Pathogenesis of FOLFOX induced sinusoidal obstruction syndrome in a murine chemotherapy model

Abstract

Background & aims: Sinusoidal obstruction syndrome (SOS) following oxaliplatin based chemotherapy can have a significant impact on post-operative outcome following resection of colorectal liver metastases. To date no relevant experimental models of oxaliplatin induced SOS have been described. The aim of this project was to establish a rodent model which could be utilised to investigate mechanisms underlying SOS to aid the development of therapeutic strategies.

Methods: C57Bl/6 mice, maintained on a purified diet, were treated with intra-peritoneal FOLFOX (n=10), or vehicle (n=10), weekly for five weeks and culled one week following final treatment. Sections of the liver and spleen were fixed in formalin and paraffin embedded for histological analysis. The role of oxidative stress on experimental-induced SOS was determined by dietary supplementation with butylated hydroxyanisole and N-acetylcysteine.

Results: FOLFOX treatment was associated with the development of sinusoidal dilatation and hepatocyte atrophy on H&E stained sections of the liver in keeping with SOS. Immunohistochemistry for p21 demonstrated the presence of replicative senescence within the sinusoidal endothelium. FOLFOX induced endothelial damage leads to a pro-thrombotic state within the liver associated with upregulation of PAI-1 (p<0.001), vWF (p<0.01) and Factor X (p<0.001), which may contribute to the propagation of liver injury. Dietary supplementation with the antioxidant BHA prevented the development of significant SOS.

Conclusions: We have developed the first reproducible model of chemotherapy induced SOS that reflects the pathogenesis of this disease in patients. It appears that the use of antioxidants alongside oxaliplatin based chemotherapy may be of value in preventing the development of SOS in patients with colorectal liver metastases.

Keywords: ALP; ALT; AST; BHA; CRLM; CXCL1/2; Chemotherapy induced liver injury; Colorectal liver metastases; GAPDH; H&E; HPF; IL-6; MCP1; N-acetylcysteine; NAC; NAD(P)H dehydrogenase 1; NQO1; NRF2; Oxaliplatin; PAI-1; PAR 1/2; PCNA; SOS; STAT3; Sinusoidal obstruction syndrome; TXN1; VEGF-A/B/C; VEGFR-1/2; alanine aminotransferase; alkaline phosphatase; aspartate aminotransferase; butylated hydroxyanisole; chemokine (C-X-C motif) ligand 1/2; colorectal liver metastases; gyceraldehyde 3-phosphate dehydrogenase; haematoxylin and eosin; high powered field; i.p.; interleukin 6; intraperitoneal; monocyte chemotactic protein-1; nuclear factor (erythroid-derived 2)-like 2; phosphorylated form of the H2A histone family, member X; plasminogen activatior inhibitor 1; proliferating cell nuclear antigen; protease activated receptor 1/2; signal transducer and activator of transcription 3; sinusoidal obstruction syndrome; thioredoxin 1; vWF; vascular endothelial growth factor A/B/C; vascular endothelial growth factor receptor ½; von Willebrand factor; γH2AX.

Fig. 1

Endothelial senescence in FOLFOX induced SOS. (A) Mice maintained on a purified diet and treated with intraperitoneal FOLFOX for 5 weeks (n = 9 per group) demonstrate histological changes of SOS on H&E stained sections of the liver. (B) To identify potential biological processes implicated in the pathogenesis of FOLFOX induced SOS, a microarray was performed using mRNA isolated from 6 animals in each group, a heat map of which is shown. (B) Cellular senescence was identified as a key mechanism in FOLFOX induced SOS, with upregulation of p21^Cip1 at both a transcript level (C; n = 8 animals per group) and protein level (D; representative blot of n = 6 per group). (E) Immunohistochemistry revealed that p21Cip1 staining is limited to endothelial cells at the site of sinusoidal injury (yellow arrows). (F) Densitometry (n = 9 animals per group) confirmed an increased intensity of p21^Cip1 staining. (G) It is likely that endothelial senescence is driven by p53 with Western blot of whole liver protein extracts revealing increased phosphorylation of this transcription factor at serine 15 (representative blot of n = 6 per group). ^∗p <0.05; ^∗∗∗p <0.001. (This figure appears in colour on the web.)

Fig. 2

Cytokine response to sinusoidal injury. (A) Microarray suggested upregulation of the senescence associated chemokine CXCL1 in the liver of FOLFOX treated mice, which was confirmed by qRT-PCR (n = 8 animals per group). (B) Furthermore, CXCL1 was demonstrated to be elevated within the serum of mice with FOLFOX induced SOS (n = 9 animals per group). (C) qRT-PCR demonstrated upregulation of other senescence associated inflammatory mediators including CXCL2, MCP-1, and IL-6 (n = 8 animals per group). (D) IL-6 is more typically described as a pro-proliferative cytokine implicated in liver regeneration where it signals through activation of the transcription factor STAT3. Microarray and subsequent qRT-PCR demonstrated increased expression of this transcription factor in FOLFOX treated animals (n = 8 animals per group). Western blot demonstrated activation of this transcription factor by phosphorylation (E; representative blot of n = 6 per group), which immunohistochemistry revealed to be occurring predominantly in hepatocytes around portal tracts (F). Densitometry confirmed the increase in phosphorylated STAT3 seen on Western blot (F; n = 9 animals per group). ^∗p <0.05; ^∗∗p <0.01; ^∗∗∗p <0.001. (This figure appears in colour on the web.)

Fig. 3

Oxidative stress in FOLFOX induced SOS. (A) To assess the role of oxidative stress in the development of SOS, the experiment was repeated, but this time supplementing the diet with 0.7% BHA with the purified diet serving as a control. Supplementation with 0.7% BHA prevented the development of sinusoidal injury. (B) In keeping with this, there was decreased expression of p21^Cip1 and PAI-1 transcript (n = 5 per group, n = 4 in the FOLFOX and BHA group) in the livers of these animals. (C) The reduction in p21 expression was confirmed at a protein level by Western blot (representative blot of n = 2 per group). (D) FOLFOX induced SOS is associated with diminished expression of the antioxidant transcription factor NRF2 which is prevented when BHA is administered (representative blot of n = 2 per group). (E) BHA supplementation is associated with increased expression of the NRF2-regulated antioxidant genes TXN1 and NQO1 (n = 5 per group, n = 4 in the FOLFOX and BHA group). ^∗p <0.05; ^∗∗p <0.01; n.s, not significant. (This figure appears in colour on the web.)

Fig. 4

Angiogenesis in FOLFOX induced SOS. (A) FOLFOX induced SOS is associated with upregulation of the angiogenic factors VEGF-A and VEGF-C, but not VEGF-B, at the transcript level within the liver (n = 8 per group). (B) This is reflected in an increase in serum VEGF-A levels in mice with FOLFOX induced SOS (n = 9 per group). (C) In addition there is increased expression of the VEGF receptors VEGFR-1 and VEGFR-2 within the liver of FOLFOX treated animals (n = 8 per group). Previous reports have suggested that, in the context of SOS, VEGF mediated signalling results in phosphorylation of JNK leading to increased expression of MMP-9 . In support of a role for this mechanism in FOLFOX induced SOS, (D) Western blot revealed increased phosphorylation of JNK within the liver of FOLFOX treated animals (representative blot of n = 6 per group) with (E) an associated increase in MMP-9 transcript (n = 8 per group). (F) BHA treatment protects against the FOLFOX induced increase in both VEGF-A and VEGFR-1 transcript expression (n = 5 per group, n = 4 in the FOLFOX and BHA group). ^∗∗p <0.01; ^∗∗∗p <0.001.