Luteolin, MTT and dimethyl sulfoxide were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). RPMI-1640 medium, fetal bovine serum (FBS), penicillin and streptomycin were purchased from Gibco (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Annexin V-FITC apoptosis detection kit and anti-cytochrome c antibody (catalogue no. 556433) were purchased from BD Biosciences (San Jose, CA, USA). Primary antibodies, including anti-p-PI3K (Y607; catalogue no. YP0765), anti-p-mTOR (S2448; catalogue no. YP0176) and anti-β-actin (catalogue no. YT0099) antibodies, were from ImmunoWay Biotechnology Company (Plano, TX, USA), while anti-p-AKT (Ser473; catalogue no. 4051), anti-p-p38 (Thr180/Tyr182; catalogue no. 9216), anti-p-ERK1/2 (Thr202/Tyr204; catalogue no. 9106), anti-p-c-Jun N-terminal kinase (JNK) (Thr183/Tyr185; catalogue no. 4668), anti-B-cell lymphoma (Bcl)-2 (catalogue no. 15071), anti-Bcl-2 associated X protein (Bax) (catalogue no. 2772), anti-caspase-3 (catalogue no. 9668) and anti-caspase-9 (catalogue no. 9508) antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Caspase-3 (3G2, catalogue no. 9668) is mouse monoclonal antibody that detects endogenous levels of full length (35 kDa) and the large fragment (17/19 kDa) of caspase-3 resulting from cleavage at aspartic acid 175. Caspase-9 (C9, catalogue no. 9508) is mouse monoclonal antibody that detects endogenous levels of the pro form and cleaved fragments of caspase-9 (47,37 and 35 kDa). The horseradish peroxidase-conjugated secondary antibodies anti-rabbit immunoglobulin (Ig)G (catalogue no. sc-2357) and anti-mouse IgG (catalogue no. sc-516102) were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). U0126 (catalogue no. S1102) and LY294002 (catalogue no. S1105) were purchased from Selleck Chemicals (Houston, TX, USA). All other chemicals were purchased from Sangon Biology Engineering Technology Service, Ltd. (Shanghai, China) and were of analytical grade.

The human gastric cancer cell line BGC-823 was obtained from the Cell Center at Chinese Academy of Medical Sciences and Peking Union Medical College (Beijing, China). The cells were maintained in RPMI-1640 medium supplemented with 10% FBS, 100 U/ml penicillin and 100 µg/ml streptomycin at 37°C in a 5% CO₂ incubator. The cells were sub-cultured every 2 or 3 days and routinely checked visually under an inverted microscope (Leica Microsystems GmbH, Wetzlar, Germany) for potential contamination. No contamination was identified.

Cells (1.0×10⁶) were seeded in 10-cm dishes. When cells were in logarithmic growth phase, they were treated with luteolin at the 0, 20, 40 and 60 µM for 48 h. Subsequently, BGC-823 cells were washed twice with PBS (pH 7.4) and lysed in 100 µl radioimmunoprecipitation assay buffer (AR0102) that purchased from Boster Biotech (Wuhan, China). The lysed cells were removed from the culture dish by gentle scraping with a cell scraper (#3010; Corning Incorporated, USA) and transferred to a microcentrifuge tube. The samples were centrifuged at 13,000 rpm for 5 min at 4°C, and the supernatant was then transferred to a new tube. Total protein concentration was determined using the Pierce BCA Protein assay kit (Thermo Fisher Scientific, Inc.). Proteins were separated by 10% SDS-PAGE and electrotransferred to a polyvinylidene fluoride membrane (0.2 µm; Merck KGaA, Darmstadt, Germany) using a Trans-Blot SD Semi-Dry Transfer Cell (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Blocking was carried out for 2 h in TBST [Tris-buffered saline (TBS) containing 1‰ Tween-20, v/v] with 5% non-fat milk at room temperature. The primary antibodies (1:1,000) were incubated with the membrane overnight at 4°C. Following three washes in TBST, secondary antibodies (1:2,000) were added and incubated at room temperature for 2 h. The blots were washed with TBST three times, and detection was then performed using Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific, Inc.).

After BGC-823 cells were exposed to 0, 20, 40 and 60 µM luteolin for 48 h, total RNA was extracted using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. RNA concentration and purity were determined based on the measurement of absorbance at 260 and 280 nm. RNase-free DNase I (Takara Bio, Inc., Japan) was used to remove the DNA contamination. M-MLV Reverse Transcriptase (Fermentas, Thermo Fisher Scientific, Inc.; Pittsburgh, PA, USA) was used according to the manufacturer's protocol to treat 2 µg total RNA for synthesizing first-strand complementary DNA (cDNA). The cDNA was then subjected to qPCR for evaluation of the relative messenger RNA (mRNA) levels. Gene-specific amplification was performed using an StepOne™ (96 wells) Real-Time PCR system (Applied Biosystems^® ABI; Thermo Fisher Scientific, Inc.) with a 20 µl PCR reaction mixture containing 1 µl cDNA (synthesized as described above), 10 µl 2X Fast SYBR-Green Master Mix (Applied Biosystems^® ABI, USA), forward and reverse primers with a final concentration of 0.25 µM. The amplification conditions were 95°C for 15 min, followed by 40 cycles of 95°C for 15 sec, 56°C for 20 sec and 72°C for 30 sec. The relative expression levels of the target genes were normalized to the geometric mean of the internal control gene, GAPDH. Each gene was performed in a set of three replicates. No template control was included in all batches. The Cq (threshold cycles) values were used to calculate the mRNA levels by the formula 2^−∆∆Ct = 2^{−[∆Ct treatment−∆Ct control]} (31). The primers used in qPCR analysis were obtained from Sangon Biology Engineering Technology Service, Ltd. (Shanghai, China), and their sequences are reported in Table I.

The data were expressed as the mean ± standard deviation (n=3). All calculations were performed with SPSS version 16 (SPSS, Inc., Chicago, IL, USA). Statistical significance was analyzed by one-way analysis of variance. P<0.05 was considered to indicate a statistically significant difference.

Our group previously reported that luteolin inhibited BGC-823 cell proliferation and induced apoptosis in a dose-dependent manner (32). In the present study, to further examine whether luteolin-induced apoptosis resulted from activating caspase enzymes, the activities of initiator caspase (caspase-9) and effector caspase (caspase-3) (33,34) were measured by western blotting in BGC-823 cells. The results revealed that luteolin treatment increased the expression of cleaved caspase-9 and caspase-3 in a dose-dependent manner (Fig. 1). Next, the level of cytochrome c in the cytoplasm was detected (35–38), as this is a key step in the process of caspase activation during apoptosis. As shown in Fig. 1, luteolin induced an increase of cytoplasmatic cytochrome c in a dose-dependent manner. These results implied that luteolin-induced apoptosis in BGC-823 cells may occur through the intrinsic pathway.

The involvement of the intrinsic pathway in apoptosis is regulated by proteins of the Bcl-2 family, which comprises anti-apoptotic (e.g., Bcl-2) and pro-apoptotic (e.g., Bax) proteins (39). The ratio of Bax/Bcl-2 could determine whether the cell undergoes apoptosis (40,41). In the present study, luteolin treatment of BGC-823 cells resulted in decreased of Bcl-2 in a dose-dependent manner (Fig. 2A). Compared with that in control cells, the ratio of Bax/Bcl-2 significantly increased at the highest concentrations of luteolin in treated cells (Fig. 2B). These data further suggested that luteolin induced apoptosis via the intrinsic pathway in BGC-823 cells.

The key elements of the PI3K and MAPK signaling pathways were evaluated by western blotting in BGC-823 cells exposed to luteolin at 0, 20, 40 and 60 µM for 48 h. As shown in Fig. 3A, luteolin reduced the expression level of p-PI3K, p-AKT and p-mTOR in a dose-dependent manner. These results suggested that luteolin treatment suppressed the PI3K signaling pathway.

With regards to the MAPK signaling pathway, after BGC-823 cells were treated with luteolin at 0, 20, 40 and 60 µM for 48 h, the results of western blotting indicated that the levels of p-ERK1/2 were reduced markedly in a dose-dependent manner, while the levels of p-p38 and p-JNK exhibited no significant change (P>0.05). These results suggested that luteolin treatment suppressed the ERK1/2 signaling pathway, but not the JNK or p38 signaling pathways, in BGC-823 cells (Fig. 3B).

To confirm the roles of the ERK1/2 or PI3K signaling pathways in luteolin-induced apoptosis in BGC-823 cells, the ERK inhibitor U0126 (42) and the AKT inhibitor LY294002 (43) were used for treating the cells in the absence or presence of 60 µM luteolin. The effects were examined according to the ratio of Bax to Bcl-2. The results indicated that exposure of BGC-823 cells to U0126 or LY294002 alone did not significantly alter the Bax/Bcl-2 ratio compared with that of control cells (Fig. 3C and D; P>0.05). The Bax/Bcl-2 ratio in the combination U0126 plus luteolin group increased compared with that in the control group, while such ratio was lower than that observed upon luteolin treatment alone (P<0.05). The Bax/Bcl-2 ratio in the combination LY294002 plus luteolin group increased compared with that in the control group, being much higher than that observed following luteolin treatment alone (P<0.05). Taken together, these results indicated that the ERK and PI3K signaling pathways were involved in the apoptosis induced by luteolin in BGC-823 cells. Notably, the Bax/Bcl-2 ratio in the LY294002 plus luteolin group was markedly higher than that in the U0126 plus luteolin group (~3.7 vs. 1.6), respectively, indicating that the PI3K signaling pathway has much stronger effects on luteolin-induced apoptosis than the MAPK signaling pathway.

Dual-specificity phosphatases (DUSPs) are a heterogeneous group of protein phosphatases that have ability to dephosphorylate both tyrosine and serine/threonine residues (44) and serve a critical role in the inactivation of different isoforms of MAPK (45) DUSPs share common features, including a cluster of basic amino acids as part of the kinase interactive motif (KIM) on their amino terminus. The KIM confers substrate specificity and is the least homologous region demonstrating individual substrate preferences (46,47). Therefore, the transcription level of DUSP1-DUSP10 was examined. The mRNA levels of DUSP1, 2, 4 and 5 were upregulated in luteolin-treated BGC-823 cells compared with those in controls cells, while the mRNA levels of DUSP6, 7, 9 and 10 did not change during the treatment (Fig. 4). DUSP1, 2, 4 and 5 prefer to use the ERK as their substrates. This result was consistent with the decrease of p-ERK protein in the western blotting results of the present study, indicating that luteolin exhibits the potential to regulate the expression of specific DUSP genes, resulting in an attenuation of the MAPK signaling pathway.

Chalabi-Dchar et al (48) reported that blocking CXCL16 activity abrogated activation of the PI3K/AKT pathway, implying the CXCL16 axis may regulate the activity of PI3K pathway. Therefore, the expression of CXCL16 was monitored at an mRNA level. The results showed that the CXCL16 mRNA level was greatly downregulated in a dose manner after treatment with luteolin, suggesting that luteolin efficiently suppressed the mRNA level of CXCL16. Hence, the attenuation of PI3K pathway may be ascribed to the suppression of CXCL16 in BGC-823 cell lines following luteolin treatment.