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. 2013 Jan;58(1):257-64.
doi: 10.1007/s10620-012-2325-y. Epub 2012 Aug 24.

Antifibrotic activity of sorafenib in experimental hepatic fibrosis: refinement of inhibitory targets, dosing, and window of efficacy in vivo

Feng Hong  1 , Hsini Chou, Maria Isabel Fiel, Scott L Friedman
Affiliations

Antifibrotic activity of sorafenib in experimental hepatic fibrosis: refinement of inhibitory targets, dosing, and window of efficacy in vivo

Feng Hong et al. Dig Dis Sci. 2013 Jan.

Abstract

Background: Sorafenib, which is approved for treatment of HCC, has also shown promising antifibrotic activity, and therefore refinement of its dosing requirements and window of efficacy are important goals prior to antifibrotic clinical trials.

Aim: The purpose of this study was to determine the minimal effective dose and optimal timing of sorafenib therapy in cultured human stellate cells and in rats with experimental hepatic fibrosis.

Methods: Effects of sorafenib were assessed in a human stellate cell line (LX-2). In vivo, rats were treated for 8 weeks with TAA three times per week (150 mg/kg IP), and with either PBS or sorafenib administered daily at doses of 1.25, 5 or 7 mg/kg/day gavage either at the beginning of TAA administration for 8 weeks, during weeks 4-8, or from weeks 8-12.

Results: Sorafenib treatment significantly inhibited LX-2 proliferation by >75% (7.5 or 15 μM). Treatment with 7.5-μM sorafenib for 12 h markedly inhibited expression of TGFβ1, TIMP-1, collagen I, and MMP2 mRNAs, but not of β-PDGFR or type I TGFβR. In vivo, sorafenib significantly inhibited liver fibrosis when started concurrently with TAA and during weeks 4-8 with TAA. In contrast, there was no significant effect of sorafenib on fibrogenic gene expression or fibrosis when begun after cirrhosis was already established.

Conclusion: Sorafenib is anti-proliferative and antifibrotic towards human HSCs in culture, and is a potent antifibrotic agent in TAA-induced hepatic fibrosis in rats. The drug is effective at relatively low doses at the early stage of liver fibrosis, but is not effective when cirrhosis is already established.

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Figures

Figure 1
Figure 1. Sorafenib inhibits HSC proliferation
LX-2 cells were incubated with 7.5 or 15 µM of sorafenib for 24 and 48 hours. Cell proliferation was assessed via 3H-thymidine was added 4 hour prior to assessment of incorporation. Sorafenib significantly inhibited DNA synthesis at both concentrations for 12 and 24 hours. Values are means ± SD (n=3). *p<0.001 in comparison with control group.
Figure 2
Figure 2. Sorafenib induces apoptosis in LX-2 cells
LX-2 cells were treated with 15 µM of sorafenib- or vehicle-treated and DNA was isolated. At 48 hours, DNA fragments of ~150 kb were observed in cells treated with sorafenib.
Figure 3
Figure 3. Sorafenib affects cell viability in LX-2 cells
LX-2 cells were treated with 7.5 and 15 µM for 12 and 24 hours. Cell viability was assessed using alamarBlue® and fluorescence was measured. Cell viability was reduced up to 70% at 24 hours and up to 80% at 48 hours at both concentrations.
Figure 4
Figure 4. Sorafenib down-regulates expression of fibrogenic genes in LX-2 cells
LX-2 cells were incubated with 7.5 µM of sorafenib for 12, 24, and 48 hours. Sorafenib reduced fibrogenic mRNA expression of Collα1, TIMP1, MMP2. Expression of α-SMA, β-PDGFR and TGFβR1 mRNA were not affected to some extent. At 48 hours, there was a slight but significant decrease in mRNA expression for TGFβR1. n=3. *p<0.001.
Figure 5
Figure 5. Collagen 1 but not alpha smooth muscle actin protein expression is reduced in LX-2 and human primary cells treated with sorafenib
Total protein was extracted from LX-2 (5a) or primary human stellate cells (5b) treated with 7.5 and 15 µM for 24 hours. A reduction in Collagen 1 protein is observed at higher concentrations of 15 µM. There was a slight reduction in α-SMA protein detected in primary human stellate cells. GAPDH is used as a reference gene.
Figure 6
Figure 6. Down-regulation of fibrogenic mRNA expression by sorafenib in TAA-induced hepatic fibrosis in rats
Real-time PCR was performed to quantify expression of key genes associated with fibrogenesis. Consistent down-regulation of TGF-β1, a-SMA, collagen I were observed while MMP2, TIMP1 and β-PDGFR were down regulated at lower dosages. MMP14 was not affected significantly. n=3. *p<0.001.
Figure 7
Figure 7. Liver fibrosis was greatly reduced in rat liver tissue in ‘therapeutic’ treatment group
7a) Histology of rat liver with H&E and Sirius Red in representative liver section from animals in Group 3 at 100X and 200X magnifications. Relative fibrosis area (expressed as a percentage of total liver area) was assessed by analyzing 36 Sirius red-stained liver sections per animal. Each field was acquired at 100X magnification and analyzed using a computerized Bioquant morphometry system. There was a statistically significant difference in fibrosis in the livers of animals treated with 7b) 1.25 mg/kg/day 7c) 5 mg/kg/day and 7d) 7 mg/kg/day of sorafenib. *p <0.001
Figure 7
Figure 7. Liver fibrosis was greatly reduced in rat liver tissue in ‘therapeutic’ treatment group
7a) Histology of rat liver with H&E and Sirius Red in representative liver section from animals in Group 3 at 100X and 200X magnifications. Relative fibrosis area (expressed as a percentage of total liver area) was assessed by analyzing 36 Sirius red-stained liver sections per animal. Each field was acquired at 100X magnification and analyzed using a computerized Bioquant morphometry system. There was a statistically significant difference in fibrosis in the livers of animals treated with 7b) 1.25 mg/kg/day 7c) 5 mg/kg/day and 7d) 7 mg/kg/day of sorafenib. *p <0.001
Figure 7
Figure 7. Liver fibrosis was greatly reduced in rat liver tissue in ‘therapeutic’ treatment group
7a) Histology of rat liver with H&E and Sirius Red in representative liver section from animals in Group 3 at 100X and 200X magnifications. Relative fibrosis area (expressed as a percentage of total liver area) was assessed by analyzing 36 Sirius red-stained liver sections per animal. Each field was acquired at 100X magnification and analyzed using a computerized Bioquant morphometry system. There was a statistically significant difference in fibrosis in the livers of animals treated with 7b) 1.25 mg/kg/day 7c) 5 mg/kg/day and 7d) 7 mg/kg/day of sorafenib. *p <0.001
Figure 7
Figure 7. Liver fibrosis was greatly reduced in rat liver tissue in ‘therapeutic’ treatment group
7a) Histology of rat liver with H&E and Sirius Red in representative liver section from animals in Group 3 at 100X and 200X magnifications. Relative fibrosis area (expressed as a percentage of total liver area) was assessed by analyzing 36 Sirius red-stained liver sections per animal. Each field was acquired at 100X magnification and analyzed using a computerized Bioquant morphometry system. There was a statistically significant difference in fibrosis in the livers of animals treated with 7b) 1.25 mg/kg/day 7c) 5 mg/kg/day and 7d) 7 mg/kg/day of sorafenib. *p <0.001
Figure 8
Figure 8. Sorafenib inhibits expression of α-SMA protein in TAA-treated rat liver
Expression of a-SMA protein in the liver was analyzed by western blot analysis in rats treated with sorafenib at; a) the beginning of TAA treatment, Group 2 and b) during weeks 4–8, Group 3. a-SMA protein expression was reduced significantly compared with rats treated with vehicle.
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