YD-38 human GSCC cells were purchased from the Cell Bank of Chinese Academy of Sciences (Shanghai, China), and were cultured according to the manufacturer's protocol. Cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.). The HGF-1 cell line (CRL-2014) was purchased from American Type Culture Collection (ATCC) and was also cultured in DMEM supplemented with 10% FBS. The culture media were replaced every 2 days, unless otherwise specified. Cells were incubated at 37°C in a humidified environment containing 5% CO₂.

The P21 luciferase reporter plasmid was constructed, according to the following procedures. The pGL3-basic vector (Promega Corporation) was digested using KpnI and HindIII restriction enzymes. Primers (forward, 5′-GACTCTGTGATCAATTTCTT-3′ and reverse, 5′-TTGTATCTCTGCAGGCG-3′) were used to clone the P21 promoter from THLE-2 normal liver cells (ATCC), to which the KpnI and HindIII restriction enzymes were added (21). After PCR, double digestion and DNA gel (1% agarose) extraction (22), the fragments were ligated together. Sequencing was used to ensure that the P21 luciferase reporter plasmid was successfully constructed. siRNAs were designed and synthesized by Shanghai GenePharma Co., Ltd. and were transfected into cells at a concentration of 10 µM. The sequences were as follows: siNC, sense 5′-ACUUACGAGUGACAGUAGA-3′, antisense 5′-UCUACUGUCACUCGUAAGU-3′; and siP21, sense 5′-CCAACUCAUUCUCCAAGUA-3′ and antisense 5′-UACUUGGAGAAUGAGUUGG-3′. Briefly, YD-38 cells were seeded into 96-well plates at a density of 2,000 cells/well and transfection was performed using Lipofectamine^® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocols. A total of 48 h post-transfection, subsequent experiments were conducted.

Luciferase reporter assays were performed using the Dual-Luciferase^® Reporter assay system (cat. no. E1910; Promega Corporation) according to the manufacturer's protocol. Briefly, YD-38 cells seeded into a 24-well plate at a density of 30,000 cells/well were transfected by Lipofectamine^® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.) with P21 luciferase plasmid (1 µg/well) and Renilla vector (100 ng/well) and were treated with various natural products from the compound library at the Chinese Academy of Sciences (working concentration, 10 mg/ml for each compound) for 24 h at 37°C. Cells were then lysed with lysis buffer (Beyotime Institute of Biotechnology) for 20 min at room temperature with agitation (150 rpm). Subsequently, the lysate was incubated with luciferase assay reagent II and the absorbance of all wavelengths was immediately analyzed. Finally, the Stop & Glo reagent was added to the cells and absorbance was measured again (Tecan). Luciferase activities were determined by obtaining the ratio of the two readings; luciferase activity was normalized to Renilla luciferase activity.

YD-38 cells were seeded into 96-well plates (3,000 cells/well) and allowed to grow overnight. Cells transfected with siP21 (10 µM) were subsequently treated with lapiferin (10 µg/ml), followed by incubation in DMEM for a further 72 h. Cell proliferation was determined for 2 consecutive days using the MTT assay. Briefly, 2 mg/ml MTT solution was added to each well and the cells were incubated for 4 h at 37°C. Subsequently, media were removed and 200 µl dimethyl sulfoxide was added. The plate was agitated for 5 min and the optical density was subsequently determined at 490 nm using a spectrophotometer.

Total RNA was extracted from cultured GSCC cells using the RNA kit (Qiagen, Inc.) according to the manufacturer's protocol. Total RNA was reverse transcribed into cDNA using QuantiTect Reverse Transcription kit (Takara Bio, Inc.), according to the manufacturer's protocol. Primer sequences were as follow: p21, forward 5′-GAAAAGGAGAACACGGGATG-3′, reverse 5′-AAAGTCACTAAGAATCATTTATTG-3′; and β-actin, forward 5′-CGGAGTCAACGGATTTGGTC-3′ and reverse 5′-AGCCTTCTCCATGGTCGTGA-3′. RT-qPCR was performed using a TaqMan miRNA RT-qPCR assay (Takara Bio, Inc.) equipped with ABI-Prism 7300 system (Applied Biosystems; Thermo Fisher Scientific, Inc.). RT-qPCR thermocycling conditions were performed as follows: Denaturation at 95°C for 30 sec, followed by 45 cycles at 95°C for 3 sec and 60°C for 30 sec. Expression was quantified using the 2^−ΔΔCq method (23).

The Annexin V/propidium iodide (PI) assay was performed according to the manufacturer's protocol (Invitrogen; Thermo Fisher Scientific, Inc.). Briefly, YD-38 cells (1×10⁵/well) were plated into 6-well plates and treated with lapiferin (10 µg/ml) in the presence or absence of siP21 for 48 h. Subsequently, cells were washed with pre-chilled PBS, trypsinized and resuspended in 100 µl binding buffer containing 2.5 µl FITC-conjugated Annexin V and 1 µl PI (100 µg/ml). Cells were then incubated at room temperature for 15 min in the dark. Finally, ≥10,000 cells were collected and analyzed by flow cytometry (FACSCanto II; BD Biosciences) with FlowJo VX software (FlowJo, LLC). Cell apoptotic rates were assessed as follows: (Apoptotic cells in the control group-apoptotic cells in the lapiferin group)/apoptotic cells in the control group × 100.

Total proteins were extracted from cultured YD-38 cells. Briefly, cells were grown until they reached 95% confluence; after washing with PBS, cells were lysed using RIPA lysis buffer (Beyotime Institute of Biotechnology) to obtain the total protein lysate. Equal amounts of protein (30 µg/lane), the concentration of which was determined by the BCA method (Thermo Fisher Scientific, Inc.), were then subjected to 12 or 15% SDS-PAGE. Subsequently, the proteins were transferred to a PVDF membrane (0.22 µm; EMD Millipore,) and blocked with 5% milk dissolved in TBS-Tween (0.1%) for 1 h at room temperature. GAPDH was detected as a loading control. Immunoreactivity was determined using enhanced chemiluminescence autoradiography (Thermo Fisher Scientific, Inc.). The membrane was incubated with primary antibodies overnight at 4°C and with secondary antibodies at room temperature for 1 h. The following antibodies were used for western blotting: P21 (cat. no. ab109520; 1:1,000; Abcam), proliferating cell nuclear antigen (PCNA; cat. no. 10205-2-AP; 1:1,000; ProteinTech Group, Inc.), Ki67 (cat. no. ab15580; 1:1,000; Abcam), cell division cycle 25C (Cdc 25C; cat. no. ab32444; 1:1,000; Abcam), cyclin B1 (cat. no. ab72; 1:1,000; Abcam), cleaved (cl)-caspase-3 (cat. no. 9664; 1:1,000; Cell Signaling Technology, Inc.), cl-caspase-9 (cat. no. 20750; 1:1,000; Cell Signaling Technology, Inc.), Bax (cat. no. ab32503; 1:1,000; Abcam), cytochrome c (cyto. C; cat. no. ab13575; 1:1,000; Abcam), cl-poly(ADP-ribose) polymerase (PARP; cat. no. ab32064; 1:1,000; Abcam), GAPDH (cat. no. sc-47724; 1:2,000; Santa Cruz Biotechnology, Inc.), and horseradish peroxidase-conjugated secondary antibodies (cat. nos. sc-2004 and sc-2005; 1:5,000; Santa Cruz Biotechnology, Inc.). Each experiment was repeated at least three times.

Data are expressed as the mean ± standard deviation. One-way analysis of variance was used for comparisons among multiple groups (≥3 groups), followed by a least significant difference post hoc test. P<0.05 was considered to indicate a statistically significant difference. All experiments were repeated at least three times, unless otherwise stated.

To identify natural products that target P21, natural compounds from the Chinese Academy of Sciences compound library, which consists of 480 natural products, were purchased. YD-38 cells were transfected with P21 luciferase plasmid and Renilla control plasmid for 24 h in 96-well plates, after which the natural products were added to each well and mixed gently. As shown in Fig. 1, ~30% of the natural products inhibited the luciferase activities of P21 (green) and 53% of the natural products promoted the luciferase activities of P21in GSCC cells (red); the remaining compounds had no effect on P21 transcriptional activities. Of all the effective natural compounds, lapiferin increased P21 activity to the highest level compared with in untreated cells (6.32-fold). Therefore, this natural product was selected for subsequent analysis.

The present study examined the detailed effects of lapiferin on human GSCC cell proliferation. As shown in Fig. 2A, when YD-38 cells were treated with lapiferin, the proliferative rates remained stable in response to a low dose of lapiferin (2.5 µg/ml), but gradually decreased as the concentration of lapiferin increased (5, 10 and 20 µg/ml). In addition, cell proliferation was determined at various time points. Similarly, cell proliferation was stable in response to lapiferin treatment for 12 h; however, the cell proliferative rates were decreased to 75% when YD-38 cells were stimulated with lapiferin for 24 h; the suppressive effects were further enhanced as the treatment time was extended (Fig. 2B). These findings suggested that lapiferin decreased cell proliferation in a dose- and time-dependent manner in human GSCC cells.

Increased cell proliferation and decreased cell apoptosis are the two main manifestations of human malignancies. Therefore, the role of lapiferin in human GSCC cell apoptosis was determined. The results revealed that when cells were treated with lapiferin at a concentration of 5 µg/ml, the cell apoptotic rates were significantly increased to 2.2-fold compared with in the control YD-38 group (Fig. 3A). Furthermore, cell apoptosis was assessed when cells were treated with lapiferin for various durations. As shown in Fig. 3B, the cell apoptotic rates remained stable at 12 h and were markedly increased when YD-38 cells were treated with lapiferin for increased durations. Cell apoptotic rates peaked (3.5-fold) when cells were treated with lapiferin for 48 h. These results suggested that lapiferin increased cell apoptosis in a dose- and time-dependent manner in GSCC.

As shown in Fig. 1, lapiferin increased the luciferase activity of P21. The present study subsequently examined the effects of lapiferin by western blotting. GAPDH was included as an internal control. As shown in Fig. 4, when YD-38 cells were treated with lapiferin for 24 h, the protein expression levels of P21 were markedly increased, which was consistent with the findings presented in Fig. 1. However, PCNA and Ki67, two cell proliferation markers, and Cdc 25C and Cyclin B1, key cell cycle regulators, were downregulated by lapiferin; these findings are concordant with those presented in Fig. 2. These data further suggested that treatment of cells with lapiferin decreased cell proliferation.

The expression levels of cell apoptosis-related proteins were also examined by western blot analysis. As shown in Fig. 5, when the human GSCC cell line YD-38 was stimulated with lapiferin for 24 h at a concentration of 10 µg/ml, the protein expression levels of cl-caspase-3, cl-caspase-9, Bax, cyto. C and cl-PARP were markedly increased; expression of the internal control GAPDH remained stable. These data indicated that lapiferin increased the expression of cell apoptosis-associated proteins in human GSCC.

Furthermore, the detailed mechanism underlying the role of lapiferin in human GSCC was explored. Firstly, the expression of P21 was knocked down in YD-38 cells using a specific siRNA against P21. As shown in Fig. 6A, the basal expression levels of P21 were low and treatment with lapiferin markedly increased P21; however, when cells were transfected with siP21, the mRNA expression levels of P21 returned to basal levels. Accordingly, cell proliferation was decreased (Fig. 6B) and cell apoptosis was increased in response to treatment with lapiferin for 24 h (Fig. 6C). Notably, cell proliferation and apoptosis returned to a level comparable to the control when cells were transfected with siP21 (Fig. 6B and C). Taken together, these results suggested that lapiferin decreased cell proliferation and increased cell apoptosis through regulating P21 in human GSCC.