Fig 2: Bestatin suppresses the BLT1-ERK1/2 signaling pathway in CRC patients. (a–c) mRNA levels of BLT1, c-Myc, and Ki-67 in human CRC patient tissues were analyzed by quantitative PCR (qPCR). Relative mRNA levels were normalized against GAPDH. The data are shown as mean values ± S.E. from triplicate experiments and the asterisks (**, ***) indicate a significant (p < .01, p < .001) decrease in tissues from the group treated with bestatin compared with the untreated group. (d) Immunohistochemistry analysis was used to determine the expression of BLT1, p-ERK1/2 (Thr202/Tyr204), c-Myc,and Ki-67 in patients treated with bestatin compared with untreated patients. The asterisks (*, **, ***) indicate a significant (p < .05, p < .01, p < .001, respectively) decrease in protein expression in the bestatin-treated group compared with the untreated group (scale bar, 100 µm). (e) The expression of BLT1, p-MEK (Ser217/221), p-ERK1/2 (Thr202/Tyr204), c-Myc, and Ki-67 was also determined by Western blot.

Fig 3: Bestatin prevents intestinal adenoma formation in ApcMin/+ mice. (a) Representative photographs show polyp formation in ApcMin/+ mice treated or not treated with bestatin. (b) Polyp number was measured in mice. The asterisks (***) indicate a significant (p < .001) decrease in polyp formation in mice treated with bestatin compared with the control group. The data are shown as mean values ± S.E., (n = 6). (c) LTB4 production was measured in these groups. The data are shown as mean values ± S.E. and the asterisks (*, **) indicate a significant (p < .01, p < .001, respectively) decrease in LTB4 production in bestatin-treated mice compared with the control group. (d) Immunohistochemistry analysis was used to determine the level of BLT1, Ki-67, and phosphorylation of ERK1/2 (Thr202/Tyr204) and MEK (Ser217/221) in the control group compared with the bestatin-treated group. Representative photographs for each protein and each group are shown. The integrated optical density (IOD) was evaluated using the Image-Pro Premier software (v.9.0) program. The asterisks (*, **, ***) indicate a significant (p < .05, p < .01, p < .001, respectively) decrease in protein expression compared with the control group (Scale bar, 100 µm). (e) Representative western blot analysis confirmed that bestatin inhibited expression of BLT1 and downstream signaling pathways.

Fig 4: Bestatin inhibits tumor growth in a PDX mouse model. (a) Mice bearing PDX tumors were treated or not treated with bestatin. The asterisks (*, **) indicate a significant (p < .05, p < .01, respectively) decrease in tumor volume in mice treated with bestatin compared with the control group. (b) Tumor weight was measured at the end of the study. The asterisk (*) indicates a significant (p < .05) decrease in tumor weight in bestatin-treated mice compared with control mice. The data are shown as mean values ± S.E., (n = 6). (c) LTB4 concentration was measured in mouse serum. The asterisks (*, **) indicate a significant (p < .05, p < .01, respectively) decrease in LTB4 concentration in bestatin-treated mice compared with control mice. (d) Photographs of tumors from each group are shown. (e) Bestatin treatment has no effect on the body weight of mice. (f) Immunohistochemistry analysis was used to determine the levels of BLT1, p-ERK1/2 (Thr202/Tyr204), c-Myc, and Ki-67 in bestatin-treated and control mice. Representative photographs for each protein and each group are shown. The integrated optical density (IOD) was evaluated using the Image-Pro Premier software (v.9.0) program (scale bar, 100 µm). The asterisks (*, **) indicate a significant (p < .05, p < .01, respectively) decrease in protein expression compared with the control group.

Fig 5: LTA4H and BLT1 are overexpressed in human colon adenocarcinoma tissues and colitis and are associated with CRC patient survival probability. (a) LTA4H levels in human colon tissue were analyzed by immunohistochemistry and the density score from each sample was determined (right panel). Representative cases are shown (left panels). The asterisk (*, ***) indicates a significant (p < .05, p < .001, respectively) increase in LTA4H expression in colon adenocarcinoma tissue compared with normal tissue (scale bar, 100 µm). (b) LTB4 concentration in human serum was detected by ELISA assay. The asterisks (**) indicate a significant (p < .01) difference in CRC patients compared with normal adult subjects. (c) LTA4H levels in human colitis were analyzed by immunohistochemistry. The asterisks (***) indicate a significant (p < .001) increase in LTA4H expression in colitis tissue compared with normal tissue (scale bar, 100 µm). (d) Kaplan-Meier survival curves showed the life span of CRC patients with LTA4H high expression (n = 32) or LTA4H low expression (n = 31). (e) BLT1 levels in human colon tissue were analyzed by immunohistochemistry. The asterisks (**, ***) indicate a significant (p < .01, p < .001, respectively) increase in BLT1 expression in colon adenocarcinoma tissue compared with normal tissue (scale bar, 100 µm). (f) BLT1 levels in human colitis were analyzed by immunohistochemistry. The asterisks (***) indicate a significant (p < .001) increase in BLT1 expression in colitis tissue compared with normal tissue (scale bar, 100 µm). (g) Kaplan-Meier survival curves relative to BLT1 expression were analyzed for CRC patients.