*2.4. Invasion (Transwell Assay)*

The invasive capacity of cells was evaluated by gelatin-coated transwell assay. As presented in Figure 4, the invasive ability of the EO-treated cells was significantly decreased after 24 h of treatment. Compared with the untreated cells, the relative invasion ratio of EO-treated HepG2 cells was 0.58 ± 0.046. There were significant differences between the treated and untreated cells, and EO significantly decreased the invasive capacity of HepG2 liver cancer cell lines.

**Figure 3.** Effect of EO from *A. lanatum* on the migration of HepG2 liver cancer cell lines (scratch wound assay). (**A**) EO inhibits HepG2 human liver cancer cell proliferation and migration. HepG2 cell monolayers were scratched, and cells were supplemented with EO (10 and 25 μg/mL) for 24 h. Migration was determined using an optical microscope (200× magnification) by wound-healing assay. (**B**) Scratch distance was used to calculate the area of the wound and assess wound closure. The data are shown as the mean ± SD of triplicate values; \* *p* < 0.05 when evaluated with respect to the control (PBS, phosphate-buffered saline).

**Figure 4.** Effect of EO from *A. lanatum* on the inhibition of invasion and angiogenic capacity of HepG2 liver cancer cells. (**A**) EO inhibits human liver cancer cell migration and invasion. HepG2 liver cancer cell lines were supplemented with EO (10 and 25 μg/mL) and loaded to the upper chambers of matrigel-coated transwells. Invasion was determined by total cell counting. Cells invading the lower chamber after 24 h were counted. (**B**) The inhibition percentage of invasion was quantified and expressed relative to the control (untreated cells), whose level of invasion was set at 100%. Invading cells quantified using manual counting and values are noted as fold changes, compared to control. The data are shown as the mean ± SD of triplicate values; \* *p* < 0.05 when evaluated against control (PBS, phosphate-buffered saline).

### *2.5. Immunoblotting and Localization of Cytochrome-c*

Liver cancer markers are important tools for the evaluation of the migration and invasion of tumor cells. The apoptotic regulatory markers BCL-2 and CASPASE-3 were reciprocally regulated by the EO. Angiogenesis was lost through the regulation of CYP-1A1 and NFκB. EO negatively regulated the NFκB markers and increased CYP-1A1 expression levels in both mRNA and protein markers. These results demonstrate that EO regulates protein expression levels in HepG2 liver cancer cell lines; see Figure 5. The apoptotic marker CASPASE-3 was significantly up-regulated (2.5 ± 0.6-fold; *p* ≤ 0.05), whereas the angiogenic marker NFκB was down-regulated in EO-treated HepG2 cells (0.65 ± 0.1-fold; *p* ≤ 0.05). Furthermore, the metabolic marker CYP-1A1 was significantly up-regulated in EO-treated cells (3.2 ± 0.5-fold; *p* ≤ 0.05). EO-treated HepG2 cells showed a loss of mitochondrial membrane potential (MMP) integrity at both concentrations (10 and 25 μg/mL). Untreated cells showed high MMP integrity by an emission of orange-red fluorescence. On the other hand, apoptotic cells stained with JC-1 showed green fluorescence. Consequently, the EO-treated cells demonstrated an emission of green fluorescence, indicating a loss of mitochondrial membrane integrity and release of the mitochondrial contents, including cytochrome-c (cyt-c), in the cytoplasm (Figure 5D,E). These changes revealed the possible participation of EO in induction of the apoptotic pathway. Therefore, it was suggested that EO treatment (at 25 μg/mL) decreased the migration and invasion abilities of HepG2 liver cancer cells by reciprocal regulation of angiogenesis and apoptotic markers.

**Figure 5.** Effects of EO from *A. lanatum* on the mRNA and protein markers of HepG2 liver cancer cells. The effects of EO on the inhibition of apoptotic and angiogenic markers were evaluated by real-time PCR. (**A**) HepG2 liver cancer cells were supplemented with EO (10 and 25 μg/mL). The mRNA of apoptotic and angiogenic markers that was altered in EO-treated cells was quantified using quantitative real-time PCR. GAPDH was used as an internal mRNA control. (**B**,**C**) Alterations in the status of metastasis-associated proteins in response to EO supplementation were inspected using Western blot. HepG2 cells were supplemented with EO (10 and 25 μg/mL) for 24 h. β-actin was utilized as a control. (**D**,**E**) The mitochondrial membrane potential (MMP) was estimated for in EO-treated (10 and 25 μg/mL) HepG2 cell lines after an incubation period of 24 h. The mitochondrial membrane integrity was analyzed using the emission of green fluorescent by ImageJ software. The experimental data are shown as the mean ± SD of triplicate values; \* *p* < 0.05 when evaluated against control (PBS; phosphate-buffered saline).
