The Food and Drug Administration (FDA) approved the kinase inhibitor sorafenib for treatment of advanced renal cell (2005) and hepatocellular carcinomas (2007). Regorafenib, as approved in 2012 for colorectal cancer. Although these two compounds are closely related, their clinical adverse events differ. For example, regorafenib may produce fatal hepatotoxicity, a complication that has not been linked to sorafenib [6
This study employed structural (hepatocyte histology and EM) and functional (hepatocyte respiration, ATP, caspase activity, and urea synthesis) surrogate biomarkers to investigate the toxic effects of sorafenib and regorafenib in vitro
. The measured functional biomarkers were similar in treated and untreated liver specimens (Table 1). The ultrastructural changes, most notably loss of rER integrity and detachment of ribosomes, were more evident with regorafenib than sorafenib (Figures 2A and B). At 0 h, the untreated liver specimen showed well preserved hepatocyte architecture. At 4 h, the hepatocytes demonstrated only minimal
mitochondrial distension and rER disintegration. Sorafenib treatment (2.5 μM for 4 h) produced mild
mitochondrial distension and rER disintegration. The rER changes were more noticeable with regorafenib treatment (2.5 μM for 4 h), Figure 2A. Higher sorafenib dose (50 μM for 3 h) showed only mitochondrial distension with relatively preserved rER. Significant disruption of the rER and focal detachments of the ribosomes were evident in the tissue treated with 50 μM regorafenib at 3 h (Figure 2B).
Biologic activities of sorafenib and regorafenib can be demonstrated in vitro
within a few hours of exposure to 0.1-10 μM of the drugs [8
]. The cytotoxicity is cell specific and includes alterations in multiple signaling pathways, execution of apoptosis, induction of ER stress, and inhibition of protein synthesis. At 5 to 50 μM, for example, sorafenib inhibited the proliferation of hepatocellular carcinoma cell lines; the degree of inhibition was dependent on pERK expression [10
]. Exposure of human leukemia cells to 10 μM sorafenib produced cytotoxicity that involved inducing ER stress and generation of ROS [12
]. At 3 to 20 μM, sorafenib induced apoptosis in melanoma cells in 4 h, mainly by nuclear translocation of the apoptosis-inducing factor [14
]. In cell lines, apoptosis is induced via caspase dependent (e.g., caspase-2 and caspase-4 processing) and independent (e.g., nuclear translocation of the apoptosis-inducing factor) pathways.Regorafenib also inhibited the proliferation of human hepatocellular carcinoma cell lines, but the cells regrew after drug removal [13
In contrast to these malignant cells, the findings here show high doses of sorafenib and regorafenib (50 μM) do not alter normal liver caspase-3 activity (Figure 4) or GSH content (Figure 5). The results also show hepatocyte bioenergetics (respiration and ATP content) following in vitro
exposure to sorafenib or regorafenib for several hours is similar to that of untreated tissue (Figure 3 and Table 1). Consistently, hepatocyte urea synthesis is similar with and without the drugs (Table 1). By contrast, both compounds produce subtle derangements in hepatocyte ultrastructure (Figure 2). The rER changes, however, are more prominent with regorafenib, perhaps accounting for its potential hepatotoxicity (Figures 2A and 2B). Of note, the mitochondrial swelling is relatively similar in samples treated with sorafenib or regorafenib (Figures 2A and 2B).
Sorafenib and regorafenib are tested at their therapeutic concentrations (2.5 and 5 μM, respectively [6
] and at a 10- to 20-fold higher than the therapeutic concentration (50 μM). The first objective of using 50 μM was to investigate potential concentration-dependent hepatotoxicity. Of note, a few of the fatal regorafenib-associated hepatotoxicity were in patients with dehydration, a complication that increased serum drug concentration. The second aim was to compensate for the relatively short drug exposure (3-4 h).
In contrast to previous toxicology studies that were performed on isolated hepatocytes [23
], this study utilized viable liver fragments. Advantages of our approach include minimum tissue handling and avoiding extensive collagenase digestion required for single cell preparations. Successful liver fragment collection, however, requires rapid sampling of thin (<0.2 mm) slices, preferably <20 mg, while the liver is still perfused [24
].The specimens should be immediately immersed in appropriate buffer supplemented with protease inhibitors.
Important limitation of this study is deterioration of the measured biomarkers with time in KH buffer (Table 1) and RPMI medium (Figure 6S, Supplementary Material). The biomarker values at t
=0 (immediately after tissue collection) corresponded to the best possible results (Table 1). At 180 ≤ t
≤ 240 min, hepatocyte respiration decreased by 30%, ATP decreased by 66%, caspase-3 activity increased by 47%, urea synthesis decreased by 36%, and GSH decreased by 59% (Table 1). This limitation prevented extending the incubation beyond 4-5 h.
Intracellular caspase activity is measured on viable liver fragments, using Ac-DEVD-AMC. This substrate is cleaved by several caspases, including caspase-3 (kcat
), caspase-7 (kcat
), caspase-1/interleukin-1 converting enzyme (kcat
), caspase-6 (kcat
), and caspase-4 (kcat
]. Of note, ER stress triggers a specific cascade involving caspase-12, -9, and -3 in a cytochrome c-independent manner [26
]. Consistently, caspase-3 labeling at 4 h was similar in treated and untreated specimens; the few caspase-3 positive cells were mostly localized to Kupffer cells (Figure 1S, Supplementary Material). Similarly, compared with untreated specimens, cytochrome c, BAX, caspase-9 and annexin A2 labeling showed no significant drug effects at 4 h (Figure 2S – 5S, Supplementary Material).
The concentrations used in this study were therapeutics and 10 to 20-fold higher than therapeutics. These drug levels produced structural and ultrastructural changes in the liver (Figures 1 and 2). It is unclear, however, whether the observed adverse effects were due to multikinase inhibition (e.g., VEGFR-2, PDGFR, Raf kinase, FLT3, Ret, and cKit) or “off” target effects. Further studies are needed to address this important issue.
In conclusion, this in vitro
study shows murine hepatocyte bioenergetics, caspase-3 activity, urea synthesis and GSH are not significantly affected by sorafenib or regorafenib. Altered hepatocyte rER is more noticeable with regorafenib. Thus, these data demonstrate ultrastructural changes with regorafenib treatment, justifying its Boxed Warning of hepatotoxicity. The findings call for novel methods that allow early detection of regorafenib and sorafenib hepatotoxicities.