2.5.2. Integrin Human ITG α5β1

The test was performed using competitive ELISA human integrin α5β1 (MyBiosource, San Diego, CA, USA). The procedure was carried out according to the manual in the kit (MyBiosource, San Diego, CA, USA). A total of 100 mL of standard and lysate sample was added to each, then 10 mL of balanced solution was added and homogenized; no bubbles were formed. A total of 50 mL of the conjugate was added in the well, then homogenized, and incubated at 37 ◦C for 60 min. After 60 min, we drained the liquid on the plate and washed it with wash buffer 5 times, for 1 minute each time. Volumes of 50 mL of substrates A and B were added to the wells. The plates were then closed tightly and incubated at 37 ◦C for 15 min. Stop solution (50 mL) was added, and the well plate was immediately read at a wavelength of 450 nm (BioRad, Hercules, CA, USA).

#### 2.5.3. Enzyme-Linked Immunosorbent Assay (ELISA) Data Analysis

ELISA data analysis of human ITG αvβ3, human ITG α5β1, and vascular endothelial growth factor (VEGF) was carried out quantitatively using an ELISA reader to determine the optical density value of each test, then the concentration values were calculated based on the standard value. Data were analyzed via one-way ANOVA using the GraphPad Prism 7 software (La Jolla, CA, USA).

#### *2.6. Scratch Wound Healing Assay*

The scratch wound healing assay procedure was carried out based on [11]. A-549 cells at 2.5 × 104 cells/500 mL were grown on a culture test 12-well plate (Greiner, Frickenhausen, Germany). Cells were incubated for 24 h. Cells were rinsed with DMEM high-glucose three times. A sterile 200 mL pipette tip (Vertex, Boston, MA, USA) was used to make a scratch on the cell surface, then they were treated in groups, namely non-treated (NT); AP3 80 μg/mL; ethanol extract of *Ocimum sanctum* Linn. (EEOS) at graded concentrations of 50, 70, 100, and 200 g/mL; and cisplatin 9 g/mL. The treatments were incubated for 24 h in a CO2 incubator at 37 ◦C, then observed after 24 h via inverted microscopy. Data analysis of the scratch wound healing assay to determine cell migration was carried out by measuring the surface area of the treated cells. Areas were calculated using the free software ImageJ (https://imagej.nih.gov/ij/, accessed on 4 April 2022) (National institute of Health-NIH, Bethesda, MD, USA). We then calculated the percentage of the area covered according to the following formula:

$$\text{closing area} = \frac{(\text{area of 0th hour} - 24 \text{th hour area})}{\text{area of 0th hour}} \times 100\%$$

The final data were analyzed via one-way ANOVA using GraphPad Prism 7 software (La Jolla, CA, USA).

#### **3. Results**

#### *3.1. EEOS Inhibited the Adhesion of A549 (Non-Small Cell Lung Carcinoma)*

To analyze the ability of EEOS to inhibit cell attachment, A459 cells were cultured on a well plate and treated for 24 h with different concentrations of EEOS (50, 70, 100, and 200 μg/mL). Cisplatin was used as a positive control and to provide a comparison with commercial drugs. Our results show that EEOS inhibited the cell attachment of A549 cells in a dose-dependent manner. EEOS showed significant inhibition at the optimum concentration of 200 μg/mL, but the inhibition was not significant with 50 μg/mL of EEOS (Figure 1).

**Figure 1.** The ethanolic extract of *Ocimum sanctum* Linn. (EEOS) inhibited the adhesion of A549 cells (non-small cell lung carcinoma). The cells were cultivated in the presence of an inhibitor (AP3) as the positive control, cisplatin as the commercial drug comparison, and EEOS at concentrations of 50, 70, 100, and 200 μg/mL. After 24 h, EEOS's inhibitory effect was visualized using MTT reagent at a wavelength of 450 nm (NT: non-treated; \* significant *p* = 0.0332; \*\* significant *p* = 0.026; \*\*\*\* significant *p* < 0.0001; ns = not significant).

#### *3.2. EEOS Inhibited the Cell Migration of A549 (Non-Small Cell Lung Carcinoma) after 24 h of Treatment*

The scratch wound healing assay is one of the most commonly used assays for assessing therapeutic impacts on cell migration. In this study, we found that EEOS significantly suppressed the cell migration of NSCLC (A549 cell line). We examined cell migration in response to the mechanical scratch wound. The cells were cultured in a well plate, and after confluence, the cells were treated with EEOS. After 24 h, the cell culture was observed under inverted microscopy. Images of scratch areas after 24 h (Figure 2) indicate that the untreated wounds were half closed within 24 h. To quantify the effects of putative migration inhibitors, the percentage of the open wound area after 24 h was determined (Figure 2). Our data clearly show that treatment with EEOS caused a significant inhibition of cell migration in a concentration-dependent manner.

**Figure 2.** (**A**) Photomicroscopic images of A549 cells during the scratch wound healing assay. The cells were cultivated under normal conditions as non-treated cells (**a**), in the presence of an inhibitor (AP3) as the positive control (**b**), with cisplatin as the commercial drug comparison (**c**), and with EEOS at concentrations of 50 (**d**), 70 (**e**), 100 (**f**), and 200 g/mL (**e**). The wound healing was observed at the 0th hour and after 24 h. (**B**) Ethanolic extract of *Ocimum sanctum* Linn. reduced the migration ability of non-small cell lung carcinoma (A549), as shown by the scratch wound assay. The cells were cultivated in the presence of an inhibitor (AP3) as the positive control, cisplatin as the commercial drug comparison, and EEOS at concentrations of 50, 70, 100, and 200 g/mL. (**a**). The wound healing was observed at 0 h and after 24 h. (**b**). The wound healing after 24 h. Statistical analysis was performed via one-way ANOVA, followed by post hoc Tukey test (NT: non-treated; \*\* significant *p* < 0.0060; \*\*\* significant *p* < 0.0009; \*\*\*\* significant *p* < 0.0001; ns = not significant).

*3.3. EEOS Inhibited Cell Migration of the A549 Cell Line (Non-Small Cell Line Carcinoma) by Suppressing the Concentrations of Integrin αvβ3, Integrin α5β1, and Vascular Endothelial Growth Factor (VEGF)*

To strengthen the evidence regarding the effect of EEOS on cell migration, we performed ELISA on A549 cell lysates. The representative parameters observed were integrin αvβ3, integrin α5β1, and VEGF. The untreated A549 cells produced the highest concentration of integrin αvβ3, integrin α5β1, and VEGF. Additional treatment of A549 with EEOS diminished the integrin αvβ3, integrin α5β1, and VEGF concentrations in a dosedependent manner. The integrin αvβ3 (Figure 3A), integrin α5β1 (Figure 3B), and VEGF (Figure 3C) concentrations were significantly suppressed under the optimum concentration of EEOS (200 μg/mL) and under cisplatin.

**Figure 3.** (**A**) The ethanolic extract of *Ocimum sanctum* Linn. decreased expression of non-small cell lung cancer (A549) integrin αvβ3, as shown by sandwich ELISA. The cells were cultivated in the presence of an inhibitor (AP3) as the positive control, cisplatin as the commercial drug comparison, and EEOS at concentrations of 50, 70, 100, and 200 g/mL for 24 h. A549 cells were then lysed and analyzed via ELISA for the concentration of αvβ3 integrin (ug/mL). Statistical analysis was performed via one-way ANOVA, followed by post hoc Tukey test (NT: non-treated; \*\*, \*\*\*, and \*\*\*\* indicate statistically significant values for the non-treated group as a negative control compared with treatment, with *p*-values of 0.0085, 0.0004, and <0.0001, respectively; ns = not significant). (**B**) Ethanolic extract of *Ocimum sanctum* Linn. decreased expression of integrin α5β1 in non-small cell lung cancer (A549), as shown by competitive ELISA. The cells were cultivated in the presence of an inhibitor (AP3) as the positive control, cisplatin as the commercial drug comparison, and EEOS at concentrations of 50, 70, 100, and 200 g/mL for 24 h. A549 cells were then lysed and analyzed via ELISA for the concentration of α5β1 integrin (ug/mL). Statistical analysis was performed via one-way ANOVA, followed by post hoc Tukey test (NT: non-treated; \*, \*\*, and \*\*\*\* indicate statistically significant values for the non-treated group as a negative control compared with treatment, with *p*-values of 0.0348, 0.0027, and <0.0001, respectively; ns = not significant). (**C**) Ethanolic extract of *Ocimum sanctum* Linn. decreased expression of the non-small cell lung cancer (A549) integrin VEGF, as shown by sandwich ELISA. The cells were cultivated in the presence of an inhibitor (AP3) as the positive control, cisplatin as the commercial drug comparison, and EEOS at concentrations of 50, 70, 100, and 200 g/mL for 24 h. A549 cells were then lysed and analyzed via ELISA for the concentration of VEGF ((ug/mL). Statistical analysis was performed via one-way ANOVA, followed by post hoc Tukey test (NT: non-treated; \*\*, \*\*\* and \*\*\*\* indicate statistical significance of the non-treated group as a negative control compared with treatment, with *p*-values of 0.0012, 0.0002, and <0.0001, respectively; ns = not significant).

#### **4. Discussion**

Cancer has properties such as evading cell death, sustaining proliferation, inducing vasculature, and activating invasion and metastasis [12]. In the process of malignancy, tumor cells will migrate to other organs through blood vessels and lymph vessels and grow in the appropriate organs; this process is called metastasis. Cell–cell and cell–extracellular matrix (ECM) adhesions play a fundamental role in governing the structural integrity of healthy tissue and in regulating cellular morphology, migration, proliferation, survival, and differentiation events [13]. In the classic view of malignant transformation in the epithelium, cells lose their dependence on integrin-mediated interactions with the extracellular matrix and the resulting signaling [14]. In the process of metastasis, tumor cells will migrate to find the best place to maintain their function. Cell migration, invasion, and adhesion are pivotal steps in this process [15,16].

In this study, we observed the ability of EEOS to prevent the adhesion of the A549 cell line. The CCK-8 test chart showed a decrease in the adhesion ability of A549 cells treated with EEOS (Figure 1). The results of this study add to the information from previous studies that EEOS can reduce the adhesion ability of A549 cells, as shown via adhesion assay [8]. We also performed scratch wound healing assay to investigate the migration ability of the A549 cell line. Our data show that EEOS also has the ability to inhibit A549 cell migration (Figure 2A,B). The ability of tumor cells to adhere and migrate is closely related to the process of tumor progression and metastasis, which is responsible for 90% of cancerrelated deaths [17]. The phytochemical compounds in EEOS were previously dialyzed using thin-layer chromatography (TLS) and UV–vis spectrophotometry. The results of the analysis showed that EEOS contains several active compounds, such as flavonoids, phenols, saponins, alkaloids, tannins, terpenoids, and steroids [18]. Flavonoids and phenols have important roles as anti-cancer and cytotoxic agents, inducing apoptosis in cancer cells [19]. In silico molecular docking was also performed to predict the chemical binding between active compounds and protein. In silico molecular docking analysis of the flavonoid compounds (quercetin) and flavonoids (eugenol) showed that these active compounds can bind to the active site of integrins and VEGF, thereby inhibiting the activity of integrins and VEGF for adhesion, cell spread, and blood vessel formation [20]. The inhibition of active compounds with integrin complexes will have an impact on the inhibition of the extracellular matrix (ECM) adhesion process and result in a decrease in tumor cell invasion. In vitro results on the cell line A549 also showed consistent results that the content of active compounds in EEOS can reduce the viability of the A549 cell line.

To elucidate this mechanism, we also examined the expression of integrin αvβ3, integrin α5β1, and VEGF as biochemical cues for blood vessel formation, adhesion, and migration of cancer cells. We found that EEOS reduced the concentrations of integrin αvβ3, integrin α5β1, and VEGF in the A549 cell line (Figure 3A–C). Integrins are transmembrane adhesion receptors for the extracellular matrix (ECM) and have essential roles, including sensing and adhering to the extracellular environment to maintain global tissue architecture and multicellularity [21]. Integrins are the major class of receptors in adhesive events, acting by bi-directionally (inside-out and outside-in) transducing biochemical signals and mechanical force across the plasma membrane [22]. Integrins play a key role in single-cell migration and act via conformational changes in the extracellular matrix (outside-in) or intracellular protein that are triggered by altering the affinity of integrins (inside-out). These changes recruit cytoskeletal linker proteins to remodel nascent or focal adhesions and generate tension; these adherent structures generate forces of cellular movement. There are several pathways by which integrin can mediate cell spreading and migration and one of them involves focal adhesion kinase and the capacity of tyrosine-protein kinase Src to up-regulate integrin expression [23].

The integrins αvβ3 and α5β1 have roles as adhesion molecules in cell-to-cell interactions and motility-supporting roles that promote cell migration during nervous system development, and they also promote metastatic spread [24]. Integrin αvβ3 is mostly expressed on angiogenic endothelial cells in remodeling and pathological tissues. Expression of the αvβ3 integrin by endothelial cells promotes cell adhesion to the ECM, cell migration, and angiogenesis, along with angiogenic growth factors, including VEGF/VEGFR [25]. The α5β1 integrin is also overexpressed in, and closely related with, metastatic events. In normal endothelial cells, α5β1 will be expressed at very low levels, but this expression will be significantly increased in endothelial cells during cancer cell angiogenesis [26]. Integrin expression and activation directly influence human malignancies. Due to their broad impact in malignant transformations, they are considered potential targets for cancer therapy [27]. Integrins are considered as pharmacological targets for drugs by inhibiting several key processes in cancer development, such as cell proliferation, survival, and migration. Targeting integrins to enhance the delivery of anti-tumor agents or to delineate cancerous lesions is a new and promising approach. Integrin-inhibiting anticancer drugs have been conceived for their ability to impair ligand binding [28].

In addition to integrin expression, VEGF expression has been confirmed to be a critical pathological factor in the occurrence of NSCLC by increasing vascular permeability and increasing angiogenesis [29]. This study confirmed that EEOS has the ability to inhibit A549 cell angiogenesis by inhibiting tube formation, as shown through the angiogenesis assay [9] and reducing VEGF concentrations. During angiogenesis, VEGF has an associated mechanism with integrins, as integrins are overexpressed on the endothelial cell surface to facilitate the growth and survival of new vessels [25]. The supply of oxygen and nutrients to cells through blood vessels is the most important aspect in the survival of cells, including cancer cells. Vascular endothelial growth factor (VEGF) is a homodimeric glycoprotein from the endothelial growth factor family and is an important factor in the formation and regulation of angiogenesis processes [30]; in addition, VEGF has biological roles in the regulation of vascular permeability, metabolism, immune system, inflammation, and neurological function [31]. Tumors can generate their own vascular system. VEGF acts as an angiogenic factor by promoting their proliferation, migration, adhesion, and survival. VEGF may, thus, play a role in vascular invasion [32]. Furthermore, VEGF also play role in targeting other cells in the tumor microenvironment, as well as initiating the function of growth factors and integrin, mainly svb3 and a5b1 [33–35]. In recent years, the inhibition expression of VEGF has been utilized in tumor-targeted therapy [34].

Taken together, our findings underline the ability of the ethanolic extract of *Ocimum sanctum* Linn. to prevent the migration and metastasis of human lung adenocarcinoma cells (A549); however, more research and discussion are required, since our research was limited only to the role of integrin αvβ3, integrin α5β1, and VEGF. Moreover, the data derived from the in vitro analysis demonstrate the direct impact on the cells (Figure 4). Furthermore, in vivo experiments are needed as basic data to complete the preclinical phase of this analysis.

**Figure 4.** Schematic overview of the mechanism of *Ocimum sanctum* Linn. ethanolic extract, inhibiting the adhesion, proliferation, and migration of A549 human lung adenocarcinoma, mediated by the downregulation of αvβ3, α5β1, and VEGF. Overall, inhibition by EEOS will mitigate angiogenesis and metastasis of human lung adenocarcinoma cells.
