BS-IV was generously provided by Professor Hee-Jun Park (Sangji University, Wonju, Korea) (10). Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), 1% antibiotic-antimycotic mixture, phosphate-buffered saline (PBS), and trypsin-EDTA were purchased from Gibco; Thermo Fisher Scientific, Inc. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), Triton X-100, bovine serum albumin (BSA), and Tween-80 were purchased from Sigma-Aldrich; Merck KGaA and protease inhibitor cocktail was purchased from Roche Diagnostics GmbH. Z-VAD-FMK (cat. no. 627610), MG132 (cat. no. 474790), and calpeptin (cat. no. 03-34-0051) were purchased from Calbiochem; Merck KGaA. Antibodies were obtained from the following sources: Bax (cat. no. sc-7480) and Bcl-2 (cat. no. sc-7382) antibodies and horseradish peroxidase (HRP)-conjugated secondary antibodies from Santa Cruz Biotechnology, Inc.; caspase-3 (cat. no. 14220), caspase-9 (cat. no. 9508), poly (ADP-ribose) polymerase (PARP) (cat. no. 9542), Akt (cat. no. 4685) and phosphorylated (p)-Akt antibodies (cat. no. 4060) from Cell Signaling Technology, Inc.; NAG-1/GDF15 antibody (cat. no. ab180929) from Abcam; integrin α₂β₁ (cat. no. MAB1998), α₂ (cat. no. AB1936) and β₁ (cat. no. AB1952) antibodies from Chemicon International; Thermo Fisher Scientific, Inc.; focal adhesion kinase (FAK; cat. no. 610088) and p-FAK (Tyr397; cat. no. 611807) antibodies from BD Transduction Laboratories; β-actin antibody (cat. no. A1978) from Sigma-Aldrich; Merck KGaA; and secondary fluorescein isothiocyanate (FITC)-conjugated antibody (cat. no. 6400-08) from SouthernBiotech. All reagents used in the present study were of analytical grade.

Human colorectal cancer HT-29 cells (cat. no. 30038) and murine colorectal cancer CT-26 cells (cat. no. 80009; both from Korea Cell Line Bank) were cultured in DMEM containing 10% FBS and a 1% antibiotic-antimycotic mixture in a humidified 5% CO₂ incubator at 37°C. Both cell lines were authenticated by STR profiling and screened periodically for mycoplasma contamination using a Mycoplasma Detection kit (MycoAlert^™; Lonza Group, Ltd.).

HT-29 cells (5×10³ cells/well) were seeded into 96-well plates and incubated in DMEM with various concentrations of BS-IV for 2 h at 37°C. Cell viability was measured using an MTT assay as previously described (13). The purple formazan product was dissolved with DMSO and absorbance was detected at 570 nm.

HT-29 cells were treated with 4, 5 and 6 µM of BS-IV. After 2 h, the cells were harvested, and DNA was extracted as previously described (13). Extracted DNA was separated on a 1.8% agarose gel containing ethidium bromide at 50 V and then visualized at 300 nm by ultraviolet transillumination.

HT-29 cells (1×10⁶ cells) were treated for 2 h with 4, 5 and 6 µM of BS-IV. HT-29 cells were also treated for 2 h with proteasome inhibitor MG132 (5 µM), calpain inhibitor calpeptin (10 µM), caspase-3 inhibitor Z-VAD-FMK (5 µM) in the absence or presence of 6 µM BS-IV. Cells were lysed using RIPA buffer containing protease inhibitor cocktail (Santa Cruz Biotechnology, Inc.), and the protein concentration was determined using a BCA protein assay kit (Pierce; Thermo Fisher Scientific, Inc.) as previously described (13). Protein from the cell lysates (50 µg) was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluoride membrane (MilliporeSigma). Membranes were blocked in 5% skim milk in Tris-buffered saline (10 mM Tris, pH 8.0 and 150 mM NaCl) with 0.1% Tween-20 (TBS-T) for 2 h at room temperature and then incubated with primary antibodies (1:1,000) against target molecules overnight at 4°C. After washing, the blots were incubated with a 1:5,000 dilution of the respective HRP-conjugated secondary antibodies for 1 h at room temperature. The targeted proteins were visualized using an enhanced chemiluminescence detection kit (Amersham; Cytiva) according to the manufacturer's protocol. The Band intensities were quantified using an ImageJ analyzer (version 1.8.0; National Institutes of Health) and normalized to the intensity of β-actin, a loading control.

HT-29 cells were treated with 4, 5 and 6 µM of BS-IV for 2 h, and caspase-3 activity was measured using a commercially available caspase activity assay kit (cat. no. E-CK-A311; Enzo Life Sciences) according to the manufacturer's instructions. Absorbance was measured at 405 nm using an ELISA plate reader.

HT-29 cells (3×10⁶ cells/well) were plated in culture dishes coated with solution of 1% polyHEMA (Sigma-Aldrich; Merck KGaA) in 95% ethanol and treated with 4, 5 and 6 µM of BS-IV for 2 h. The attached cells and suspended cells in culture media were harvested and stained with 0.4% trypan blue solution. After 3 min incubation at room temperature, cells were observed and counted under a light microscope at a magnification of ×10.

HT-29 cells (7.5×10⁴ cells) were seeded into CytoMatrixTM cell adhesion strips coated with human collagen type I (cat. no. ECM104) or IV (cat. no. ECM105; both from Sigma-Aldrich; Merck KGaA) and treated with 4, 5 and 6 µM of BS-IV or anti-α₂β₁ integrin antibody. Cell adhesion to BSA-coated strips served as a negative control. After 1 h of incubation at 37°C, attached cells were stained with 0.2% crystal violet in 10% ethanol for 5 min at room temperature. The stained cells were gently washed with PBS, and the cell-bound stain was completely solubilized using solubilization buffer (a 50/50 mixture of 0.1 M NaH₂PO₄, pH 4.5 and 50% ethanol) for 5 min at room temperature. Absorbance was measured at 570 nm on a Benchmark microplate reader (Bio-Rad Laboratories, Inc.).

HT-29 cells were treated with 4, 5 and 6 µM of BS-IV for 2 h. The cells were collected, washed with PBS containing 1% BSA, and incubated with anti-α₂β₁ integrin antibody (10 µg/ml) for 1 h at room temperature. After washing, the cells were resuspended in wash buffer (Mg²⁺ and Ca²⁺-free PBS containing 1% BSA) and incubated with a 1:50 dilution of FITC-conjugated secondary antibody for 30 min at 4°C. The cells were suspended in 200 ml of wash buffer and finally analyzed using a FACSCalibur equipped with CellQuest software (version 7.5.3; Becton-Dickinson, Inc.).

HT-29 cells (1×10⁶ cells) were treated with 5 µM BS-IV for 2 h. Cell lysates were treated with peptide: N-glycosidase F (PNGase F; cat.no. P0704S; New England BioLabs, Inc.) according to the manufacturer's instructions and then incubated overnight at 37°C. The cell lysates were subjected to western blotting using a monoclonal antibody against β₁ integrin to assess the effects of deglycosylation.

Animal studies were approved (approval no. 2020-0193) by the animal ethics committee of Yonsei University College of Dentistry (Seoul, Korea) and conducted in accordance with the approved guidelines of the regional authorities according to Yonsei University animal care regulations. Male BALB/c mice (n=30, 5 weeks-old, 18±3 g; Orient Co. Ltd.) were provided a standard laboratory diet and water ad libitum and were maintained at a temperature of 22±2°C under a 12 h light/dark cycle. Male BALB/c nude mice were randomly divided into 5 groups, with 5 mice per group. Mouse colorectal cancer CT-26 cells (1×10⁵ cells/200 µl PBS) were injected into the tail vein of Balb/c male mice (n=5) using a 27-gauge needle. Vehicle (PBS containing 1% DMSO and 1% Tween-80) and BS-IV at 0.01, 0.1, 1, and 2 mg/kg body weight (BW) were intraperitoneally administered to mice 30 min before cancer cell injection and once daily for 14 days after injection. Control mice received PBS and vehicle. The mice were euthanized by rapid cervical dislocation, and the lobes of the lungs were separated and fixed in Bouin's solution for 4 h at room temperature (Sigma-Aldrich; Merck KGaA). The number of tumor nodules on the lung surfaces was counted under an ocular micrometer, and the lung weights were also recorded.

Data are expressed as the mean ± standard error (SE) of three independent experiments. Student's t-test was used to compare the differences between two groups and one-way ANOVA followed by a Turkey's post hoc test was used to compare differences between multiple groups using SPSS version 19.0 (IBM Corp.). P<0.05 was considered to indicate a statistically significant difference.

Treatment with BS-IV for 2 h inhibited the viability of HT-29 colorectal cancer cells by 23.6% at 2 µM, 60.3% at 3 µM, and 80.5% at 4 µM (Fig. 1B). Treatment with BS-IV for 2 h displayed a characteristic ladder pattern of discontinuous DNA fragmentation, a distinct biochemical hallmark of apoptosis (Fig. 1C). Western blot analysis showed that treatment with BS-IV for 2 h weakly reduced pro-apoptotic Bax expression but significantly suppressed antiapoptotic Bcl-2 expression, resulting in an increased ratio of Bax to Bcl-2 in HT-29 cells in a dose-dependent manner (Fig. 1D). Expression levels of procaspase-9, procaspase-3, and full-length PARP as a substrate of caspase-3 were reduced, and cleaved PARP level was increased in BS-IV-treated HT-29 cells (Fig. 1E). In addition, BS-IV treatment induced dose-dependent increase in NAG-1 expression (Fig. 1F) and increased caspase-3 activity (Fig. 1G). These results indicated that BS-IV induces apoptosis via a mitochondrial-dependent pathway and expression of NAG-1 in HT-29 human colorectal cancer cells.

It was also detected that the number of attached cells decreased and the number of floating cells increased in BS-IV-treated HT-29 cells (Fig. 2A). It was next investigated whether BS-IV affected the attachment of HT-29 cells to the extracellular matrix (ECM). The attachment of HT-29 cells was significantly increased in collagen type I- or type IV-coated plates compared with BSA-coated plates. BS-IV treatment reduced the attachment of HT-29 cells to collagen types I and IV to the level of cell attachment to BSA, whereas the addition of anti-α₂β₁ integrin antibody at 10 µg/ml blocked cell attachment to collagen types I and IV by 75 and 51%, respectively (Fig. 2B). The molecular mechanism by which BS-IV inhibits the attachment of HT-29 cells to collagen types I and IV was further explored. Flow cytometric analysis demonstrated that treatment with BS-IV inhibited cell surface expression of α₂β₁ integrin in HT-29 cells in a dose-dependent manner (Fig. 2C). Western blot analysis indicated that treatment with BS-IV reduced cellular levels of the α₂ subunit and β₁ subunit mature form (Fig. 2D). The β₁ integrin subunit is synthesized as an 86 kDa polypeptide and sequentially glycosylated to a precursor form of 105 kDa followed by a mature form of 125 kDa in the endoplasmic reticulum and Golgi apparatus. The maturation of β1 integrin is important for its transport to the cell surface and/or binding to ECM ligands. Cell surface α integrins are transported to the cell surface after binding to β₁ integrin in cells (16). Treatment with BS-IV and then PNGase F, which completely removes oligosaccharide chains from glycoproteins, converted both the precursor and mature forms of β₁ integrin to the β₁ integrin core of an 86 kDa polypeptide. β₁ integrin core levels were not reduced by BS-IV treatment (Fig. 2E). These results indicated that BS-IV inhibits the adhesion of HT-29 cells on ECM components and that the reduced cell adhesion to collagen types I and IV in response to BS-IV treatment may be attributed to reduced levels of α₂β₁, the cellular receptor of collagen types I and IV, which appears to occur by reducing expression of the α₂ subunit and blocking the maturation of β₁ integrin precursors via glycosylation.

It was further examined whether BS-IV affected the activation of FAK and Akt, major signaling molecules that play important roles in numerous fundamental cellular functions, including cell adhesion (17). Treatment with BS-IV reduced the expression and phosphorylation of Akt, as well as the expression and Tyr³⁹⁷ phosphorylation of FAK (Fig. 3A). The reduced FAK and Akt expression levels were recovered in the presence of the caspase-3 inhibitor Z-VAD-FMK but not the proteasome inhibitor MG132 or the calpain inhibitor calpeptin in HT-29 cells treated with 6 µM BS-IV (Fig. 3B). These results indicated that caspase-3 activated in response to BS-IV treatment may reduce the expression and phosphorylation levels of Akt and FAK by degrading them.

Intravenous injection of CT-26 murine colorectal cancer cells into mice caused the formation of tumor nodules in the lung, resulting in a significant increase in lung weight. Intraperitoneally injected BS-IV significantly reduced the number of lung tumor nodules and lung weight in a dose-dependent manner (Fig. 4A). Administration of BS-IV at 2 mg/kg BW reduced the formation of lung tumor nodules by 54% and lung weight by 33%. These results indicated that BS-IV suppresses lung metastasis of murine colon carcinoma CT-26 cells.