Kim JL , Park SH , Jeong S , Kim BR , Na YJ , Jo MJ , Jeong YA , Yun HK , Kim DY , Kim BG , You S , Oh SC , Lee DH .

	Figure 1. Sea cucumber (SC) F2 significantly activated TNF-related apoptosis-inducing ligand (TRAIL)-induced apoptosis of human colorectal cancer (CRC) cell lines. (A) Cytotoxicity of SC F2 (B) Cytotoxicity of TRAIL (C) Cytotoxicity of either substance alone, or the combination of SC F2 and TRAIL in DLD-1 cells (mean ± SD, n = 5) was evaluated using MTT (3-(4,5-dimethylthiazol-2-ly)-2,5-diphenyl tetrazolium bromide) assay. Cells were treated with Dimethyla sulfoxide (DMSO) (mock control) or SC F2 at various concentrations (0–300 μg/mL) for 20 h. Cells were treated with TRAIL at various concentrations (0–100 ng/mL) for 4 h. To check the cytotoxic effect of the combination of SC F2 and TRAIL in DLD-1 cells, they were cultured in the presence or absence of TRAIL (20 ng/mL) and/or SC F2 (200 μg/mL) for 24 h. (D) Western blotting of cleaved PARP-1, cleaved caspase-9 and cleaved caspase-3 antibodies. (E) DLD-1 cells were treated with SC F2 or TRAIL alone or SC F2 in combination with TRAIL for 24 h. Cell morphology was observed using the optical microscope. Scale bar: 100 μm. (F) DLD-1 cells plated in 6-well cell culture plates were treated with 200 μg/mL SC F2, 20 ng/mL TRAIL, or their combination. After 12 days, the plates were stained with crystal violet dye, and colonies were visualized and counted using a digital camera. Quantitative analysis of data. (G) DLD-1 cells were stained with annexin V and propidium iodide (PI), for flow cytometry analysis. Percentage of apoptosis in cells treated with SC F2 or TRAIL alone or in combination. Experiments were performed at least three times. Error bars represent standard error of the mean (SEM) from three independent experiments. ** p < 0.05, *** p < 0.01.
	Figure 2. Suppression of XIAP by SC F2 sensitizes cells to TRAIL-mediated apoptosis. (A) DLD-1 cells were treated with the indicated concentrations of SC F2 for 20 h. Western blotting of death receptor (DR4, DR5), anti-apoptotic and pro-apoptotic protein. (B) DLD-1 cells were treated with indicated SC F2 doses for 20 h. Cell lysates were analyzed by Western blotting using anti-caspase 9, anti-caspase 3, and anti-cleaved PARP-1 antibodies. (C) DLD-1 and HCT116 cells were exposed to 200 µg/mL of SC F2 for the indicated time intervals. Cell lysates were analyzed by Western blotting using an anti-XIAP antibody. (D) XIAP was silenced by XIAP siRNA in DLD-1 cells. The cells were then treated with TRAIL for 4 h followed by flow cytometry analysis. Error bars represent standard error of the mean (SEM) from three separate experiments. “*” represents a statistically significant difference between TRAIL + siCon-treated and TRAIL + siXIAP-treated cells at p < 0.05. (E) XIAP was silenced by XIAP siRNA in DLD-1 cells. The cells were then treated with TRAIL for 4 h followed by Western blotting. ** p < 0.005.
	Figure 3. SC F2-induced C/EBP-homologous protein (CHOP) up-regulation is mediated through selective induction of IRE1 α-JNK endoplasmic reticulum (ER) stress signaling. DLD-1 cells were treated with indicated concentrations of SC F2 for 20 h (A), or treated with SC F2 (200 μg/mL) for indicated time points (B). The protein expression levels of the ER stress pathway were measured by immunoblotting using corresponding antibodies. (C) DLD-1 cells were transfected with siRNA against CHOP or control. After 24 h, cells were treated with SC F2 (200 μg/mL) for 20 h followed by flow cytometry analysis. Error bars represent standard error of the mean (SEM) from three separate experiments. (D) CHOP was silenced by CHOP siRNA in DLD-1 cells. The cells were then treated with SC F2 (200 μg/mL) for 20 h followed by Western blotting using an anti-cleaved PARP-1 antibody. (E) DLD-1 cells were transiently transfected with CHOP siRNA (siCHOP). Twenty-four hours after transfection, cells were pretreated with or without SC F2 (200 μg/mL) for 20 h and then TRAIL (20 ng/mL) for 4 h. “*” or “**” represents a statistically significant difference between untreated control cells and drug treated cells at p < 0.05 or p < 0.01, respectively.
	Figure 4. SC F2-induced ROS generation is mediated through selective decrease of catalase. (A) ROS production was assessed by DCFH-DA, following 1 h pre-treatment with 200 μg/mL of SC F2 in DLD-1 cells. (B) DLD-1 cells were treated with SC F2 (200 μg/mL) for 20 h, cells were stained with FITC probe DCFH-DA (10 μM) for 30 min at 37 °C, and visualized under a confocal microscope. (C) DLD-1 cells were treated with SC F2 (200 μg/mL) for 20 h. The protein expression levels of ROS-related proteins were measured by immunoblotting using corresponding antibodies. (D) DLD-1 cells were pre-treated with 10 mM N-acetyl-l-cysteine (NAC) for 1 h, followed by treatment with or without SC F2 (200 μg/mL). Cells were then lysed and subjected to Western blotting. “*” or “**” represents a statistically significant difference between untreated control cells and drug treated cells at p < 0.05 or p < 0.01, respectively.
	Figure 5. XIAP plays a role in the sensitization function of SC F2. DLD-1 cells were treated with SC F2 at indicated doses for 20 h. The mRNA expression levels of XIAP and actin were measured by reverse transcriptase-PCR (A) and real time-PCR (B). (C) SC F2-treated DLD-1 cells were treated in the presence or absence of MG132 and Western blot analysis using anti-XIAP and anti-actin antibodies was carried out. (D) SC F2-treated DLD-1 cells were subjected to immunoprecipitation with an anti-XIAP antibody and immunoblotted for anti-Ub. (E) Empty vector (0.5 μg) or XIAP vector (0.5 μg) transiently over-expressing. DLD-1 cells were treated with 200 μg/mL SC F2 for 20 h and immunoprecipitated with the anti-XIAP antibody or with IgG, and immunoblotted for Ub. Western blot analysis showing XIAP and actin. (F) Measurement of apoptotic index in Myc-control and Myc-XIAP-treated DLD-1 cells by MTT assay after SC F2 and TRAIL treatment. Error bars represent standard error of the mean (SEM) from three separate experiments. “**” represents a statistically significant difference between TRAIL treated on empty vector transfected cells and TRAIL treated on Myc-XIAP vector transfected cells at p < 0.01.
	Figure 6. SC F2 enhanced TRAIL-induced apoptosis in vivo. (A–C) DLD-1 cells were inoculated into nude mice (n = 5 per group) at 5 × 106 per mouse subcutaneously. Mice received 4 ng/kg TRAIL and 50 mg/kg SC F2 either alone or in combination at day 19 after tumor implantation. Representative tumors of each group are shown. Tumor volume was calculated every 2 days for 19 days according to the following equation: tumor volume (mm3) = π/6 × length × (width) 2. Maximum tumor area and its corresponding section were calculated using MetaMorph software (Molecular Devices). (D) The average tumor weight in nude mice after administration of TRAIL and SC F2 alone or in combination. (E) Schematic diagram for a working model of SC F2 sensitizing TRAIL-induced apoptosis. *** p < 0.001.

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