**1. Introduction**

Lung cancer is one of the most common malignant tumors and a leading cause of cancer-related death in the worldwide in both males and females, excluding sex-specific cancers [1]. Lung cancer is heterogenous in terms of pathological features: small-cell lung cancer (SCLC) accounts for ~14% of lung cancer patients and non-small cell lung cancer (NSCLC) accounts for ~82% of lung cancer patients [2,3]. The emergence of novel therapeutic methods has significantly improved the treatment of NSCLC, but the prognosis is still not good, and the overall 5-year survival rate of NSCLC patients is only 19.3% [4,5]. Various anticancer drugs, such as pemetrexed and cisplatin, have been used for the treatment of lung cancer, but these anticancer drugs cause acute kidney damage through nephrotoxicity and oxidative damage and cause side effects due to toxicity [6–9]. Therefore, the development of new therapeutic drugs with fewer side effects is necessary.

Autophagy is the process of removing damaged proteins and organelles within cells and recycling intracellular materials and energy, and this mechanism plays an important role in maintaining intracellular homeostasis [10–14]. Autophagy is activated in injury

**Citation:** Ku, J.M.; Kim, M.J.;

Choi, Y.-J.; Lee, S.Y.; Im, J.-Y.; Jo, Y.-K.; Yoon, S.; Kim, J.-H.; Cha, J.W.; Shin, Y.C.; et al. JI017 Induces Cell Autophagy and Apoptosis via Elevated Levels of Reactive Oxygen Species in Human Lung Cancer Cells. *Int. J. Mol. Sci.* **2023**, *24*, 7528. https://doi.org/10.3390/ ijms24087528

Academic Editors: Barbara De Filippis, Marialuigia Fantacuzzi and Alessandra Ammazzalorso

Received: 5 April 2023 Revised: 15 April 2023 Accepted: 18 April 2023 Published: 19 April 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

and disease in response to stresses such as nutrient deprivation, infection, and certain signaling pathways and contributes to cell survival [15,16]. Excessive autophagy is known to induce apoptosis [17]. For example, combined treatment with docetaxel and curcumin induces the apoptosis of malignant esophageal squamous cell carcinoma cells by increasing autophagy [18]. LC3 is a major participant in autophagy, and at initiation, the protein complex LC3-I is degraded to LC3-II, recruited into autophagosomes, and interacts with p62 [19]. Then, the formation of the lysosomal complex then leads to total proteolysis [20].

Reactive oxygen species (ROS) are highly reactive molecules formed from diatomic oxygen that are natural byproducts of normal oxygen metabolism, and they play an important role in homeostasis in the body [21]. Cellular stress caused by changes in the external environment dramatically increases intracellular ROS concentrations, which can cause severe damage to cellular structures, resulting in oxidative damage. Additionally, increased ROS levels are accompanied by apoptosis [22–24]. Therefore, ROS are an indicator of cellular stress and apoptosis [25,26]. Moreover, they are known to be important regulators of autophagy activation, and targeting increased autophagy and ROS production has been identified as a novel therapeutic approach for the treatment of several types of cancer [27,28].

Recently, studies on the anticancer effects of natural products have been conducted, and interest in natural product-derived medicines is increasing [29–32]. According to our previous report, *Angelica gigas* (*Ag*), *Zingiber officinale Roscoe* (*Zo*), and *Aconitum carmichaeli* (*Ac*) showed anticancer effects in the cell lines of several cancers, including brain, breast, prostate, colorectal, skin, and pancreatic cancer [33–38]. Furthermore, it is known that natural treatments for various inflammatory diseases and obesity have neuroprotective effects [39–41]. Decursin-inhibited tumor progression in head and neck squamous cell, as well as the active compound of *Ag*, induces apoptosis by inhibiting the PI3K–Akt axis in HeLa cells [42,43]. Among the active compounds of *Zo*, 6-gingerol suppresses tumor cell proliferation by blocking the nuclear translocation of HIF-1α in lung cancer [44].

Although the anticancer effects of JI017 are known, the efficacy of JI017 treatment for NSCLC has not yet been evaluated. In this study, we investigated whether JI017 exhibits NSCLC cell death and cell growth inhibitory effects. In addition, we analyzed the protein signaling pathway to elucidate the mechanism by which JI017 treats NSCLC.

#### **2. Results**

#### *2.1. JI017-Induced Apoptosis and Inhibited Proliferation in Lung Cancer Cells*

JI017 is known to be an effective anticancer drug for prostate, ovarian, and breast cancer [45–48]. Therefore, we investigated the effect of JI017 treatment on cell viability in several lung cancer cell lines. A549, H460, and H1299 cells were treated with different concentrations of JI017 for 24 h. Cell viability was then measured by MTS assay. We found that JI017 treatment significantly suppressed cell growth in a dose-dependent manner (Figure 1A). Additionally, JI017 treatment significantly reduced colony formation (Figure 1B). Moreover, cell migration was decreased in the JI017 treatment group compared to the control group (Figure 1C).

To investigate whether JI017 induces apoptosis, we performed an annexin V-FITC/PI assay in A549, H460, and H1299 cells. As expected, we found that the JI017 treatment group had an increased apoptosis rate in A549, H460, and H1299 cells, with apoptotic cell ratios of 9 to 56%, 10 to 88%, and 11 to 71%, respectively (Figure 1D). To confirm that caspase activation is induced by JI017 and is involved in apoptosis, we measured the expression of apoptotic molecules through Western blot analysis. We found that JI017 decreased the levels of Bcl-2 in A549, H460, and H1299 cells. Additionally, we found that JI017 increased the levels of Bax, cleaved caspase-3, cleaved caspase-8, cleaved caspase-9, and cleaved PARP in A549, H460, and H1299 cells (Figure 1E). These results confirmed that JI017 induced apoptosis through the apoptotic mechanisms of Bax, Bcl-2, caspase-3, caspase-8, and PARP in A549, H460, and H1299 cells.

**Figure 1.** Effect of JI017 on A549, H460, and H1299 cell viability. (**A**) Cells were treated with different concentrations of JI017 for 24 h. (**B**) Cell viability was then measured using the MTS assay. H460, A549, and H1299 cells were treated with JI017 and exposed for 7 days, then the effect on cell growth was assessed using a colony formation assay. (**C**) The migration of JI017-treated cells was assessed using a wound-healing assay. Intervals between cells are marked with red lines. Scale bar = 400 μm. (**D**) H460 and H1299 cells were treated with JI017 for 24 h, stained with Annexin V/PI, and analyzed by flow cytometry. (**E**) Whole cell lysates were analyzed by Western blotting with anti-PARP; anti-cleaved caspase 3, -8, and -9; and anti-Bax, anti-Bcl-2, and anti-Actin antibodies. Data are presented as the mean ± SEM. \*\*\* *p* < 0.001 compared to untreated cells.

#### *2.2. JI017 Increased the Generation of Intracellular ROS*

ROS is a small molecule, and it is primarily involved in several signaling pathways, and excessive ROS accumulation can induce lipid, nucleic acid, protein, and DNA damage and affect cancer cells to promote apoptosis [23,24]. We used the fluorescent dye

2- ,7- -dichlorofluorescein diacetate (DCFH-DA) to measure ROS levels in lung cancer cells. The cells were treated with JI017 (150 μg/mL) or pretreatment with the ROS inhibitor NAC and JI017 for 24 h and labeled with DCFH-DA. The analysis measured signal intensity using a flow cytometer. JI017 treatment increased the DCFH-DA signal intensity in lung cancer cells. JI017 treatment combined with NAC pretreatment decreased the signal intensity of DCFH-DA in H460 and H1299 cells (Figure 2A). As a result, we confirmed that JI017 induced intracellular ROS generation. Additionally, we investigated whether ROS generated by JI017 mediated apoptosis. We added JI017 (150 μg/mL) to H460 and H1299 cells pretreated with NAC and performed MTS. JI017 treatment combined with NAC pretreatment decreased cell death was compared with that of JI017 treatment alone in H460 and H1299 cells (Figure 2B). Moreover, the cells treated with both JI017 and NAC showed decreased levels of the apoptosis markers cleaved caspase3 and cleaved PARP compared with those in the cells treated with JI017 alone (Figure 2C). These results suggested that JI017 induced apoptosis by increasing ROS production in lung cancer cells.

**Figure 2.** Effect of JI017 in A549, H460, and H1299 ROS accumulation. (**A**) Treatment with JI017 (150 μg/mL) in H460 and H1299 cells for 24 h; they were labeled with DCFH-DA (10 μM) for 30 min. Additionally, intracellular ROS levels were determined by flow cytometry. Pretreatment with NAC (15 mM) for 1 h was followed by treatment with various concentrations of JI017. (**B**) Cell viability was measured using the MTS assay. (**C**) Whole cell lysates were analyzed by Western blotting with anti-PARP, anti-cleaved caspase 3, and anti-GAPDH antibodies. Data are presented as the mean ± SEM. \* *p* < 0.05 and \*\*\* *p* < 0.001 compared to untreated cells.

#### *2.3. JI017-Induced Autophagy by Increasing LC3 Levels in H460 and H1299 Cells*

We performed Western blot analysis to evaluate the expression of the PI3K-AKTmTOR pathway and IF staining analysis to evaluate the expression of LC3. JI017 treatment decreased the levels of PI3K, AKT, and mTOR in H460 and H1299 cells (Figure 3A). The inhibition of mTOR is known to increase autophagy signaling [49]. To investigate whether JI017 induces autophagy, Western blot analysis was used, and it was confirmed in H460 and H1299 cells. The activation of the LC3 protein is a marker of autophagy, and we found that JI017 treatment increased the levels of LC3 A/B and p62 (Figure 3B). Additionally, the change in the LC3 level after JI017 treatment time was examined, and it was confirmed that both LC3 and cleaved PARP levels increased after 12 h of treatment with JI017 (Figure 3C). Additionally, the IF staining experiments showed that the cytoplasmic accumulation of

LC3 was increased through JI017 treatment (Figure 3D). The autophagy pathway plays an important role in cancer cell death, and these results suggest that JI017 induces autophagy in lung cancer cells.

**Figure 3.** JI017 activated autophagy by suppressing the PI3K-AKT-mTOR pathway in H460 and H1299 cells. H460 and H1299 cells were treated with JI017 (50, 100, and 150 μg/mL) for 24 h. (**A**,**B**) Whole cell lysates were analyzed by Western blotting. (**C**) H460 and H1299 cells were treated with JI017 (150 ng/mL) for different times (4, 8, 12, and 24 h). (**D**) H460 and H1299 cells transfected with the pEGFP-LC3 vector were treated with JI017 (150 μg/mL) for 8 h. The Fluorescence microscopy analysis confirmed positive staining of LC3B puncta. LC3B puncta are indicated by white arrows. Scale bar = 20 μm.

#### *2.4. JI017 Induced Autophagy through Increased ROS Production in H460 and H1299 Cells*

3MA is known to inhibit the autophagy pathway in the early stage, and chloroquine and Bafilomycin in the late stage [50–52]. Therefore, we treated H460 and H1299 cells with 3MA (2 mM) or chloroquine (100 μM) in combination with JI017 (150 μg/mL) and performed Western blot analysis. We found that 3MA treatment reduced the elevated LC3 levels induced by JI017 administration. Moreover, cells treated with JI017 and 3MA or chloroquine exhibited reduced levels of caspase3 cleavage and PARP cleavage compared to cells treated with JI017 alone, confirming reduced apoptosis (Figure 4A). To investigate whether JI017-induced apoptosis is regulated by autophagy inhibition, we compared cell viability after treatment with 3MA, bafilomycin, and chloroquine. Compared with JI017 treatment alone, JI017 treatment combined with 3MA or Bafilomycin or chloroquine decreased cell death by approximately 20% in H460 and H1299 cells (Figure 4B). Moreover, cells treated with JI017 combined with NAC decreased the level of LC3 compared with the cells treated with JI017 alone (Figure 2C). These results suggested that JI017 promoted apoptosis through ROS-induced autophagy in H460 and H1299 cells.

**Figure 4.** JI017 induced apoptosis through an autophagy pathway and ROS generation. (**A**) H460 and H1299 cells were treated with 3MA (2 mM) or chloroquine (100 μM) combined with JI017 (150 μg/mL). 3MA, chloroquine, or bafilomycin treatment was followed by treatment with JI017. (**B**) Cell viability was measured using MTS assay. (**C**) H460 and H1299 cells were treated with NAC (15 mM) and JI017 (150 μg/mL). Whole cell lysates were analyzed by Western blotting with anti-LC3 and anti-GAPDH antibodies. Data are presented as the mean ± SEM. \*\*\* *p* < 0.001 compared to untreated cells.
