Roswell Park Memorial Institute (RPMI)-1640 medium was purchased from Hyclone (Logan, UT, USA). Fetal bovine serum (FBS) and antibiotics (streptomycin/penicillin) were obtained from Gibco (BRL Life Technologies, Grand Island, NY, USA). 3-(4,5-dimethyl-thiazol-2- yl)-2,5-diphenyltetrazolium bromide (MTT) and Naringin (Fig. 1A) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Materials and chemicals used for electrophoresis were obtained from Bio-Rad (Hercules, CA, USA). Primary antibodies ERK1/2, p-ERK1/2 (Thr²⁰²/Tyr²⁰⁴), JNK, p-JNK (Thr¹⁸³/tyr¹⁸⁵), p38, p-p38 (Thr¹⁸⁰/Try¹⁸²), p-PI3K (Tyr⁴⁵⁸/Tyr¹⁹⁹), p-Akt (Ser⁴⁷³), mTOR, p-mTOR (Ser²⁴⁴⁸), LC3B, Beclin 1, Bcl-xL and PI3K inhibitor LY294002 were purchased from Cell Signaling (Beverly, MA, USA). Akt (H-136) and Bax (P-19) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). PI3K p110δ antibody was obtained from Enzo Life Sciences. Anti-p21^WAF1/CIP1 and β-actin were purchased from Millipore (Billerica, MA, USA). Anti-rabbit IgG horseradish conjugate secondary antibody was purchased from Enzo Life Sciences. All the chemicals used were of the highest grade commercially available.

AGS cancer cell line, which is a gastric adenocarcinoma, was obtained from the Korean Cell Line Bank (Seoul, Korea). AGS cells were maintained in RPMI-1640 supplemented with 10% heat inactivated FBS and 1% penicillin/streptomycin at 37°C in a 5% CO₂ incubator. Cells were treated with vehicle alone (DMSO) or a series of concentrations of Naringin dissolved in 1% DMSO.

Cell proliferation of AGS cancer cells was assessed using MTT. Cells were seeded at density of 2.5×10⁴ cells per well in a 24-well plate, incubated overnight at 37°C in a 5% CO₂ incubator and treated with various concentrations of Naringin (1, 2 and 3 mM) or vehicle alone (DMSO) for 24 and 48 h. After treatment, MTT solution (5 mg_/ml in 1X PBS) was added followed by incubation for 3 h at 37°C in the dark. The formazan crystals formed were solubilized by incubating cells with 500 μl of DMSO. Cell absorbance was read by enzyme-linked immunosorbent assay (ELISA) plate reader (BioTek Instruments Co., Korea) at 540 nm. Cell proliferation was quantified as a percentage compared to the control group (untreated cells), which was set at 100%.

DNA was isolated with little modification following DNA extraction protocol (32). Briefly, untreated and Naringin-treated cells incubated for 24 h were harvested and were lysed with cell lysis buffer for 30 sec at room temperature (RT). The supernatant was collected after centrifugation at 3,000 rpm for 5 min followed by incubation at 56°C for 2 h after adding 10% SDS solution and RNase A. Proteinase K 25 mg/ml was added and incubated overnight till complete lysis at 37°C. After adding saturated NaCl and absolute ethanol to the samples, the mixture was incubated at −80°C for precipitation. Centrifuging for 20 min at 12,000 rpm followed by washing the white pellet with 80% ice cold ethanol and air-dried at RT. The obtained pellets were dissolved in 1X TE buffer. The total DNA solutions were then subjected to 1.5% agarose gel electrophoresis at 100 V for 45 min at room temperature. Tris acetate EDTA was used as the electrophoresis running buffer and DNA bands were visualized by UV light and documented by photography.

For the transmission electron microscopy analysis (TEM) the cells were seeded in a 100-mm dish and incubated with vehicle or 2 mM Naringin for 24 h. The cells were harvested and fixed in 4% formaldehyde and 1% glutaraldehyde phosphate buffer (1:1) for 48 h at 4°C. The fixative was pipetted and replaced with 8% sucrose in 1X PBS, followed by post-fixation with 1% osmium tetraoxide for 1 h at 4°C. The cells were then washed with 1X PBS three times for 10 min. After dehydration in 50–100% ethanol, the cells were embedded in Poly/Bed 812 resin (Pelco, Redding, CA, USA). The cells were polymerized overnight at 60°C. Ultrathin sections were stained with lead citrate and examined on Tecnai 12, FEI transmission electron microscope.

Briefly, AGS cells treated with 1 mM and 2 mM Naringin or vehicle (as control) for 24 h were lysed overnight with lysis buffer (RIPA) containing phosphatase inhibitor cocktail along with protease inhibitor and EDTA (Thermo Scientific, MA, USA). The extracted proteins were then centrifuged at 14000 rpm for 30 min at 4°C to remove debris. The proteins were resolved using 8–15% SDS-PAGE and subsequently transferred to polyvinylidene difluoride (PVDF) membrane (Immunobilon-P, 0.45 mm; Millipore) using the TE 77 Semi-Dry Transfer Unit (GE Healthcare Life Sciences, Buckinghamshire, UK). The membranes were blocked with 5% non-fat milk in Tris-buffered saline containing 1% Tween-20 (TBS-T, pH 7.4) or 1X phospho-blocking solution (TransLab, Biosciences in Korea) at RT for 1 h. Blots were probed with a 1:500 or 1:1,000 dilutions of the respective primary antibodies at 4°C overnight. After washing five times with TBS-T, the membranes were incubated with a 1:1,000 diluted enzyme-linked secondary antibodies at RT for 3 h. The immune blots were visualized using an enhanced chemiluminescence (ECL) kit and Western Blotting Detection Reagents (GE Healthcare Life Sciences). Each protein band was quantified using ImageJ software (http://rsb.info.nih.gov) followed by densitometry reading, undertaken after normalization by β-actin expression.

To explore the effect of PI3K as upstream targets of PI3K/Akt/mTOR signaling pathway on Naringin induced autophagy in AGS cell growth inhibition, 10 μmol/ LY294002 (a PI3K specific inhibitor) were pre-treated for 2 h prior to the addition of 2 mM Naringin followed by 24-h incubation. The protein expression was analyzed by immunoblotting as described above against the p-PI3K and LC3B antibodies.

The obtained results were expressed as the mean ± standard deviation (SD) of a minimum three replicates in independent experiments. The data were analyzed by unpaired, two tailed Student's t-test using SPSS version 10.0 for Windows (SPSS, Chicago, IL, USA). The p-value of <0.05 and <0.01 was considered statistically significant.

In order to assess the potential anti-proliferative activity of Naringin on AGS cancer cells, MTT assay was conducted. The anti-proliferative effect of Naringin on AGS cancer cells were examined dose- and time-dependently. It has been observed that treatment with different doses of Naringin (0–3 mM) or vehicle alone at two time points (24 and 48 h) exhibited significantly decreased cell proliferation (Fig. 1B). Besides, >50% cell growth inhibition was observed at 3 mM dose within 24-h, but the opted effective concentration (EC) of Naringin was 2 mM based on the morphological observation of treated AGS cells with visible increased vacuolization. Moreover, the cell growth was efficiently attenuated dose- and time-dependently followed by 59.8, 54.8 and 49.5% in 24 h and 57.4, 52.5 and 41.02% in 48-h durations respectively at subsequent doses of Naringin.

DNA fragmentation has been considered as hallmark for apoptotic cell death which proceeds before the onset of morphological changes during apoptosis. On the contrary, Naringin attenuated AGS cell growth, investigation was conducted to confirm the induction of apoptotic cell death by 1.5% agarose gel electrophoresis analysis. DNA ladder assay presented no apparent DNA inter-nucleosomal fragmentation in the 2 mM Naringin-treated AGS cells for 24 h compared with control (Fig. 2A), suggesting the cell death occurrence was not due to apoptosis. Further confirmation was done to observe the potential role of apoptosis related proteins in Naringin-treated cells. Western blot analysis for Bax and Bcl-xL (Bax/Bcl-xL) relative ratio revealed a gradual decreasing trend in Naringin-treated AGS cells (Fig. 2B). Thus, collectively these results supported non-apoptotic cell death in the Naringin-treated AGS cell line.

In human cancer cells, activated Akt and mTOR stimulates cell growth through activation of PI3K. The present experimental results determined that phosphorylation of PI3K and its activated downstream targets p-Akt and p-mTOR are significantly decreased at 2 mM in Naringin-treated AGS cells, observed by immunoblot analysis (Fig. 3A). To further validate the effect of Naringin on cell growth inhibition via PI3K pathway, pre-treatment of AGS cells with 10 μM of LY294002 as described earlier was done by western blot analysis (Fig. 3B). It was observed that pre-treatment with LY294002 inhibited the expression of PI3K in Naringin-treated cells. The above data represent the anti-proliferative role of Naringin in AGS cancer cells.

Electron microscopic investigation is still the most reliable method for monitoring autophagic morphology (33). The TEM observation reports revealed formation of double-membrane vesicles containing subcellular materials, representing formation of phagophore in 2 mM Naringin-treated cells when compared with the non-treated AGS cells (Fig. 4) showing the vesicle formation in 2 mM Naringin treated cells with damaged organelles, such as swollen mitochondria/lysosomes surrounded by double-membrane vacuoles, which further formed autophagosomes.

To further elucidate the molecular mechanisms that underlie Naringin-induced autophagosome, examination was done to assess the expression of vacuolar protein Beclin1 and microtubule-associated protein light chain 3 (LC3) in AGS cancer cells. The observed data represented a gradual increase of Beclin 1 protein, including an increased conversion of cytosolic LC3-I protein to autophagic isoform LC3-II (LC3 II/LC3 I ratio) that was significant at 2 mM Naringin-treated AGS cells compared with control (Fig. 5A). In addition to confirming the effect of Naringin on LC3B conversion, examination was done with 10 μM of LY294002 pre-treatment. Western blot analysis result (Fig. 5B) revealed that the conversion of LC3B was inhibited in the presence of LY294002. Taken together, these results indicated that Naringin induces autophagy in AGS cancer cells.

MAPK signaling pathways play an important role in cell growth inhibition and upregulation of autophagic protein followed by autophagy-mediated cell death. To further investigate the role of MAPK family proteins in Naringin-treated AGS cells inducing autophagy, western blot analysis was done. As shown (Fig. 6), Naringin induced significant activation of p-ERK1/2, p-p38 at 2 mM and p-JNK at 1 mM concentration in AGS cells during 24-h incubation. Taken together, these results demonstrated that MAPK signaling pathways are involved in Naringin-induced autophagic cell growth inhibition in AGS cells.

A marked overexpression of p21^WAF1/CIP1 in Naringin-treated cancer cells inducing apoptotic cell death in breast cancer and cell cycle arrest in bladder cell carcinoma have been reported (34,35). To further assess the cell anti-proliferative mechanism by p21^WAF1/CIP1 protein, expression study was determined by immunoblot analysis. The result demonstrated a significant increase of p21^WAF1/CIP1 expression at 2 mM in Naringin-treated AGS cells (Fig. 7). The observed data could be correlated with the induction of autophagy depicting the role of p21 in Naringin-inducing cell death in AGS cancer cells.