The human colorectal cancer cell lines, SW480 and HT-29, were purchased from the American Type Culture Collection. The cells were cultured in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin/streptomycin (Sigma-Aldrich; Merck KGaA), and maintained at 37°C in a humidified incubator with 5% CO₂. The cells were treated with TCG (purity, ≥95%), which was kindly gifted by Professor HaoFu Dai (Chinese Academy of Tropical Agricultural Sciences, Haikou, China).

The PI3K inhibitor, 3-methyladenine (3-MA; cat. no. 189490) and chloroquine (CQ; cat. no. C6628) were purchased from Sigma-Aldrich (Merck KGaA). Verteporfin (VP; cat. no. HY-B0146) was purchased from MedChemExpress. The cells were treated with 10 mM 3-MA, 5 µM CQ, or 10 µM VP at 37°C for 24 h.

Cell cytotoxicity was measured using a MTT assay. Briefly, SW480 or HT-29 cells were seeded (1×10⁶/well) into 96-well plates and treated with indicated concentrations of TCG or DMSO (or 10 mM 3-MA, 5 µM CQ and 10 µM VP) for 24 h at 37°C. Following incubation, 10 µl MTT was added to each well and the samples were incubated for a further 4 h at 37°C. Then, 200 µl DMSO was added to each well to dissolve purple formazan. The absorbance was measured at 570 nm using an ELISA reader (Bio-Rad Laboratories, Inc.).

The SW480 cells were seeded (1×10⁶/well) into 96-well plates and treated with indicated concentrations of TCG or DMSO (or 10 mM 3-MA and 10 µM VP) for 24 h. Following incubation, cell proliferation was determined using a BrdU Cell Proliferation ELISA kit (cat. no. ab126556; Abcam) according to the manufacturer's protocol.

The SW480 cells were seeded (1×10³/well) into 24-well plates and treated with indicated concentrations of TCG or DMSO (or 3-MA according to each experiment requires). Following 7 days of incubation, the colonies were stained with Giemsa for 15 min at 37°C, then washed three times with PBS. The visible colonies were visualized using a Molecular Imager Gel Do XR+ system (Bio-Rad Laboratories, Inc.) and counted using ImageJ 1.47 software (National Institutes of Health).

The SW480 cells were seeded (1×10⁶/well) into 96-well plates and treated with indicated concentrations of TCG or DMSO (or 10 mM 3-MA and 10 µM VP) for 24 h. Following incubation, the cytotoxicity was measured using a LDH release kit (cat. no. C0016; Beyotime Institute of Biotechnology) according to the manufacturer's protocol.

Briefly, the SW480 cells were seeded (3×10⁵/well) into 6-well plates and treated with TCG (0, 0.2 and 0.4 µM) for 24 h. Following incubation, the cells were harvested and washed with PBS, then resuspended in PI/AnnexinV solution (Nanjing KeyGen Biotech Co., Ltd.). Apoptosis was subsequently analyzed using a FACSCalibur flow cytometer (BD Biosciences) and FlowJo v7.6.1 software (FlowJo LLC).

Total protein was extracted from the SW480 cells treated with 0, 0.2 and 0.4 µM TCG (or control, 0.4 µM TCG, 10 mM 3-MA, 0.4 µM TCG+10 mM 3-MA; or control, 0.4 µM TCG, 5 µM CQ, 0.4 µM TCG+5 µM CQ) using RIPA lysis buffer (Thermo Fisher Scientific, Inc.) and quantified using a BCA protein assay kit (Thermo Fisher Scientific, Inc.). The proteins were separated using 10% SDS-PAGE and transferred onto PVDF membranes. After blocking with 5% BSA (Sigma-Aldrich; Merck KGaA) for 30 min at 37°C, the membranes were incubated at 4°C overnight with the following primary antibodies: Anti-poly (ADP-ribose) polymerase 1 (PARP; cat. no. ab191217; 1:1,000; Abcam), anti-caspase-3 (cat. no. ab32351; 1:1,000; Abcam), anti-LC3B (cat. no. L7543; 1:2,000; Sigma-Aldrich; Merck KGaA), anti-Beclin1 (cat. no. ab210498; 1:1,000; Abcam), anti-autophagy related 5 (ATG5; cat. no. ab108327; 1:1,000; Abcam), anti-P62 (cat. no. ab109012; 1:1,000; Abcam), anti-phosphorylated (p)-YAP (cat. no. ab76252; 1:1,000; Abcam), anti-YAP (cat. no. ab52771; 1:1,000; Abcam), anti-LATS1 (cat. no. 3477; 1:1,000; Cell Signaling Technology, Inc.) and anti-β-actin (cat. no. ab8226; 1:2,000; Abcam). Following incubation with the primary antibodies, the membranes were incubated with secondary antibodies (anti-rabbit IgG; cat. no. ab6721; 1:2,000; Abcam) at room temperature for 2 h. The protein bands were visualized using an enhanced chemiluminescence reagent (cat. no. WBKLS0100; MilliporeSigma) and the protein ratios were calculated following densitometric analysis using ImageJ v1.47 software (National Institutes of Health).

Total RNA was extracted from the SW480 cells using TRIzol^® (Invitrogen; Thermo Fisher Scientific, Inc.). Total RNA was reverse transcribed into cDNA using reverse transcriptase and random hexamers from a RevertAid First Strand cDNA Synthesis kit (Thermo Fisher Scientific, Inc.). The following temperature protocol was used: Priming at 25°C for 5 min, RT at 42°C for 60 min, then inactivation at 70°C for 5 min. qPCR was subsequently performed by mixing cDNA, gene-specific primers and IQ SYRB Green Supermix (Agilent Technologies, Inc.) and detected using a Mx3005P Real-Time PCR system (Agilent Technologies, Inc.) according to the manufacturer's protocol. The following primers were used for the qPCR: CTGF forward, 5′-AAAAGTGCATCCGTACTCCCA-3′ and reverse, 5′-CCGTCGGTACATACTCCACAG-3′; CCN1 forward, 5′-AGCCTCGCATCCTATACAACC-3′ and reverse, 5′-TTCTTTCACAAGGCGGCACTC-3′; and GAPDH forward, 5′-GAGCGAGATCCCTCCAAAAT-3′ and reverse, 5′-GGCTGTTGTCATACTTCTCATGG-3′. The following thermocycling conditions were used for qPCR: Initial denaturation at 95°C for 2 min, followed by 40 cycles of denaturation at 94°C for 15 sec, and annealing/extension at 60°C for 1 min. Expression levels were quantified using the 2^−∆∆Cq method and fold changes were obtained by normalization to GAPDH expression (27).

The SW480 cells treated with 0.4 µM TCG or control (or 5 µM CQ) were fixed with 4% paraformaldehyde at room temperature for 30 min, washed with PBS, then incubated with 0.1% Triton X-100 for permeabilization. Non-specific binding was performed by blocking with 5% BSA (Sigma-Aldrich; Merck KGaA) for 30 min at 37°C and the cells were then incubated with anti-LC3B (1:200; cat. no. L7543; Sigma-Aldrich; Merck KGaA), anti-lysosomal associated membrane protein 2 (LAMP2; 1:200; cat. no. sc-20004; Santa Cruz Biotechnology, Inc.) and anti-YAP (1:200; cat. no. ab52771; Abcam) primary antibodies overnight at 4°C. Following incubation with the primary antibody, the cells were incubated with an Alexa Fluor 488-conjugated goat anti-rabbit IgG (1:1,000; cat. no. ab150077; Abcam) or an Alexa Fluor 594-conjugated donkey anti-mouse IgG (1:1,000; cat. no. ab150108; Abcam) secondary antibody at room temperature for 1 h. Nuclei were stained with 10 ug/ml DAPI for 5 min at 37°C. The stained cells were visualized using a confocal laser scanning microscope (FV1000; Olympus Corporation).

Small interfering (si) RNA targeting Atg5 and YAP, and scramble siRNA were synthesized by Shanghai GenePharma Co., Ltd. The sequences of the siRNAs are as follows: Atg5 siRNA, 5′-GCAACUCUGGAUGGGAUUGTT-3′; YAP siRNA, 5′-CGAGAUGAGAGCACAGACAdTdT-3′; negative control (NC) of Atg5 (siScramble#1; 5′-GGAAAGAGCUGCAUAUUAATT-3′); and NC of YAP, (siScramble#2; 5′-UAAGGCUAUGAAGAGAUAC-3′). The SW480 cells were transfected with the siRNAs (50 nmol/l) at 37°C for 48 h using Lipofectamine^® 3000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. At 72 h post-transfection, the cells were harvested and used for subsequent experiments.

The pEGFP-LC3 plasmid was kindly gifted by Dr Lu Zhang (Sichuan University, Chengdu, China). The SW480 cells were transfected with the pEGFP-LC3 plasmid (2.5 µg) at 37°C using Lipofectamine^® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. After 36 h at 37°C, the cells were treated with DMSO or TCG (0.4 µM) for another 36 h at 37°C. Formation of EGFP-LC3 puncta was visualized using fluorescence microscopy (FV1000; Olympus Corporation).

Statistical analysis was performed using GraphPad Prism v6.0 software (GraphPad Software, Inc.). Statistical differences between groups were determined using a one-way ANOVA followed by Tukey's post hoc test or an unpaired Student's t-test. The data are presented as the mean ± SD. P<0.05 was considered to indicate a statistically significant difference.

To determine whether TCG exhibits anticancer effects against colorectal cancer cells, cell viability was determined using a MTT assay. As shown in Fig. 1A, TCG treatment decreased the cytotoxicity of the colorectal cancer cells in a dose-dependent manner compared with that in the control group. Consistent with this finding, the results of the colony formation and BrdU incorporation assays revealed that TCG treatment inhibited SW480 cell proliferation compared with that in the control group (Fig. 1B and C). Furthermore, the LDH release assay showed that TCG treatment increased SW480 cell death compared with that in the control group (Fig. 1D). Taken together, these results indicated that TCG may inhibit SW480 cell proliferation and viability in vitro.

To further evaluate whether apoptosis was associated with the anticancer effect of TCG, the apoptotic ratio was analyzed using flow cytometry. As shown in Fig. 1E, TCG treatment for 24 h did not significantly increase the levels of apoptosis in SW480 cells compared with that in the control group. Consistent with this observation, the expression levels of cleaved PARP and cleaved caspase-3 were not altered between TCG-treated and control cells (Fig. 1F). These findings indicated that TCG may not induce apoptosis in the SW480 cell line. Taken together, these data suggested that TCG may inhibit SW480 cell viability and proliferation, independent of apoptosis.

Accumulating evidence has indicated that autophagy is involved in drug-mediated anticancer therapy (10,28,29); therefore, the present study investigated whether TCG induced autophagy in the SW480 cell line. First, LC3-II accumulation, a hallmark of autophagy, and the levels of Beclin 1 and Atg5, two autophagy-related proteins, were analyzed. As shown in Fig. 2A, TCG treatment markedly increased LC3-II expression in the SW480 cell lines. In addition, the expression levels of Beclin1 and Atg5 were found to be increased in the TCG-treated cells. To confirm this observation, a GFP-tagged LC3B plasmid was used, and a marked increase in GFP-tagged LC3B puncta was observed in TCG-treated cells compared with that in the control cells (Fig. 2B). Next, the distribution of endogenous LC3 puncta, another classical marker of autophagy, was examined. The results revealed that TCG treatment significantly increased the number of endogenous LC3 puncta in the SW480 cell lines (Fig. 2C and D). Furthermore, treatment with 3-MA (an autophagy inhibitor) markedly decreased LC3-II expression in the TCG-treated cells (Fig. 2E). Taken together, these findings indicated that TCG may induce autophagy in SW480 cells.

In addition to autophagy initiation, LC3-II accumulation may result from impaired autophagic flux (30). Therefore, the present study investigated whether TCG induced complete autophagic flux in the SW480 cell line. The protein expression levels of LC3-II and P62 (an autophagy-specific substrate) were investigated by adding CQ (a lysosomal inhibitor) to the TCG-treated cells to CQ. The results revealed that both LC3-II and P62 were significantly increased in TCG-treated cells, but expression was not further increased upon combined treatment with CQ (Fig. 3A). Furthermore, the colocalization of LC3B with LAMP2 (a lysosomal marker) was not observed in the TCG-treated cells (Fig. 3B), suggesting that TCG hinders the fusion of autophagosomes with lysosomes. These results indicated that TCG may induce autophagosome accumulation by blocking autophagosome-lysosome fusion.

To determine whether autophagy was involved in the TCG-mediated inhibition of cell viability and proliferation, the SW480 cell line was treated with TCG in combination with 3-MA or CQ. As shown in Fig. 4A and B, 3-MA treatment markedly restored the TCG-mediated inhibition of cell viability, whereas CQ treatment did not affect cell viability. The results of the BrdU incorporation and colony formation assays showed that 3-MA treatment significantly restored the TCG-mediated suppression of cell proliferation (Fig. 4C and D). In addition, 3-MA treatment markedly decreased TCG-induced cytotoxicity, as evidenced by the LDH release assay (Fig. 4E). Atg5 knockdown significantly decreased Atg5 protein expression levels in the SW480 cell line compared with that in the control group (Fig. 4F and G). Knockdown of Atg5 also prevented the TCG-mediated suppression of cell viability, as evidenced by MTT and LDH release assays (Fig. 4H and I). Taken together, these results suggested that TCG may inhibit SW480 cell viability and proliferation by promoting lethal autophagosome accumulation.

As the Hippo signaling pathway has been found to play a key role in the development of numerous types of human cancer (31), the present study aimed to determine whether the Hippo signaling pathway was associated with the TCG-mediated inhibition of SW480 cell viability and proliferation. The results demonstrated that TCG treatment promoted YAP dephosphorylation at serine 127 in the SW480 cell line (Fig. 5A). In addition, YAP nuclear localization was found to be elevated in the TCG-treated cells (Fig. 5B and C). The mRNA expression levels of downstream target genes of YAP were also investigated. As shown in Fig. 5D, TCG treatment markedly upregulated the mRNA expression levels of CTGF and CCN1. Furthermore, TCG treatment significantly downregulated LATS1 protein expression level in the SW480 cell line (Fig. 5E). Collectively, these results indicated that TCG may enhance YAP dephosphorylation and nuclear localization, and downstream target gene expression, suggesting that YAP may be activated in the TCG-treated SW480 cell line.

To determine the role of YAP in the TCG-mediated inhibition of cell viability and proliferation, cell viability and proliferation were measured following treatment with the YAP inhibitor, VP. The results of the MTT assay showed that VP treatment significantly decreased cell viability compared with that in cells treated with TCG alone (Fig. 6A). In addition, VP treatment further suppressed cell proliferation in TCG-treated cells, as demonstrated by the BrdU incorporation assay (Fig. 6B). Furthermore, VP treatment markedly increased TCG-induced cytotoxicity, which was evidenced using a LDH release assay (Fig. 6C). YAP knockdown significantly decreased YAP protein expression levels in the SW480 cell line compared with that in the control group (Fig. 6D and E). Knockdown of YAP further enhanced the TCG-mediated inhibition of cell viability and proliferation, which was evidenced by MTT, BrdU incorporation and LDH release assays (Fig. 6F-H). Taken together, these findings indicated that TCG-induced YAP activation may play a protective role against the TCG-mediated inhibition of cell viability and proliferation.