein cleaved caspase-3. Furthermore, this knockdown enhanced the HMT-mediated anticancer effects by inhibiting the PAK-1/AKT pathway. Consistently with our data, PAK-1 inhibition using shRNA or siRNA strategies induced apoptosis and CDK4/6 inhibition [39; 40; 41]. HMT is mainly prepared using extracts from _Hippocampus abdominals_, _Angelica ggas Nakai_, _Rhem palmatum_, _and Critri unshius Markovich_. The _Hippocampus_ extract contains steroids and fatty acids and possesses various pharmacological properties including antitumor effects [42; 43] and anti-prostatic hyperplasia activity [44; 45]. _Rhem palmatum_ contains emodin, which has been reported to target the AR directly and induce apoptosis [46; 47]. Moreover, emodin has been reported to inhibit tumor cell migration through suppression of the PI3-K-cdc42/Rac1 pathway in MDA-MB-231 cells [48]. _Rhem palmatum_ contains catechin and epicatechin. Green tea catechins have been reported to possess anticancer effects by many researchers [49; 50; 51; 52]. The therapeutic mechanisms of catechins, especially in prostate cancer, include direct action on cancer cells and indirect action on tumor-associated inflammation [53]. _Critri unshius Markovich_ contains hesperidin and naringin. According to a recent study, naringin inhibits cell viability and induces apoptosis in PC-3, LNCaP, and DU145 cells.