Inhibition of induced-hepatic cancer in vivo through IQGAP1-shRNA gene therapy and modulation of TRAIL-induced apoptosis pathway

Abstract

Background: Liver cancer is the deadliest malignancy among common tumors. It is the top cause of cancer-related deaths in Egypt, and it is characterized by increasing occurrence among the population. The objective of this study was to determine the outcome of pre-treatment of IQGAP1-shRNA on induced mouse hepatocellular carcinoma model and evaluate the potency of this IQGAP1-shRNA plasmid to recover hepatic cancer as a new tool of cancer therapy. Therefore, we will use RNA interference (RNAi) technology to silence IQGAP1 oncogene to completely recover the chemically induced models for hepatic cancer by designing short RNAi specific for IQGAP1 gene in HCC cells in vivo and construct new vectors suitable for this purpose. We assigned mice into three groups: the first negative control group (NC) was injected with saline, the second control group was injected with shRNA (shNC), the third positive control group was injected with diethylnitrosamine (DENAA), and the fourth group was treated with the IQGAP1-shRNA prior to its exposure to DENA.

Results: Our results revealed that the treated group with IQGAP1-shRNA with DENA developed very few cases of hepatic cancer when compared with the positive control group. The positive control group exhibited significant increases in the liver function level as well as a decrease in serum albumin levels when compared to both the treated and the negative control groups. The altered levels of the serum α-fetoprotein as well as of the tumor necrosis factor-alpha, and interleukin-4 in DENA-treated mice were significantly ameliorated by IQGAP1-shRNA administration. Flow cytometer analyses have indicated that the silencing of IQGAP1 cannot significantly modulate DENA-induced apoptosis in the circulating blood cells. Moreover, the elevated mRNA expression levels of IQGAP1, IQGAP3, KRas, HRas, interleukin-8, nuclear factor kappa B, caspase-3, caspase-9 and Bcl-2, were significantly decreased by the IQGAP1-shRNA treatment. However, the IQGAP2, DR4, DR5, p53 and BAX genes were found to be significantly up-regulated post-therapy. In agreement with these findings, IQGAP1-shRNA was able to modulate the DENA-induced histological changes in the mice liver which were represented by severe necrosis and hydropic degenerative changes.

Conclusion: Our study revealed that IQGAP1-shRNA was able to preserve hepatocyte integrity and the liver histological architecture through the regulation of the expression of IQGAPs, Ras, TRAILs and IL-8 receptors, as well as of pro-apoptotic and anti-apoptotic genes. Therefore, the silencing of IQGAP1 could be part of a promising therapeutic strategy against hepatic cancer.

Keywords: IQGAP1; in vivo; liver cancer; mouse; shRNA.

Figure 1

Histopathology changes. (A) A photomicrograph of a section of control liver showing the normal structure of the hepatic lobule. The central vein lies at the center of the lobule and intact with the hepatocytes with strong stained eosinophilic granulated cytoplasm, and distinct nuclei. In between the strands of hepatocytes, the hepatic sinusoids are shown (B) A micrograph of liver section from mice that given DENA only showing complete destruction of the sinusoidal architectural pattern. Hepatocellular carcinoma: a mononuclear inflammatory cell infiltrate extends from portal areas and disrupts the limiting plate of hepatocytes, which are undergoing apoptosis (C) A micrograph of a section of positive-control liver mice that given DENA and treated with IQGAP1-shRNAL plasmid showing improvements in the structure of liver that appeared more or less like normal one. Complete arrangement of the sinusoidal architectural patterns, no mononuclear inflammatory cells are shown (H & E stain). (D) Relative liver weight (%) to body weight. (E) Histopathology changes in normal, induced, and treated tissues.a indicated high significantly difference at P <0.05 between treated mouse and normal control.

Figure 2

The RT-qPCR validation of mRNA expression for IQGAP1, IQGAP2, IQGAP3, KRas, HRas, IL-8, CXCR3, caspase-3, caspase-9, Bcl-2, BAX, DR4 (TRAIL1), DR5 (TRAIL2), DecoyR1, DecoyR2, p53, and NF-kB (A-F) in liver tissue among groups of control, DENA and IQGAP1-shRNA plasmid. NC; normal control and ShNC; IQGAP1-shRNA control. ^a indicated high significantly difference at P <0.01 between DENA-induced mouse and normal control. ^b indicated significantly difference at P <0.05 between DENA-induced mouse and normal control. ^c indicated significantly difference at P <0.05 between IQGAP1-shRNA treated group and DENA-induced mouse. Error bars represents standard error of mean (SEM). Means comparisons were performed by using One-Way ANOVA test.

Figure 3

Protein expression of both IQGAP1 and IL-8 in liver tissues by western blot. ^a indicated high significantly difference at P <0.01 between DEN-induced mouse and normal control. ^c indicated significantly difference at P <0.05 between IQGAP1-shRNA treated group and DEN-induced mouse. Error bars represents standard error of mean (SEM). Means comparisons were performed by using One-Way ANOVA test.

Figure 4

Flow cytometry analysis using Annexin V FITC and propidium iodide (PI) for apoptosis measurements. Apoptosis of the untreated (A), DENA injected (B), and IQGAP1-shRNA vector treated circulating cells in the blood stream were measured in mice (C). Charts in each column represent cells underwent early, late apoptosis, and necrosis.