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Hydrocortisone Suppresses Early Paraneoplastic Inflammation And Angiogenesis To Attenuate Early Hepatocellular Carcinoma Progression In Rats

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Hydrocortisone Suppresses Early Paraneoplastic Inflammation And Angiogenesis To Attenuate Early Hepatocellular Carcinoma Progression In Rats

Xiaolong Liu et al. Onco Targets Ther.

Abstract

Background: Inflammation is implicated in both hepatic cirrhosis development and hepatocellular carcinogenesis, and treatment with long-acting glucocorticoid dexamethasone prevented liver carcinogenesis in mice. However, it is unclear whether glucocorticoids have anti-inflammatory effect on hepatocellular carcinoma (HCC) and if short-acting glucocorticoids (with fewer adverse effects) inhibit paraneoplastic inflammation and HCC progression.

Methods: To investigate whether different types of anti-inflammatory agents attenuate HCC progression, the current study compared effects of treatments with hydrocortisone (a short-acting glucocorticoid) or aspirin on HCC progression. HCC was induced in diethylnitrosamine-treated rats which were randomly divided into 4 groups (n=8), respectively receiving orally once daily vehicle, glucuronolactone, glucuronolactone+hydrocortisone, and glucuronolactone+aspirin. Diethylnitrosamine (DEN) was given to rats in drinking water (100mg/L) to induce HCC. At weeks 12 and 16 post-induction, effects were compared on HCC nodule formation, microvessel density, and macrophage infiltration, and levels of paraneoplastic protein expression of tumor necrosis factor (TNF)-α, p38 mitogen-activated protein kinase (p38), phosphorylated p38 (p-p38), nuclear factor (NF)-κB, interleukin (IL)-10, hepatocyte growth factor (HGF), transforming growth factor (TGF)-β1 and vascular endothelial growth factor (VEGF).

Results: Compared to the model and glucuronolactone alone groups, HCC nodule number and microvessel density in the glucuronolactone+hydrocortisone group were significantly lower at week 12. At week 12 but not week 16, significantly lower levels of macrophages, TNF-α, p-p38, NF-κB, IL-10, HGF, TGF-β1 and VEGF were observed in the paraneoplastic tissue of the glucuronolactone+hydrocortisone group when compared with the control and glucuronolactone groups.

Conclusion: The results suggest that hydrocortisone treatment reduces macrophage polarization, expression of inflammatory and anti-inflammatory cytokines, and angiogenesis in paraneoplastic tissue, and attenuates early HCC progression. Although hydrocortisone did not have attenuation effect on advanced solid tumor, the current study shows the potential benefits and supports potential clinical use of hydrocortisone in attenuating early progression of HCC, which is through suppressing paraneoplastic inflammation and angiogenesis.

Keywords: angiogenesis; aspirin; hepatocellular carcinoma; inflammation; macrophage; steroid.

Conflict of interest statement

The authors report no conflicts of interest in this work.

Figures

Figure 1
Figure 1
HCC nodules and pathological analyses at 12 and 16 weeks after HCC induction and treatment with glucuronolactone (GLU) or together with hydrocortisone (HYD) or aspirin (ASP) when compared with the model group (Control). (A) Obvious liver cirrhosis and HCC nodules found in gross examination of specimens in the Control group at Week 12 and in all groups at Week 16. (B) Fewer HCC nodules in the GLU+HYD and GLU+ASP groups than the Control and GLU groups at Week 12. (C) At Week 12, all groups developed HCC and obvious cirrhosis, but Grade II lesions were found only in the Control and GLU groups. In Week 16, all groups developed Grade I and II HCC (hematoxylin and eosin, 400×). Red arrows indicate HCC lesions. **P<0.01 versus the Control group; ††P < 0.01 versus the GLU group.
Figure 2
Figure 2
Effects of treatment with glucuronolactone (GLU) or together with hydrocortisone (HYD) or aspirin (ASP) compared with the model group (Control) on microvessel densities and protein expression levels of vascular endothelial growth factor (VEGF) in paraneoplastic tissue at 12- or 16-weeks post-HCC induction. (A) CD34-positive vessels (red arrows) in paraneoplastic tissue (200×). (B) Comparison of CD34-positive vessels per measured area. (C–D) Western blot analyses for VEGF: representative blots and quantitative results. 1. Control; 2. GLU; 3. GLU + HYD; 4. GLU +ASP. Results are means ± SD, n=3–5 animals in each group. **P<0.01 versus the Control group; ††P < 0.01 versus the GLU group; ‡‡P < 0.01 versus the GLU+ASP group.
Figure 3
Figure 3
Effects of treatment with glucuronolactone (GLU) or together with hydrocortisone (HYD) or aspirin (ASP) compared with the model group (Control) on numbers of CD68 or CD206-positive macrophages in paraneoplastic tissue at Week 12 or 16 post-HCC induction. (A) CD68-positive cells (red arrows) in paraneoplastic tissue (200×) and comparison of positive cell density (cells per field, 200×). (B) CD206-positive cells (red arrows) in paraneoplastic tissue (200×) and comparison of positive cell density (cells per field, 200×). Values are expressed as means ± SD (n=3–5 animals in each group). **P<0.01 versus the Control group; ††P < 0.01 versus the GLU group; ‡‡P < 0.01 versus the GLU+ASP group.
Figure 4
Figure 4
Effects of treatment with glucuronolactone (GLU) or together with hydrocortisone (HYD) or aspirin (ASP) compared with the model group (Control) on protein expression levels of inflammatory and anti-inflammatory cytokines and inflammation regulatory factors in hepatic paraneoplastic tissue at the ends of weeks 12 and 16. (A) Representative Western blots for tumor necrosis factor-α (TNF-α), p38 mitogen-activated protein kinase (p38 MAPK, p38), phosphorylated (p)-p38, nuclear factor-κB (NF-κB, P65 subunit), and interleukin-10 (IL-10). Lanes 1, Control; 2, GLU; 3, GLU + HYD; 4, GLU + ASP. (B–F) Quantitative results of Western blots. Values are expressed as means ± SD (n=3–5 animals in each group). **P<0.01 versus the Control group; ††P < 0.01 versus the GLU group; ‡‡P < 0.01 versus the GLU+ASP group.
Figure 5
Figure 5
Effects of treatment with glucuronolactone (GLU) or together with hydrocortisone (HYD) or aspirin (ASP) compared with the model group (Control) on protein expression levels of transforming growth factor-β (TGF-β1) and hepatocyte growth factor (HGF) in hepatic paraneoplastic tissue at Week 12 and 16 post-HCC induction. (A) Representative Western blot assay for TGF-β1 and HGF. Lanes 1, Control; 2, GLU; 3, GLU + HYD; 4, GLU + ASP. (B) Quantitative results of Western blots. Values are expressed as means ± SD (n=3–5 animals in each group). **P<0.01 versus the Control group; ††P < 0.01 versus the GLU group; ‡‡P < 0.01 versus the GLU+ASP group.
Figure 6
Figure 6
The proposed mechanism for hydrocortisone treatment-induced attenuation of early-stage progression of HCC by suppressing macrophage-regulated chronic inflammation and angiogenesis in paraneoplastic tissue. Immune cells, stromal cells and HCC colonies in early-stage compose of a cascade initiated by exogenous chemicals (eg, DEN) or viral damage. The immune modulator, macrophage, is located at the key point of the cascade. Inflammatory cytokine, TNF-α, from activated macrophages induces overexpression of HGF and TGF-β1 from stromal cells and fibroblasts which in turn participate in the carcinoma progression. HGF has the anti-inflammatory action by promoting M2 polarization. Upregulated TGF-β1 and NF-κB promote formation of microvessels in cancerization area by increasing expression of VEGF. HGF and TGF-β1 also directly promote HCC by activating HGF/C-met and TGF-β/Smad pathways. Thus, the inflammatory cascade increases densities of the M2 polarized macrophages and microvessels (MVD) and finally promotes HCC progression. Regulation of macrophage-mediated inflammation by hydrocortisone suppresses the downstream events and inhibits formation of the HCC colonies in the early stage.

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