(+)-Terrein (Fig. 1) was isolated from A. terreus PF-26, as described previously (18). The isolated (+)-terrein was dissolved in phosphate-buffered saline (PBS, pH 7.2) for subsequent experiments. The human A549 lung adenocarcinoma epithelial cell line was provided by Dr Wei Ma (Shanghai Jiao Tong University, China), and the Bel-7402 human hepatoma cell line was obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The mentioned cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum and 100 U/ml of penicillin and streptomycin. Unless otherwise mentioned, reagents for cell cultures were purchased from Gibco/Invitrogen (New York, Grand Island, USA) and biochemical reagents were obtained from Sigma (New York, NY, USA) or Ameresco (Solon, OH, USA). The cells were grown in a 5% CO₂ atmosphere at 37°C.

An MTT assay [3-(4,5-dime-thylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] was performed with ~4×10³ cells/well in 96-well plates. The plates containing the cells were incubated at 37°C for 24 h, and the cells were treated with (+)-terrein at 37°C for 48 h. The protocol was performed using an MTT cell proliferation and cytotoxicity detection kit (KeyGen, Nanjing, China) according to the manufacturer’s instructions. Briefly, DMEM was supplemented with 50 μl of MTT reagent to each well and incubated at 37°C for 4 h. Thereafter, the MTT solution was removed. Following the addition of 150 μl of dimethyl sulfoxide (DMSO), the plates were incubated at 37°C for 15 min to dissolve the formazan crystals. Absorbance of DMSO extracts was detected at 550 nm by using an Enspire 2300 microplate reader (PerkinElmer, Foster City, CA, USA). A total of ~3×10⁵ cells/well were inoculated in 6-well plates at 37°C for 24 h and treated with (+)-terrein at 37°C for 48 h. The PBS-treated cells served as controls. These cells were used to detect cell morphology, cell cycle, apoptosis, and RNA extraction.

Cells (3×10⁵) were cultured in 6-well plates at 37°C for 24 h. The Bel-7402 and A549 cells were then treated with 10 μM and 10 mM (+)-terrein at 37°C for 48 h, respectively, and the PBS-treated cells served as controls. The treated cells were used to observe cell morphology and apoptosis. Light microscopy images of the cells were captured using a Nikon Eclipse Ti-inverted microscope and a Nikon digital sight s-Qi1Mc camera (both from Yokohama, Japan). For each surface, three non-overlapping images were selected.

The cells were rinsed once in chilled PBS, digested with 0.25% trypsin (Gibco), and then resuspended in DMEM and 10% serum. The suspended cells were centrifuged at 2,000 × g at 4°C for 5 min and washed once in cold PBS. The cells were stained with Alexa Fluor^® 488 Annexin V and PI by using an Alexa Fluor^® 488 Annexin V/Dead cell apoptosis kit (Invitrogen, New York, USA) according to the manufacturer’s instructions. The stained cells were analyzed using flow cytometry (FACSAria-II, BD Biosciences, San Jose, CA, USA).

The Bel-7402 cells treated with 10 μM (+)-terrein were trypsinized and washed with a PBS buffer. Fifty microliters of single cell suspension was examined using a cell cycle detection kit (KeyGen). The stained cells were analyzed using flow cytometry (FACSAria II).

The Bel-7402 cells treated with (+)-terrein or PBS were trypsinized and washed with a PBS buffer. The cells were collected using centrifugation at 2,000 × g for 5 min. The cell pellet was then resuspended in RL buffer and centrifuged at 13,000 × g for 5 min. Total RNA was extracted according to the manufacturer’s instructions (CWBio, Beijing, China). The purity and concentration of RNA was confirmed by the relative absorbance ratio at 260/280 nm and 260 nm, respectively, by using NanoDrop 2000 (Thermo Fisher Scientific, Waltham, MA, USA).

Reverse transcription was performed according to the manufacturer’s instructions (Thermo Fisher Scientific). Total RNA (1 μg) was mixed with 4 μl of a 5X reaction buffer, 1 μl of oligo (dT)18 primer, 1 μl of RiboLock RNase inhibitor, 2 μl of a 10 mM dNTP mix, 1 μl of RevertAid reverse transcriptase (100 U/μl), and then, ddH₂O was added to increase the volume to 20 μl. Reverse transcription was performed at 37°C for 60 min and then at 70°C for 5 min. The resulting cDNA was stored at −70°C until use.

Table I shows that 73 potential genes involved in the cell cycle were used as the target mRNAs. GAPDH, b2-MG, β-actin, RPL27, HPRT1, and OAZ1 were used as housekeeping genes for the control cells. The primers were designed (CT Bioscience Co., Changzhou, China) to cover all of the transcripts of each gene. Table I shows the RefSeq accession IDs. All of the primers had a similar melting temperature (Tm), and it was not located in the genomic repetitive regions. The primers were selected based on criteria such as a typical amplification curve and single peak from the post-PCR melting curve. FN, gene ID 2335; N-cadherin, gene ID 1000; and vimentin, gene ID 7431 which are involved in cell morphology were also investigated.

Each cDNA was diluted to 1 ml with ddH₂O and mixed with 1 ml of 2X SYBR Premix Ex Taq™ (Takara, Dalian, China). Twenty microliters of this mixture was added to each well of a 96-well PCR array, except for genomic DNA control (GDC). The 96-well PCR plates containing gene primers were prepared by the CT Bioscience Company (Changzhou, China). The sealed PCR plate was loaded in an Eppendorf Realplex 4S (Hamburg, German). The PCR was performed under the following conditions: 95°C for 5 min, 40 cycles at 95°C for 15 sec, 60°C for 15 sec, and 72°C for 20 sec. The melting curve procedure (95°C for 15 sec, 60°C for 15 sec, and 95°C for 15 sec) was implemented to analyze the PCR specificity. Dissociation curves (DC) and melting temperatures (Tm) were recorded. Relative changes in gene expression were calculated using the threshold cycle (Ct) method (20). The formula used is presented as follows: n-fold change = 2^−ΔΔCt = (Ct target gene − Ct internal control gene) treated sample − (Ct target gene − Ct internal control gene) control sample.

Statistical product and service solutions (SPSS ver. 13.0) was used for the data analysis. All of the experiments were conducted in duplicate. The results are presented as mean ± SD (standard deviation) unless otherwise specified. The P-values were two-tailed. P≤0.05 was considered to indicate a statistically significant difference. The cell-cycle pathway is a highly regulated process that incorporates three major checkpoints including the participation of several genes (21). The functional pathways associated with the set of differentially expressed genes were analyzed using the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis (http://www.kegg.jp/kegg/pathway.html). Differentially expressed gene with >2-fold change were analyzed in the cell-cycle pathway (22). The pathway map was created using the Pathview™ package (23).

The in vitro toxicity of (+)-terrein against Bel-7402 cells and the A549 human lung adenocarcinoma epithelial cell line was evaluated using the MTT method [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide] to determine the potential inhibitory concentration. MTT analysis revealed that(+)-terrein inhibited cell viability and proliferation in a concentration-dependent manner. Fig. 2A shows the inhibition of Bel-7402 cells at various (+)-terrein concentrations. The IC₅₀ (half maximal inhibitory concentration) value of Bel-7402 was calculated as 11.63 μM ±0.02. The dose-dependent inhibition of Bel-7402 indicated that 1 μM (+)-terrein was non-toxic and 10 μM (+)-terrein was cytotoxic and associated with a survival rate of 57%. The survival rate of the Bel-7402 cells treated with 100 μM and 1 mM (+)-terrein exhibited inhibitory activities of ~22 and 13%, respectively. A low dose of (+)-terrein did not substantially affect the A549 cells. As indicated in Fig. 2B, (+)-terrein at various concentrations induced inhibitory activity against the A549 cells. (+)-Terrein at concentrations of 10 μM, 100 μM, and 1 mM exhibited weak inhibitory activity, and the survival rate of the treated cells was 91, 82, and 72%, respectively. Moreover, (+)-terrein at 10 mM was cytotoxic to A549 cells, and the survival rate of the treated cells was 52%.

We observed a marked phenomenon regarding cell morphology alterations when cells were treated with (+)-terrein. Morphological changes in the Bel-7402 cells exposed to 10 μM (+)-terrein and in the A549 cells exposed to 10 mM (+)-terrein for 48 h were examined using light microscopy (Fig. 3). The Bel-7402 cell morphology alterations were from epithelial-like to spherical, when the cells were treated with 10 μM (+)-terrein (Fig. 3A and B). The morphological changes from epithelial-like to spherical of the A549 cells exposed to 10 mM (+)-terrein for 48 h were examined using light microscopy (Fig. 3C and D). However, 10 μl of (+)-terrein did not significantly affect the morphology of A549 cells (data not shown). The cells were adherent, and all of the experiments were repeated at least five times.

The cell morphology gene (FN, N-cadherin, and vimentin) expression of the Bel-7402 cells treated with 10 μM (+)-terrein was investigated. The results showed that the gene expression of FN, N-cadherin, and vimentin decreased −3.61-, −2.39-, and −2.05-fold, respectively, compared with the control (Table II).

To determine whether this reduction in cell growth induced by (+)-terrein was mediated by apoptosis, flow cytometric analysis was performed using PI and Annexin-V staining. The results indicated that the apoptotic levels of Bel-7402 and A549 cells treated with (+)-terrein (10 μM and 10 mM) did not increase (Fig. 4), but the number of cells treated with (+)-terrein was less than that of the control cells, according to cell counting (data not shown). The mean percentage ± SD (n=3) of early apoptosis for the Bel-7402 and A549 control cells was 5.33±1.05 and 6.17±0.93, respectively, but that for the Bel-7402 and A549 cells after treatment with (+)-terrein for 48 h was 2.26±0.50 and 0.53±0.31, respectively (Table III). Fig. 4 shows that (+)-terrein at 10 μM and 10 mM inhibited early apoptosis. As indicated in Table III, the mean percentage ± SD (n=3) of late apoptosis and necrosis for the Bel-7402 and A549 control cells was 1.77±0.61 and 5.43±2.55, respectively, but that for the Bel-7402 and A549 cells following treatment with (+)-terrein for 48 h was 0.83±0.23 and 1.93±1.07, respectively (Table III). Based on these results, (+)-terrein inhibited late cell apoptosis and necrosis (Fig. 4). Standard deviation was larger than the average deviation; however, for every independent experiment, the percentage value (%) of apoptosis and necrosis in the treated Bel-7402 and A549 cells was lower than that in the control cells.

Cell proliferation depends on the specific progression of the cell cycle (21). Thus, the cell cycle was analyzed to investigate the anti-proliferative mechanism of (+)-terrein against Bel-7402 cells. Provided the influence of (+)-terrein on A549 occurs at an exceedingly high concentration (>mmol), the anti-proliferative mechanism of (+)-terrein against A549 was not examined. When the Bel-7402 cells were treated with 10 μM (+)-terrein for 48 h, the proportion of the cells in the G₀/G₁ and S phases was reduced, whereas the proportion of cells in the G₂/M phase was increased (Table IV). This result suggested that the cell cycle was arrested by (+)-terrein. Thus, (+)-terrein might decrease Bel-7402 cell growth by inducing cell cycle arrest.

In this study, high-throughput gene expression analysis of 73 genes was performed (Table I). Cell cycle-related gene expression was performed by comparing the gene expression between cDNA samples of (+)-terrein-treated Bel-7402 cells and control cells. Melting curve analysis was used to assess the specificity of the array. A single product peak observed from each reaction without secondary products indicated a high specificity of PCR assay (data not shown). A subset of differentially expressed genes involved in the cell cycle was selected from all the microarray data by performing initial filtration of the P-value (P≤0.05) and expression level (>2-fold) of the 10 μM (+)-terrein-treated cells. The cell-cycle scheme from the KEGG database (http://www.kegg.jp/kegg/pathway.html) was presented. Downregulated genes were labeled in green, while no upregulated genes were overexpressed (Fig. 5). Compared with the control group, the average expression values of CCND2, CCNE2, CDKN1C, CDKN2B, ANAPC5, PKMYT1, CHEK2, and PCNA genes in the 10 μM (+)-terrein-treated group were significantly decreased by 5.52-, 3.30-, 5.32-, 2.24-, 2.52-, 2.52-, 2.31-, and 2.10-fold (>2-fold; P≤0.05), respectively (Table V), and the expression level of the remaining 65 genes was evidently unchanged (data not shown). Eight obviously downregulated genes were visualized in the cell-cycle pathway (Fig. 5). Moreover, the results of flow cytometry (Table IV) indicated that (+)-terrein arrested the cell cycle.