**3. Discussion**

In this study, we found that treatment with JI017 in H460 and H1299 cells induced apoptosis by increasing autophagy pathway signaling. Although various anticancer drugs have been developed to treat lung cancer, new therapies are needed due to their many side effects. Recently, many studies have investigated anticancer effects through autophagy activity [54,55].

Additionally, interest in natural product-derived drugs is increasing in the pharmaceutical industry because of their low side effects [29,30,32]. Thus, our drug is a good suitable candidate for the treatment of human lung cancer.

It was reported that JI017 exerts anticancer effects and, thus, has potential as a new drug that death of cancer cells and inhibits cancer cell growth [45,48]. JI017 is a combination of *Ag*, *Zo*, and *Ac*, and its anti-cancer treatment effects have been reported through various mechanisms. Unlike simply confirming the active compound of a natural product, in a new compound, as a natural product complex, various effects are shown, and as a result, the treatment mechanism is multi-faceted, so there are still many areas to be studied. Therefore, we investigated the effect of JI017 treatment on lung cancer cells. We investigated whether JI017 treatment affected cell viability in H460 and H1299 cells and found that JI017 treatment significantly inhibited cell growth. In addition, growth inhibition was accompanied by the inhibition of cell migration and colony formation. We confirmed cancer cell apoptosis using flow cytometry after staining with annexin V/PI and analyzed protein changes in the apoptosis mechanism through Western blotting experiments. We found that JI017 treatment decreased the levels of Bcl-2 in A549, H460, and H1299 cells. Additionally, we found that the JI017 treatment of A549, H460 and H1299 cells increased the levels of Bax, cleaved caspase-3, cleaved caspase-8, cleaved caspase-9, and cleaved PARP, known as the markers of apoptosis. These results suggested that JI017 induced apoptosis in lung cancer cells.

Excessive ROS accumulation is known to promote apoptosis [56]. We evaluated the level of ROS in lung cancer cells using the fluorescent dye 2- ,7- -dichlorofluorescein diacetate (DCFH-DA). We confirmed that JI017 treatment increased intracellular ROS generation. In addition, pretreatment with NAC, a ROS inhibitor, and treatment with JI017 decreased the rate of apoptosis, and we confirmed that the levels of caspase 3 and cleaved PARP, which are markers of apoptosis, were reduced in H460 and H1299 cells. These results suggested that JI017 induced apoptosis by ROS production in lung cancers.

We investigated the pathway by which JI017 induced ROS accumulation and apoptosis in H460 and H1299 cells using Western blotting. The PI3K/Akt pathway is a representative regulator of growth, proliferation, the cell cycle, metastasis, apoptosis, and autophagy [57–59]. The inhibition of the PI3K/AKT/mTOR pathway can result in cell survival or death via autophagy or apoptosis, respectively [60–64]. JI017 decreased the levels of PI3K, AKT, and mTOR in H460 and H1299 cells. Therefore, we investigated whether JI017 induced autophagy using Western blot assay and IF staining analysis. We found that JI017 regulated LC3 A/B and p62 expression. The change in the LC3 level after JI017 treatment time was examined, and it was confirmed that the LC3 level and the cleavage of PARP increased after 12 h of treatment with JI017. Additionally, through IF staining results, it was confirmed that JI017 treatment increased the cytoplasmic accumulation of LC3 in cells. These results suggest that JI017 treatment induces autophagy and results in apoptosis in lung cancer. We treated JI017 with 3MA and chloroquine, which are autophagy inhibitors, to verify that JI017 induced apoptosis through the autophagy pathway. We found that 3MA reduced LC3 levels that increased with JI017 treatment. Moreover, cells treated with JI017 combined with 3MA or Bafilomycin or chloroquine showed decreased levels of apoptosis markers and cell death compared with those in cells treated with JI017 alone. In addition, cells treated with JI017 in combination with NAC exhibited reduced levels of LC3 compared to those in cells treated with JI017 alone, suggesting that JI017 promoted apoptosis through ROS-induced autophagy in H460 and H1299 cells (Figure 6). Moreover, we subcutaneously injected H460 cells into nude mice to confirm that JI017 has an inhibitory effect on lung cancer in vivo. Compared to control mice, the xenograft tumors in JI017-treated mice showed a lower growth rate and a TGI value of 79.2%. We found that JI017 treatment increased the MDA concentration in tumor tissues in vivo, and as a result, we determined that it induced ROS generation. Additionally, in the JI017 treatment group, the protein level of Ki-67 was decreased, and the protein levels of cleaved caspase-3 and LC3 were increased. Collectively, these findings suggested that JI017 inhibited lung cancer proliferation and tumor growth in vivo. JI017 clearly induced apoptosis in human lung cancer cells, making it a useful compound in the treatment of lung cancer. Additionally, targeting autophagy to treat lung cancer could be a useful therapeutic mechanism. Our study clearly demonstrates that the anticancer effect of JI017 in human lung cancer cells occurs through ROS-induced autophagy signaling. As a new natural compound anti-cancer treatment, we think that additional research on various mechanisms should be conducted.

**Figure 6.** Mechanistic pathway diagram for the autophagy activation potency of JI017 through AKT, ERK inhibition, and ROS induction in lung cancer.

#### **4. Materials and Methods**

#### *4.1. Reagents*

JI017 consists of *Ag*, *Ac*, and *Zo* components that were supplied by the Jaseng Hospital of Korean Medicine (Seoul, Republic of Korea). The roots were boiled for 3 h in distilled 70% ethanol. The extract was filtrated twice through Whatman grade 2 qualitative filter paper (GE Healthcare Life Sciences, Marlborough, MA, USA) to remove any insoluble materials. The filtrated extract was lyophilized to a powder using a freeze dryer (IlShinBioBase, Dongducheonsi, Gyeonggi, Republic of Korea) and stored at 4 ◦C. The dried extract was then dissolved in dimethyl sulfoxide (DMSO).

#### *4.2. Cell Culture*

A549, H460, and H1299 human lung cancer cells obtained from the American Type Culture Collection (ATCC) were maintained in RPMI 1640 or F-12K medium supplemented with 10% heat-inactivated fetal bovine serum (Invitrogen, Carlsbad, CA, USA) and 100 U/mL antibiotics–antimycotics (Invitrogen). Cells were maintained at 37 ◦C in a humidified incubator with 5% CO2.

#### *4.3. Cell Viability Assay*

An MTS assay was performed to determine cell viability. To accomplish this, cells (A549, H460, and H1299 cells) were seeded into a 96-well plate at a density of 3 × <sup>10</sup><sup>3</sup> cells per well and treated 24 h later with varying concentrations of JI017 (25–500 μg/mL) for an additional 24 h. Ten microliters of MTS solution was added to each well of the plate, which was incubated in the dark at 37 ◦C for another 1 h 30 min. Optical density was measured at 450 nm using an ELISA plate reader (Versa Max, Molecular Devices, San Jose, CA, USA).
