Naringenin inhibits migration, invasion, induces apoptosis in human lung cancer cells and arrests tumour progression in vitro

Abstract Lung cancer is one of the major cause for high‐death rate all over the world, due to increased metastasize and difficulties in diagnosis. Naringenin is naturally occurring flavonoid found in various fruits including tomatoes, citrus fruit and figs. Naringenin is known to have several therapeutic effects including anti‐atherogenic, antimicrobial, anti‐inflammatory, hepatoprotective, anticancer and anti‐mutagenic. The present study was aimed to analyse the naringenin induced anti‐proliferative and apoptosis effects in human lung cancer cells. Cells were treated with various concentrations of naringenin (10, 100 & 200 µmol/L) for 48 hours. Cisplatin (20 µg/mL) was used as positive control. Cell viability, apoptosis, migration and mRNA, and protein expression of caspase‐3, matrixmetallo proteinases‐2 (MMP‐2) and MMP‐9 were determined. The cell viability was 93.7 ± 7.5, 51.4 ± 4.4 and 32.1 ± 2.1 at 10, 100 and 200 µmol/L of naringenin respectively. Naringenin significantly increased apoptotic cells. The 100 and 200 µmol/L of naringenin significantly suppressed the larger wounds of cultured human cancer cells compared with the untreated lung cancer cells. Naringenin increased d the expression of caspase‐3 and reduced the expression of MMP‐2 and MMP‐9. Taking all these data together, it is suggested that the naringenin was effective against human lung cancer proliferation, migration and metastasis.

MMP-9. Taking all these data together, it is suggested that the naringenin was effective against human lung cancer proliferation, migration and metastasis.

K E Y W O R D S
apoptosis, lung cancer, metastasis, naringenin, proliferation Naringenin is naturally occurring flavonoid found in various fruits including tomatoes, citrus fruit and figs. [9][10][11] Naringenin is known to have several therapeutic effects including anti-atherogenic, antimicrobial, anti-inflammatory, hepatoprotective, anticancer and anti-mutagenic. 12, 13 Chin et al 14  pression of matrixmetallo proteinases-2 (MMP-2) and Akt. Lung cancer is one of the major cause for high-death rate all over the world, 22 due to increased metastasize and difficulties in diagnosis. 23 Hence, the new therapeutic drug is required to inhibit metastasis and to improve clinical symptoms against lung cancer. Thus, the present study was aimed to analyse the naringenin induced anti-proliferative and apoptosis effects in human lung cancer cells.

| Cell culture
Human A549 lung cancer cells were purchased from ATCC (Manassas, VA, USA). Later, the cells were cultured in RPMI medium, supplemented with FBS (10%) and 1% of antibiotics (penicillin/ streptomycin), and maintained in CO 2 incubator at 37°C.

| Sulforhodamine B (SRB) assay
SRB assay was used to analyse the cytotoxic effects of naringenin on human A549 lung cancer cells. 24 Briefly, A549 lung cancer cells were culture in 96-well plates at a density of 1.5 ×

| Apoptosis
Apoptosis was evaluated by the acridine orange/ethidium bromide staining (AO/EB). Briefly, A549 lung cancer cells were culture in 6-well plates at a density of 1.5 ×

| Wound healing assay
Wound healing assay on cancer cell was carried out according to previously reported method. 26 A549 lung cancer cells were cul-

| Migration assay
Migration assay was determined according to the previously reported method. 27 Briefly, A549 lung cancer cells were culture in

| RT-PCR
Total RNA was isolated from the A549 lung cancer cell homogenate by using TRIzol reagent (10296028, Thermo Fisher Scientific) according to manufacturer's instructions. Reverse-transcription reaction was carried out using cDNA synthesis kit. The specific primers used for the amplification of MMP-2, MMP-9 and caspase-3 was given in Table S1. Quantitative RT-PCR was carried out by using iQ SYBR Green Supermix (Bio-Rad, Shanghai). Relative ratio of expression was determined according to change-in-threshold (−ΔΔCT) method. 28

| Western blot analysis
A549 lung cancer cell homogenized and treated with lysis buffer at cold temperature for 30 minutes. The proteins in the extract were separated, and cell debris was removed, and supernatant was taken for the protein estimation by using standard method.
Proteins of extract were separated membrane, and membranes were incubated with non-fat (5%) to inhibit non-specific sites.

| Immunofluorescence
Immunofluorescence was carried out according to as previously reported method. 30 Briefly, A549 lung cancer cells were culture in 6-well plates at a density of 1.5 ×

| Statistical analysis
Data were given as the means ± standard error of the mean. The differences between the control and naringenin-treated groups were analysed using Student's t test and analysis of variance. P < .05 was taken statistically significant.

| RE SULTS
We analysed the naringenin induced anti-proliferative and apoptosis effects in human lung cancer cells. SRB assay was used to deter- AO/EB staining method is commonly used to analyse the effect of naringenin on apoptosis of human lung cancer cells. The effect of naringenin on cancer cell apoptosis was given in Figure 2 and Table S2. Human lung cancer cell treated with 10 µmol/L of naringenin increased the early apoptotic (1.6%) and apoptotic cells (6.7%) than control (Table S2,  In immunohistochemical analysis, naringenin treatment significantly increased the protein expression of caspase-3 19%, 74% and 101% at 10, 100 and 200 µmol/L respectively, whereas cisplatin increased 100% (Figures 6 and 7, P < .05) in human lung cancer cells.

| D ISCUSS I ON
Lung cancer is one of the major cause for high-death rate all over the world, 22 due to increased metastasize and difficulties in diagnosis. 23 Sangodkar et al 31 have reported that the poor prognosis of lung cancer and survival is due to drug resistance and metastasis. However, researchers have reported that the natural products such as naringenin were effective against lung cancer cell proliferation. 32  In this study, we analysed the anti-proliferative effect of naringenin against lung cancer cells. Naringenin significantly inhibited lung cancer cell viability, which was comparable to positive control (cisplatin), and observed effect was in dose-dependent manner. Rani et al 33 have reported that naringenin's anticancer effect is primarily attributed to its strong anti-oxidant capacity. Liu et al 34 have reported that the AO/EB staining is most reliable method to detect apoptosis. In this study, the human lung cancer cell treated with naringenin significantly increased apoptosis. Apoptosis is key cellular mechanism to kill wanted, damaged and cancer cells. 35  MMPs are secreted from the tumour cells during metastasis, which leads to extracellular matrix degradation and subsequent invasion of cancer cells to lymph vessels, and blood. 39 Ahmed 40 have reported that the association between oxidative stress and human lung cancer cell survival, proliferation, invasion and metastasis. Scarano et al 41

| CON CLUS ION
Taking all these data together, it is suggested that the naringenin was effective against human lung cancer proliferation, migration and metastasis in vitro. Therefore, we suggest that this naringenin is worthy of further investigation to assess its active mechanism and their potential as anticancer drugs.

CO N FLI C T O F I NTE R E S T
Authors declare that they have no conflict of interest.

AUTH O R CO NTR I B UTI O N S
Xingyuan Shi: Conceptualization (equal); Data curation (equal).

DATA AVA I L A B I L I T Y S TAT E M E N T
Corresponding author provide data upon valid request.