*Article* **Naringenin Induces ROS-Mediated ER Stress, Autophagy, and Apoptosis in Human Osteosarcoma Cell Lines**

**Chiang-Wen Lee 1,2,3, Cathy Chia-Yu Huang <sup>4</sup> , Miao-Ching Chi <sup>5</sup> , Kuan-Han Lee 6,7, Kuo-Ti Peng <sup>2</sup> , Mei-Ling Fang 8,9, Yao-Chang Chiang 1,\* and Ju-Fang Liu 10,11,\***


**Abstract:** Osteosarcoma, a primary bone tumor, responds poorly to chemotherapy and radiation therapy in children and young adults; hence, as the basis for an alternative treatment, this study investigated the cytotoxic and antiproliferative effects of naringenin on osteosarcoma cell lines, HOS and U2OS, by using cell counting kit-8 and colony formation assays. DNA fragmentation and the increase in the G2/M phase in HOS and U2OS cells upon treatment with various naringenin concentrations were determined by using the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay and Annexin V/propidium iodide double staining, respectively. Flow cytometry was performed, and 20 ,70 -dichlorodihydrofluorescein diacetate, JC-1, and Fluo-4 AM ester probes were examined for reactive oxygen species (ROS) generation, mitochondrial membrane potential, and intracellular calcium levels, respectively. Caspase activation, cell cycle, cytosolic and mitochondrial, and autophagy-related proteins were determined using western blotting. The results indicated that naringenin significantly inhibited viability and proliferation of osteosarcoma cells in a dose-dependent manner. In addition, naringenin induced cell cycle arrest in osteosarcoma cells by inhibiting cyclin B1 and cyclin-dependent kinase 1 expression and upregulating p21 expression. Furthermore, naringenin significantly inhibited the growth of osteosarcoma cells by increasing the intracellular ROS level. Naringenin induced endoplasmic reticulum (ER) stress-mediated apoptosis through the upregulation of ER stress markers, GRP78 and GRP94. Naringenin caused acidic vesicular organelle formation and increased autophagolysosomes, microtubule-associated protein-light chain 3-II protein levels, and autophagy. The findings suggest that the induction of cell apoptosis, cell cycle arrest, and autophagy by naringenin through mitochondrial dysfunction, ROS production, and ER stress signaling pathways contribute to the antiproliferative effect of naringenin on osteosarcoma cells.

**Keywords:** osteosarcoma; naringenin; ROS; ER stress; autophagy; apoptosis

**Citation:** Lee, C.-W.; Huang, C.C.-Y.; Chi, M.-C.; Lee, K.-H.; Peng, K.-T.; Fang, M.-L.; Chiang, Y.-C.; Liu, J.-F. Naringenin Induces ROS-Mediated ER Stress, Autophagy, and Apoptosis in Human Osteosarcoma Cell Lines. *Molecules* **2022**, *27*, 373. https:// doi.org/10.3390/molecules27020373

Academic Editors: Višnja Stepani´c and Marta Kuˇcerová-Chlupáˇcová

Received: 25 September 2021 Accepted: 28 December 2021 Published: 7 January 2022

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**Copyright:** © 2022 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/).

### **1. Introduction**

Cancer is a public health problem, with high mortality and disability rates worldwide [1]. According to global cancer statistics, 19.3 million new cancer-related cases and 10 million cancer-related deaths were reported in 2020 [2]. Osteosarcoma is the most prevalent primary bone cancer in children aged 10 to 15 years and young adults [3,4]. The 5-year survival rate of patients with localized osteosarcoma is 65–75% [5]; however, the prognosis of patients with relapse and metastasis is poor, with a 5-year survival rate of 10–20% [6,7]. Currently, treatment for osteosarcoma mainly involves surgical removal, neoadjuvant chemotherapy, and radiation therapy [8]. However, the chemotherapeutic agents currently used cause severe side effects in the majority of patients. Moreover, resistance to chemotherapeutic agents is another challenge in the treatment of osteosarcoma. Hence, new drugs with both low toxicity and high efficacy are urgently required.

Natural products have been widely used as anticancer therapeutics. Sixty percent of drugs approved by the Food and Drug Administration from 1984 to 1994 were isolated from natural sources, especially plants [9,10]. Naringenin [(2*S*)-40 ,5,7-trihydroxyflavan-4-one] is present in various herbs and fruits, especially citrus plants [11]. Naringenin possesses antimicrobial, antioxidative, and anticancer properties [12–14]. Moreover, naringenin induces cytotoxicity in different types of cancer cells [15]. In 2019, Zhao et al. reported that naringenin (250 µM; 24 h) suppressed the migration of breast cancer cells by arresting the cell cycle at the G0/G1 phase [16]. Furthermore, Song et al. indicated that naringenin (200 µM; 24 h) caused colon cancer apoptosis through p38-dependent ATF3 activation [17]. Naringenin (500 µM; 24 h) enhanced TRAIL-induced apoptosis through the induction of DR5 expression in human A549 cells [18]. However, the effect of naringenin on osteosarcoma remains unclear.

Autophagy, which refers to the intracellular degradation of cytoplasmic materials caused by vacuoles or lysosomes in eukaryotic cells, eliminates and recycles damaged proteins to prolong the lifespan of cells [19]. It is a crucial homeostasis and cell survival mechanism that responds to environmental stresses such as starvation or pathogen infection [20]. Recent accumulating evidence indicates that autophagy also occurs under pathological conditions, such as in neurodegenerative disease or tumor development [21]. Specifically, autophagy is believed to play an important role in tumor development [22]. During the early stages of tumor formation, autophagy functions as a tumor suppressor, and autophagic activity is often impaired in cancer cells. Many anticancer drugs which lead to apoptosis can also induce autophagy-related cell death in cancer cell lines [23]. In osteosarcoma, autophagy is deregulated and functions as a protumoral or antitumoral process to suppress carcinogenesis and support the growth of established tumors [19].

This study investigated the effects of and molecular mechanisms underlying naringenininduced autophagy and apoptosis and the interaction between autophagy and apoptosis in osteosarcoma cells. The findings of this study can provide the proof-of-concept for evaluating naringenin as an antiosteosarcoma agent.

### **2. Materials and Methods**

### *2.1. Material*

Primary antibodies for Bak (GTX100063), Bax (GTX109683), Bcl2 (GTX100064), BclxL (GTX105661), GRP78 (GTX113340), GRP94 (GTX103203), PARP (GTX100573), calpain I (GTX102340), calpain II (GTX102499), cytochrome c (GTX108585), beclin1 (GTX134209), p53 (GTX70214), p62 (GTX102361), LC3B (GTX127375), ATG5 (GTX102360) and a voltagedependent anion channel (VDAC; GTX104745) were purchased from GeneTex International Corporation (Hsinchu City, Taiwan). Caspase-3 (19677-1-AP) and caspase-9 (10380-1-AP) were purchased from Proteintech Group Inc. (Rosemont, IL, USA). CDK1 (MN ABE1403) and cyclin B (MM05373) was purchased from Merck KGaA, Darmstadt, Germany. Antimouse and anti-rabbit IgG-conjugated horseradish peroxidase, as well as rabbit polyclonal antibodies specific for β-actin (cat. no. SI-A5441; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) were used in study. All other chemicals were obtained from Sigma-Aldrich (St. Louis, MO, USA).

### *2.2. Cell Line and Cell Culture*

The human osteosarcoma cell lines (U2OS and HOS) and osteoblast cell line (hFOB 1.19) were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). The hFOB1.19 osteoblast cells were cultured in a DMEM/F12 medium supplemented with 10% fetal bovine serum (FBS), 2.5 mM L-glutamine, 0.3 mg/mL G418 and 100 units/mL penicillin/streptomycin. Cells were incubated in an atmosphere of 5% CO<sup>2</sup> at 34 ◦C and subcultures were changed every 48 h.

The HOS cells were maintained in an Eagle's Minimum Essential medium supplemented with 10% FBS and 100 units/mL penicillin/streptomycin. The U2OS cells were cultured in a McCoy's 5A medium supplemented with 10% FBS and 100 units/mL penicillin/streptomycin. (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA). Cells were incubated in an atmosphere of 5% CO<sup>2</sup> at 37 ◦C and subcultures were changed every 48 h.

### *2.3. Cell Viability/Proliferation Assay*

Cells were seeded (6 <sup>×</sup> <sup>10</sup><sup>3</sup> ) in triplicate in a 96-well plate, treated with naringenin at various concentrations (100, 250, and 500 µM), and incubated at 37 ◦C for 24 h under 5% CO2. After incubation, the cell counting kit-8 (CCK-8) assay (10 µL; Sigma-Aldrich, St. Louis, MO, USA) solution was added into each well, and the plate was incubated under the same conditions for 2–5 h. A microplate reader was used to measure absorbance at 450 nm (Bio-Tek Instruments, Winooski, VT, USA).

### *2.4. Colony Formation Assay*

HOS and U2OS cells (5 <sup>×</sup> <sup>10</sup><sup>4</sup> ) were seeded in six-well plates and treated with naringenin at the indicated concentrations (100, 250, and 500 µM) in a medium without 10% FBS for 24 h. Untreated cells were used as the control. The culture medium containing 10% FBS was replaced after 24 h of treatment and changed every 2 days without additional naringenin treatment. After incubation for 7 days, visible colonies were washed with PBS, fixed with 4% paraformaldehyde for 15 min, and stained with 0.25% crystal violet for 15 min. The images of colonies were captured through scanning. Subsequently, the plates were washed three times with double-distilled water and then with 33 % (*v*/*v*) acetic acid, and this was followed by the measurement of absorbance at 550 nm. The colony formation assay was repeated three times in duplicate wells.

### *2.5. Cytosolic and Mitochondrial Protein Extraction*

Cytosolic and mitochondrial proteins were extracted from untreated controls and cells treated with naringenin at various concentrations (100, 250, and 500 µM) for 8 h by using the Mitochondria/Cytosol Fractionation Kit (Cat#K256-25; BioVision Inc., Milpitas, CA, USA) according to the manufacturer's protocol. Cells were collected, washed, and centrifuged for 10 min at 4 ◦C at 1000 rpm. Cells were then resuspended in the cytosol extraction buffer mix, incubated on ice for 10 min, and repeatedly passed through a 25-gauge needle. The homogenate mix was centrifuged for 10 min at 4 ◦C at 3000× *g* rpm. The supernatant was collected and centrifuged for 30 min at 4 ◦C at 15,000× *g* rpm. The supernatant was used as the cytosolic fraction. The pellet was resuspended in a mitochondrial extraction buffer mix and used as the mitochondrial fraction.

### *2.6. Western Blot Analysis*

HOS and U2OS cells were treated with naringenin at various concentrations (100, 250, and 500 µM) for 8 h. After treatment, total protein was harvested and lysed in RIPA lysis buffer containing protease inhibitors. Protein concentrations were determined using the bicinchoninic acid assay kit (Sigma-Aldrich, St. Louis, MO, USA). Proteins were separated

through 8–15% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes. The membranes were blocked with a TBST buffer containing 4% BSA for 1 h at room temperature and subsequently incubated with primary antibodies (at a dilution of 1:1000) at 4 ◦C overnight. The membranes were washed three times with the TBST buffer and then incubated with peroxidase-conjugated secondary antibodies (at a dilution of 1:10,000) for 1 h at room temperature. The blots were visualized using an enhanced chemiluminescence system (EMD Millipore, Billerica, MA, USA) with a UVP BioImaging System (Upland, CA, USA). Each experiment was repeated at least three times.

## *2.7. DAPI Staining*

HOS and U2OS cells were treated with naringenin at various concentrations (100, 250, and 500 µM) for 24 h. After treatment, cells were washed with PBS, fixed in a 3.7% formaldehyde solution for 15 min, permeabilized with 0.1% Triton X-100 for 5 min, and stained with DAPI (1 µg/mL) for 5 min. All samples were examined and photographed using a Nikon Eclipse Ti inverted fluorescence microscope (software version 5.02.01).

### *2.8. Terminal Deoxynucleotidyl Transferase-Mediated dUTP Nick-End Labeling Assay*

Apoptotic cells were quantified using the terminal deoxynucleotidyl transferasemediated dUTP nick-end labeling (TUNEL) assay, which examines breaks in DNA strands caused during cell apoptosis by using the BD APO-DIRECT kit (BD Biosciences, San Jose, CA, USA; cat. no. 556381). HOS and U2OS cells (2 <sup>×</sup> <sup>10</sup><sup>6</sup> ) were treated with naringenin at various concentrations (100, 250, and 500 µM) for 24 h. After treatment, cells were collected and centrifuged at 950× *g* for 10 min at 4◦C. Cells were fixed with 1% paraformaldehyde for 30 min on ice and washed with PBS twice. After the removal of the fixative, 0.5 mL of ethanol was added, and the mixture was incubated at −20 ◦C for 4 h. Subsequently, ethanol was removed through centrifugation and cellular DNA was obtained. The cellular DNA was stained with TUNEL solution (3 ng/mL TdT enzyme and 0.04 nmol FITC dUTP) at 37 ◦C for 1 h. After incubation with TUNEL solution, cells were washed with a rinse buffer (1 mL; BD APO-DIRECT kit; BD Biosciences; cat. no. 556381) and centrifuged at 1425× *g* for 10 min at 4 ◦C. The fluorescein-labeled DNA strand was detected and quantified using the BD Accuri C5 flow cytometer and BD Accuri C6 software (version 1.0.264.21, BD Biosciences).

### *2.9. Annexin V and Propidium Iodide Staining*

The Annexin V/propidium iodide (PI) double staining assay was performed to examine cell apoptosis by using the Annexin V/PI detection kit (cat. no. PF00005; Proteintech Group, Inc., Rosemont, IL, USA). HOS and U2OS cells were treated with naringenin at various concentrations (100, 250, and 500 µM) for 24 h. After treatment, cells were collected and washed with PBS twice and then resuspended in a staining buffer containing PI and Annexin V–FITC at room temperature for 30 min; cells were placed in the dark prior to flow cytometry. Cells were analyzed using the BD Accuri C5 flow cytometer and BD Accuri C6 software (version 1.0.264.21, BD Biosciences).

### *2.10. Cell Cycle Analysis Using PI Staining*

Apoptotic cells were quantified by examining the cell cycle. HOS and U2OS cells were treated with naringenin at various concentrations (100, 250, and 500 µM) for 24 h and collected through centrifugation (10 min at 950× *g*). Ice-cold ethanol was added to 0.5 mL of cell suspension and the mixture was then incubated at −20 ◦C for 4 h. Ethanol was removed through centrifugation (15 min at 1425× *g*) and cells were stained with a PI solution (0.1% Triton-X 100, 100 µg/mL of DNase-free RNase A, and 10 µg/mL of PI in PBS). After staining, cells were analyzed using the BD Accuri C5 flow cytometer and BD Accuri C6 software (version 1.0.264.21, BD Biosciences).
