Expression and significance of HMGB1, TLR4 and NF-κB p65 in human epidermal tumors

Abstract

Background: High mobility group protein box 1 (HMGB1) is a DNA binding protein located in nucleus. It is released into extracellular fluid where it acts as a novel proinflammatory cytokine which interacts with Toll like receptor 4 (TLR4) to activate nuclear factor-κB (NF-κB). This sequence of events is involved in tumor growth and progression. However, the effects of HMGB1, TLR4 and NF-κB on epidermal tumors remain unclear.

Methods: Human epidermal tumor specimens were obtained from 96 patients. Immunohistochemistry was used to detect expression of HMGB1, TLR4 and NF-κB p65 in human epidermal tumor and normal skin specimens. Western blot analysis was used to detect the expression of NF-κB p65 in epithelial cell nuclei in human epidermal tumor and normal tissues.

Results: Immunohistochemistry and western blot analysis indicated a progressive but statistically significant increase in p65 expression in epithelial nuclei in benign seborrheic keratosis (SK), precancerous lesions (PCL), low malignancy basal cell carcinoma (BCC) and high malignancy squamous cell carcinoma (SCC) (P <0.01). The level of extracellular HMGB1 in SK was significantly higher than in normal skin (NS) (P <0.01), and was higher than in SCC but without statistical significance. The level of TLR4 on epithelial membranes of SCC cells was significantly higher than in SK, PCL, BCC and NS (P <0.01). There was a significant positive correlation between p65 expression in the epithelial nuclei and TLR4 expression on the epithelial cell membranes (r = 0.3212, P <0.01).

Conclusions: These findings indicate that inflammation is intensified in parallel with increasing malignancy. They also indicate that the TLR4 signaling pathway, rather than HMGB1, may be the principal mediator of inflammation in high-grade malignant epidermal tumors. Combined detection of p65 in the epithelial nuclei and TLR4 on the epithelial membranes may assist the accurate diagnosis of malignant epidermal tumors.

Figure 1

Expression of HMGB1 in epidermal tumors and normal skin by IHC EnVision (magnification × 400). (a) to (i). Positive expression of HMGB1 was located in the nucleus, cytoplasm, cell, and (or) intercellular space after stimulation of inflammation or cell necrosis. The red arrow shows HMGB1 expression in the epithelial intercellular space, the black arrow shows positive HMGB1 expression in epithelial cell nuclei, the orange arrow shows HMGB1 expression in the epithelial cell cytoplasm, the blue arrow shows HMGB1 expression in an inflammatory cell, and the green arrow shows HMGB1 expression in a vascular endothelial cell. (j). **P < 0.01 of HMGB1 in epithelial intercellular spaces in SK as compared in NS. (k). **P < 0.01 of HMGB1 in epithelial cell nuclei in NS and SK as compared in SCC. (l). **P <0.01 as compared HMGB1 in inflammatory cells in NS. The error bars show the standard error of the mean (SEM).

Figure 2

Expression of TLR4 in epidermal tumors and normal skin by IHC EnVision (magnification × 400). (a) to (e). Positive expression of TLR4 was located on the epithelial membrane (purple arrow). (f). **P <0.01 as compared TLR4 on epithelial cell membranes in SCC. The error bars represent the standard error of the mean (SEM).

Figure 3

Expression of p65 in epidermal tumors and normal skin by 96 IHC EnVision (magnification × 400). (a) to (g). Positive expression of p65 was located in the cytoplasm, cell, and (or) nucleus after activation. The black arrow shows expression of p65 in the epithelial nucleus, the orange arrow shows p65 expression in the epithelial cytoplasm, the blue arrow shows p65 expression in an inflammatory cell and the green arrow shows p65 expression in a vascular endothelial cell. (h). **P < 0.01 as compared with p65 in the epithelial nuclei in different groups (i). **P < 0.01 as compared p65 in inflammatory cell nuclei in SK. The error bars represent the standard error of the mean (SEM). (j). Western blot detection of p65 expression in epithelial nuclei, which increased gradually from NS, SK, PCL, BCC, and to SCC. Histone H3 was used as a loading control.

Figure 4

Expression of HSP70 in epidermal tumors and normal skin by IHC EnVision (magnification × 400). (a) to (e). Positive expression of HSP70 was located in the epithelial intercellular space as shown by the red arrows. (f). **P <0.01 as compared HSP70 in the epithelial intercellular spaces in SCC. The error bars represent the standard error of the mean (SEM).

Figure 5

Correlation analysis of IHC. (a)-(f). The line-charts with SEM showing expression of p65 in epithelial cell nuclei and other mediators by Spearman's correlation analysis. (g). The correlation coefficients of Spearman rho as compared p65 in epithelial nuclei. (a) and (g). p65 in epithelial nuclei as compared HMGB1 in the epithelial cell nuclei ((r = -0.3264, **P <0.01). (b) and (g). p65 in epithelial nuclei as compared p65 in the inflammatory nuclei (r = -0.2496, *P <0.05). (c) and (g). p65 in epithelial nuclei as compared TLR4 on the epithelial cell membranes (r = 0.3212, **P <0.01). (d) and (g). p65 in epithelial nuclei as compared HSP70 in the epithelial intercellular spaces (r = 0.2844, **P <0.01). (e) and (g). p65 in epithelial nuclei as compared HMGB1 in the epithelial intercellular spaces (r = -0.1641, P > 0.05). (f) and (g). p65 in epithelial nuclei as compared HMGB1 in the inflammatory cells (r = -0.0452, P > 0.05).