New Approach about the Signaling Crosstalk between IQGAPs/ NF- κB/IL-8 and PDCD5/p53/TRAIL Pathways that Modulate Malignant Transformation in Hepatocellular Carcinoma

Abstract

Objective: We aimed to investigate the signalling crosstalk of TNF-related apoptosis-inducing ligand, TRAIL death receptors, tumour protein p53, and programmed cell death (PDCD5) with IQGAPs. Also, we targeted the crosstalk between IQGAPs genes with decoy receptors, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), and interleukins -8 (IL-8) and its receptor genes in a designed model of hepatocellular carcinoma induced in male Balb/c mice.

Methods: The presence of HCC was confirmed by histological and morphological alterations. In parallel to the incidence of hepatic cancer, we found lung, heart, and kidney cancer after treatment with DEN.

Results: Our results show that the expression of mRNA of IQGAP1, TRAIL decoy receptors, NF-κB, and IL-8 genes was elevated in hepatocellular carcinoma, as compared to normal liver tissue, while their expression was further up-regulated by increasing the dose of diethylnitrosamine. The expression of IQGAP2, TRAIL death receptors, p53, and PDCD5 was significantly down-regulated in HCC (p˂0.05). For confirmation of gene expression, protein levels of both IQGAP1 and P53 were measured by western blot analysis, which showed that diethylnitrosamine enhanced protein expression of IQGAP1 and diminished that of p53.

Conclusion: IQGAPs have a direct signaling relationship with p53, IL-8, and TRAIL family. This interaction is recognized as a key signalling pathway for hepatocellular carcinogenesis.

Keywords: DEN; Hepatocellular carcinoma; IL-8; IQGAPs; TRAIL.

Figure 1

Histopathological Examination. A, Normal looking of controlled mouse hepatocytes (H&E stain,100x); B, Steatohepatitis of mouse hepatocytes treated with DEN 50 mg/kg once weekly i.p for 4 weeks showing fatty degeneration and scattered inflammatory cell infiltrate (H&E stain, 200x); C, Hepatocytes from another rat treated with DEN 75 mg/kg once weekly i.p for 4 weeks show steatohepatitis, fatty degeneration and scattered inflammatory cell infiltrate (H&E stain, 200x); D, Liver of mice treated with DEN 75 mg/kg i.p once weekly for 8 weeks shows HCC that comprises sheets and nests of large tumor cells with pleomorphic nuclei, prominent nucleoli, abundant granular eosinophilic cytoplasm, scattered Mallory bodies (dense eosinophilic cytoplasmic structures) and distinct cell borders (H&E stain, 200x).

Figure 2

a, A micrograph of section of liver of control mouse shows the architecture of the hepatic lobule. The hepatocytes showed strongly eosinophilic granulated cytoplasm, and round nuclei with peripherally dispersed chromatin and prominent nucleoli. Between the strands of hepatocytes the hepatic sinusoids are often seen. The Kupffer cells are satellite and site on the sinusoidal surface of the endothelial cell, projecting in the vascular space (H & E. stain, Scale bar: 5 µm); b, A micrograph of section of liver of DEN mouse liver showing hepatocellular carcinoma. It shows trabeculae of malignant hepatocytes separated by sinusoids (H & E. stain, Scale bar: 5 µm).

Figure 3

DEN Regulates Expression of IQGAPs, DcR1 and DcR2, DR4, DR5 and NF-ng Genes in Liver Tissues. a, mRNA amounts of IQGAP1 and 2 were quantified using RT-PCR and normalized to GAPDH housekeeping gene as in Methods; b,) mRNA amounts of DcR1 and DcR2 were quantified using RT-PCR and normalized to GAPDH housekeeping gene as in Methods. Statistical analysis was performed using ANOVA followed by t-test. Each value indicates the mean ± S.E.M of ten animals. p < 0.05 was considered significant (*) compared to the corresponding normal control (0 mg/kg) group

Figur 4

a, DEN suppresses expression of IL-8, CXCR1 and CXCR2 genes in liver tissues; b and c, DEN upregulates IQGAP1 and downregulates p⁵³ expression in DEN-treated liver cells. Balb/c mice were treated with noted concentrations of DEN 4 weeks. Liver tissues were homogenized and total protein was prepared as described in Methods. IQGAP1 and p⁵³ protein levels were determined by Western blot analysis (upper panel). Representative densitometric quantifications of IQGAP1 and p⁵³ protein levels are shown in the lower two panels. Values are presented as means ± standard error of the mean.*p<0.05, compared with control (NC).

Figure 5

Flow Cytometry Analysis Using Annexin V FITC and Propidium Iodide (PI) for Apoptosis Measurements. Blood cells were treated with DEN. Early, late apoptosis, and necrosis were detected in the stained cells with Annexin V and PI