Lipopolysaccharides Enhance Epithelial Hyperplasia and Tubular Adenoma in Intestine-Specific Expression of krasV12 in Transgenic Zebrafish

Abstract

Intestinal carcinogenesis is a multistep process that begins with epithelial hyperplasia, followed by a transition to an adenoma and then to a carcinoma. Many etiological factors, including KRAS mutations and inflammation, have been implicated in oncogenesis. However, the potential synergistic effects between KRAS mutations and inflammation as well as the potential mechanisms by which they promote intestinal carcinogenesis remain unclear. Thus, the objective of this study was to investigate the synergistic effects of krasV12, lipopolysaccharides (LPS), and/or dextran sulfate sodium (DSS) on inflammation, tumor progression, and intestinal disorders using transgenic adults and larvae of zebrafish. Histopathology and pathological staining were used to examine the intestines of krasV12 transgenic zebrafish treated with LPS and/or DSS. LPS and/or DSS treatment enhanced intestinal inflammation in krasV12 transgenic larvae with concomitant increases in the number of neutrophils and macrophages in the intestines. The expression of krasV12, combined with LPS treatment, also enhanced epithelial hyperplasia and tubular adenoma, demonstrated by histopathological examinations and by increases in cell apoptosis, cell proliferation, and downstream signaling of phosphorylated AKT serine/threonine kinase 1 (AKT), extracellular-signal-regulated kinase (ERK), and histone. We also found that krasV12 expression, combined with LPS treatment, significantly enhanced changes in intestinal morphology, specifically (1) decreases in goblet cell number, goblet cell size, villi height, and intervilli space, as well as (2) increases in villi width and smooth muscle thickness. Moreover, krasV12 transgenic larvae cotreated with DSS and LPS exhibited exacerbated intestinal inflammation. Cotreatment with DSS and LPS in krasV12-expressing transgenic adult zebrafish also enhanced epithelial hyperplasia and tubular adenoma, compared with wild-type fish that received the same cotreatment. In conclusion, our data suggest that krasV12 expression, combined with LPS and/or DSS treatment, can enhance intestinal tumor progression by activating the phosphatidylinositol-3-kinase (PI3K)/AKT signaling pathway and may provide a valuable in vivo platform to investigate tumor initiation and antitumor drugs for gastrointestinal cancers.

Effect of Lipopolysaccharides on Liver Tumor Metastasis of twist1a/krasV12 Double Transgenic Zebrafish

“…Consistent with histological observations, the percentage of proliferating liver cells increased more rapidly in kras + zebrafish and twist1a +/ kras + than in wild-type and twist1a + control zebrafish from 2 and 4 wpi ( Figure 1 F); however, at 4 wpi, the percentage of liver cells undergoing apoptosis was greater in twist1a +/ kras + zebrafish than in kras + zebrafish ( Figure 3 A,B). These observations were consistent with findings from our previous studies [ 32 , 36 , 45 ].…”

“…Dry pellets (GEMMA Micro 150 and 300, Skretting Nutreco, Tooele, UT, USA) were fed to adult zebrafish twice a day at a designated amount of approximately 3% body mass and directly proportional to the density of zebrafish within the tank. The zebrafish were fed lab-grown brine shrimp or commercial fish feed three times per day [ 35 , 36 ].…”

“…In accordance with published protocols [ 33 , 36 , 40 ], all zebrafish samples were collected following euthanization at 2- or 4-weeks post-induction (wpi). Liver tissues were then fixed and embedded in paraffin for histological analysis.…”

“…All statistical analyses were performed using GraphPad Prism 9 software (GraphPad Software, La Jolla, CA, USA), as previously described [ 36 ]. For all in-vivo experiments, a two-tailed unpaired Student’s t-test or one-way analysis of variance (ANOVA) was applied to compare experimental and control groups.…”

MicroRNA-21 Plays Multiple Oncometabolic Roles in Colitis-Associated Carcinoma and Colorectal Cancer via the PI3K/AKT, STAT3, and PDCD4/TNF-α Signaling Pathways in Zebrafish

“…The cellular composition, architectural organization, and the digestive and immune functions of the intestinal tract is conserved between zebrafish and mammals [ 36 ]. However, studies examining the transgenic zebrafish as a model for GI cancer research are scarce [ 37 , 38 , 39 ].…”