**3. Discussion**

Plant metabolites exhibit remarkable effects, including anti-cancer and other important biological activities. EOs, among the important secondary plant metabolites, through a long chain of evidence, have been shown to possess several different biological activities [1,27–29]. EOs are worth consideration in research in order to highlight their mechanisms of action and pharmacological targets. Chemically, EOs are a complex blend of hydrocarbons and oxygenated hydrocarbons, biosynthesized and arising from the isoprenoid pathways, and mainly consisting of monoterpenes and sesquiterpenes [30]. The EO obtained from *A. lanatum* was analyzed using GC-MS, and interpretation of the analysis revealed and quantified 38 components representing 94.68% of the total components, including monoterpene hydrocarbons (47.86%), oxygenated monoterpenes (16.44%), sesquiterpene hydrocarbons (22.13%), and oxygenated sesquiterpenes (6.48%); see Figure 1 and Table 1. Previous reports have discussed the molecular cytotoxicity effects of EOs, and some examples of isolated compounds from EOs towards various cancer cell lines have indicated the mediation of apoptosis, loss of mitochondrial membrane integrity, and several other mechanisms involving the anti-apoptotic factor BCL-2 and pro-apoptotic protein CASPASE-3 [30–33]. Camphor and eucalyptol have demonstrated the induction of apoptosis through the down-regulation of anti-apoptotic factor BCL-2 in a human oral epidermoid carcinoma cell line and activation of the CASPASE cascade in oral KB and colorectal cancer cell lines [30,34,35]. Linalool has been shown to lead to a reduction in BCL-2 protein expression in human dermal fibroblast cancer cell lines [36].

Few studies have investigated the anti-cancer activities of EO from *Anisosciadium*. The EO from *A. orientale* has shown antioxidant and anti-cancer activities against various human cancer cell lines [17,18]. In this study, the results from an MTT assay demonstrate that the proliferation of HepG2 was significantly inhibited by the EO of *A. lanatum*. Conversely, NIH-3T3 cell lines showed higher cell proliferation and viability, and were insignificantly inhibited up to 25 μg/mL EO. Considering these results, further studies were carried out on HepG2 liver cancer cell lines (see Figure 2). Cell migratory properties affect tumorigenesis and metastasis. Various studies have targeted cancer cell migration and invasion as potent tools for controlling the progression of cancers [37,38]. In the current study, the cellular functional results revealed that EO (25 μg/mL) from *A. lanatum* exhibited a regulation of cell migration and invasion in HepG2 liver cancer cell lines (Figures 3 and 4). Reliably, the activation of CASPASE-3 and an antagonist of BCL-2 inhibits the potential intrusion of HepG2 liver cancer cells [39]. BCL-2 is an anti-apoptotic protein that controls the release of cytochrome from mitochondria [40]. Mechanistically, the results showed that EO suppressed BCL-2 expression, which could have been reflected in the cell proliferation and survival of HepG2 liver cancer cells. As BCL-2 is a marker for tumorigenesis and neoplastic progression, it may be a potential marker for anti-cancer therapies. Several studies have confirmed that BCL-2 offers potential as a prospective drug target, as it activates the protooncogenic effect of the cancer environment [41–43]. In this study, EO potentially inhibited the BCL-2 mRNA and protein expression in HepG2 liver cancer cell lines (Figure 5). Many small molecular inhibitors of BCL-2, including ABT-737 and ABT-199, have been investigated extensively [44]. The suppressive effects of BCL-2 and CYP-1A1 are interlinked in cancer prognosis through migratory action and tumorigenesis [45]. Angiogenesis involves regulatory functions played by BCL-2 and CASPASE-3 in reverse roles [38,46]. In this study, the suppression effect of EO on the cell migration process may have been mediated by the suppression of BCL-2 and NFκB markers (Figure 5). The invasiveness activity was also inhibited in EO-treated HepG2 liver cancer cell lines. Blocking of the regulatory role of NFκB suppressed the invasiveness capacity and tumorigenesis in in vitro models. The liver cancer cells were inhibited by EO, which contributed to controlling cancer metastasis [47]. Such suppression regulates the apoptosis (also known as programmed cell death) of tumor cells, morphologically characterized by nuclear damage, chromatin condensation, cell shrinkage, and DNA endonuclease activation with apoptotic bodies [48]. Importantly, in this study, EO induced cell apoptosis in HepG2 liver cancer cells through the activation of mRNA of CASPASE-3 and CYP-1A1 (Figure 5). These genes are apoptotic markers involved in the spreading and invasiveness of cancer cells [44,48,49]. Subsequently high expression of CASPASE-3 and CYP-1A1 could be initiated due to mitochondrial membrane damage. Upon permeabilization of the mitochondrial membrane, caspase activators such as cyt-c were released from mitochondria into the cytosol [50]. The results verify the loss of mitochondrial membrane integrity. Consequently, the mitochondrial contents, including cyt-c, were released into cytoplasm and triggered an intrinsic apoptotic cascade. These findings express the possible activation of apoptosis via cyt-c, which in turn initiates other events in the programmed cell death in EO-treated HepG2 cell lines (Figure-5D, E). Further molecular modulations showed that EO increased the mRNA of CASPASE-3 expression levels, as well as increased and reciprocally regulated the protein expression levels of BCL-2 and NFκB (Figure 5). These results suggest that EO promoted apoptosis in and inhibited the invasiveness of HepG2 liver cancer cells. The previously discussed findings were further confirmed by in silico studies through the potential simulation of anti-proliferative properties of major molecules of EO (Figures 6 and 7). In some studies, *β*-sitosterol was shown to have potent binding against apoptotic regulating proteins, whereas isovitexin had the lowest binding affinity detected against BCL-2 and CASPASE-3 proteins [51]. In this study, of the major molecules, only camphor, eucalyptol, nerol, and linalool showed potential binding against BCL-2. Camphor and eucalyptol alone showed significant binding against pro-apoptotic CASPASE-3 regulatory molecules. These findings suggest that the EO from *A. lanatum* can control hepatoma HepG2 liver cancer cells through the reciprocal regulation of apoptotic markers. Taken together, the EO from *A. lanatum* was found to be able to control cancer progression and tumorigenesis, as mediated by modulatory effects on apoptotic markers.
