Fig. 7
DKC1125 restores sensitivity to 5-FU, oxaliplatin, and cetuximab in KITENIN-overexpressing CRC cells and suppresses hepatic metastasis of CRC. a CRC cells overexpressing KITENIN had a blunted chemotherapeutic response to 5-FU and oxaliplatin, and treatment of DKC1125 enhances the inhibitory effects of 5-FU and oxaliplatin on cell survival. Various CRC cells were seeded at 5 × 103 cells/well on 96-well plates, and the cytotoxicity of various concentrations of 5-FU or oxaliplatin and/or DKC1125 in these cells was examined by tetrazolium salt (WST-8) assay. The half-maximal inhibitory concentration (IC50) of 5-FU or oxaliplatin alone, or combined treatment of 5-FU or oxaliplatin with DKC1125, was obtained from cell cytotoxicity data at 48 h, as shown in Fig. S5. Data were represented as mean ± SEM (n = 3). The asterisk indicates a significant difference of changes after treatment with DKC1125, compared with non-treated IC50 value (*P < 0.05; **P < 0.01). b Treatment of DKC1125 enhances the inhibitory effects of cetuximab on cell invasiveness. Cell invasion was examined in the HCT116 cells treated with vehicle, cetuximab (5, 15, 50 μg/ml), or the combination of DKC1125 (0.5, 1.5, 4.5 μM) and 5-FU (5, 15, 50 μg/ml). Data are expressed as in Fig. 1a and Fig. 7a. IC50 values were represented as mean ± SEM (n = 3). The asterisk indicates a significant difference of changes after treatment with DKC1125, compared with non-treated IC50 value (*P < 0.05; **P < 0.01). c Increased cytotoxicity by treatment with DKC1125 in HCT116 cells under combined treatment with 5-FU and cetuximab. HCT116 cells were seeded at 5 × 103 cells/well on 96-well plates, and the effects of DKC1125 on cytotoxicity under combined treatment with various concentrations of 5-FU (0.03, 0.1, or 0.3 μg/ml) and cetuximab (5, 15, or 50 μg/ml) were examined by tetrazolium salt (WST-8) assay 48 h after seeding. Results were shown in line graphs as mean ± SEM (n = 3), and compared between groups treated or not treated with DKC1125 (1 μM). The asterisk indicates a significant difference of changes after treatment with DKC1125 in each combined dosage of 5-FU and cetuximab (*P < 0.05; **P < 0.01). d Effect of treatment with DKC1125 on tumor formation of CT-26 cells in a syngeneic mouse xenograft model. BALB/c mice were injected subcutaneously with 1 × 105 CT-26/EV or CT-26/KITENIN-V5 cells, and randomly sorted into two groups (n = number of mouse) treated with vehicle (0.1% DMSO, indicated as V) or 5 mg/kg DKC1125 (indicated as D) starting 7 days after cell injection. Tumor volumes are represented as means ± SEM. An asterisk in a line graph indicates a significant difference between groups (vehicle-treated vs DKC1125-treated; *p < 0.05, **p < 0.01). e Treatment with DKC1125 increases the inhibitory effects of 5-FU on cell invasion by KITENIN-overexpressing CT-26 cells. Cell invasion was compared between CT-26 cells transfected with empty vector (EV) or KITENIN and treated with vehicle, DKC1125 (0.5 μM), or the combination of DKC1125 (0.5 μM) and 5-FU (20 μg/ml). Data are expressed as in Fig. 1a. f DKC1125 effectively suppresses colorectal liver metastasis, and the combination of DKC1125 with 5-FU exhibits more enhanced therapeutic effect. Experimental hepatic metastasis model was prepared by performing intrasplenic inoculation of stably CT-26/KITENIN-iRFP-expressing cells in syngeneic mice and subsequent splenectomy. For 2 weeks, mice were given intraperitoneal injections of 5-FU once in the entire 2 weeks (100 mg/kg); DKC1125 (10 mg/kg), or vehicle (0.1% DMSO) 3 times/week; or the combination of DKC1125 and 5-FU, respectively. To evaluate metastasis, metastatic tumor growth was calculated as the number of nodules that migrated to the surface of the liver, multiplied by size; this yielded a metastatic score (upper). In addition, total fluorescence emitted from liver nodules expressing iRFP was measured (lower). Metastatic score (left) or total fluorescence (right) are represented as mean ± SEM. An asterisk in a line graph indicates a significant difference between the indicated groups (*p < 0.05, **p < 0.01). g Schematic showing how DKC1125 breaks down the functional KITENIN complex and thereby alters the specific cellular context induced by the upregulated complex. Through these mechanisms, DKC1125 exhibits more effective anticancer action in cancer cells expressing higher levels of KITENIN levels. SCC, specific cellular context