Article Figures & Data
Figures
- Figure 1.
Expression of CCK receptors in the murine liver and HCC cells and response to CCK. A, CCK-BRs are significantly increased in the NASH livers of mice on the CDE diet compared with livers of control mice (P = 0.008). CCK-ARs are downregulated compared with control livers in mice fed a high saturated fat CDE diet (P = 0.0014). B, Compared with normal mouse liver, CCK-BR and CC-ARs are significantly increased in mouse RIL-175 and Dt81Hepa1–6 HCC cells. C, CCK-BR immunofluorescence is detected by flow cytometry in Dt81Hepa1–6 HCC cells. †reproduced with permission from Dig Dis Sci (21). D, CCK peptide stimulates growth of Dt81Hepa1–6 HCC murine cells in cell culture with the greatest effect with 1 nmol/L, a concentration equal to the physiologic binding affinity at the CCK-BR. E, Means ± SD of Dt81Hepa1–6 wild-type, CCK-AR-KO, or CCK-BR-KO cells after 4 days of exposure to PBS or CCK (1 nmol/L). CCK peptide stimulated growth of wild-type cells and CCK-AR-KO cells but not CCK-BR-KO cells. F, CCK (1 nmol/L) stimulated growth of RIL-175 murine HCC cells compared with controls. The CCK-increased growth was blocked only by the CCK-BR antagonist, L365,260, and not the CCK-AR antagonist, L364,718. G, In vivo, Dt81Hepa1–6 tumor size was significantly less in mice treated with proglumide compared with controls. H, In vivo, weekly tumor volumes of RIL-175 murine HCC tumors were significantly less in mice that received drinking water supplemented with proglumide. Significantly different than controls: *, P < 0.05; **, P < 0.01; and ***, P < 0.001.
- Figure 2.
Proglumide therapy alters the hepatic ECM rendering it less procarcinogenic. A, Gene expressions of cytokines and chemokines are increased in NASH livers of mice on the CDE diet with regular, untreated water compared with mice on the control saturated fat diet. B, Proglumide reverses gene expression of activated cytokines and chemokines in NASH livers of CDE-fed mice compared with livers of the mice fed the CDE diet with untreated (Regular) drinking water. C, Liver glutathione levels are increased in CDE-fed mice treated with proglumide compared with CDE-fed mice with regular (untreated water). D, Gene expressions of selective heat shock proteins associated with hepatic inflammation and NASH demonstrate that HSP27 and HSP70 are significantly reduced in the CDE livers of mice treated with proglumide. Significantly different than controls: *, P < 0.05 and **, P < 0.01.
- Figure 3.
Proglumide treatment decreases influx of macrophages and reduces hepatic fibrosis. A, F4/80-positive hepatic macrophages are abundant in the NASH liver of a CDE-fed mouse. Box in the center is magnified in C. B, Fewer macrophages are found in the hepatic parenchyma from a CDE-fed mouse that was treated with proglumide. Box in the center is magnified below in D. E, Analysis of number of F4/80-positive macrophages is significantly reduced in the livers of CDE-fed mice on proglumide (N = 10) compared with that of livers of CDE-fed mice on regular untreated water (N = 10). F, Masson's trichrome stain shows marked fibrosis in a representative NASH liver section from a mouse fed the CDE diet for 18 weeks. Histologically there is macrosteatosis, balloon degeneration, inflammation, and dysplastic nodules (arrows). Box is magnified in H showing marked peri-portal fibrosis. G, Masson's trichrome stain of a representative liver from a mouse fed the CDE diet for 18 weeks and treated with proglumide. The box is magnified in I demonstrating less fibrosis. J, Quantitative analysis of fibrosis in hepatic sections comparing fibrosis in livers of CDE-fed mice receiving regular or proglumide-treated water. Significantly greater than controls: **, P < 0.01 and ****, P < 0.0001.
- Figure 4.
CCK receptors in human HCC compared with normal human liver. A, Mean qRT-PCR mRNA receptor expression is shown from the 5 normal human liver tissues for both the CCK-AR and CCK-BR. The expression of both the CCK-AR and CCK-BR is increased 3- to 4-fold in human HepG2 HCC cells. B, Colony formation was significantly increased by treatment of human HepG2 HCC cells with gastrin (0.4 nmol/L) compared with PBS controls, and the gastrin-accelerated growth was blocked by proglumide. C and D, Representative images from normal human liver from the Biomax tissue microarray reacted with the CCK-BR antibody do not show immunoreactivity. E and F, Representative images of human HCC stained with the anti-CCK-BR antibody from the human tissue microarray show increased immunoreactivity.
Tables
- Table 1.
Demographics of human subjects for the normal liver tissue obtained for CCK-receptor expression.
Subject # Age Sex Race Diagnosis Tissue Sample #1 22yr Female Asian Adenoma Liver Control #2 49y Female White Adenoma Liver Control #3 70y Male White Hemangioma Liver Control #4 77yr Female AA Adenoma Liver Control #5 66yr Female White Hemangioma Liver Control
Supplementary Data
- Supplementary Data - Supplemental Table S1 and Supplemental Figures S1-S3
Volume 14, Issue 1
