RA190 exhibits more potent HepG2 cell killing than sorafenib
HepG2 cells were seeded at a density of 2,500 cells/well in 100 mL DMEM medium supplemented with 10% FBS in 96-well plate. Twenty-four hours post seeding, the cells were treated with RA190 and sorafenib at specified concentrations. Seventy-two hours after treatment, cells were incubated according to the manufacturer’s protocol with the MTT reagent for 1 hr, and absorbance at 570 nm measured to assess inhibition of cell growth. The IC50 for RA190 of 0.15 µM was significantly lower than for sorafenib (9.7 µM) against HepG2 cells (Figure 1A). In a clonogenicity assay, HepG2 cells treated with RA190 exhibited a reduced number of tumor colonies with an IC50 of 0.1 µM (Figure 1B).
RA190 triggers rapid accumulation of polyubiquitinated proteins
Since RA190 is a 19S RP-targeted proteasome inhibitor , we examined its impact on the levels of polyubiquitinated proteins in HepG2 cells by ubiquitin immunoblot analysis. RA190 treatment of HepG2 cells for 12 hr at 1 µM or 2 µM dramatically increased the levels of polyubiquitinated proteins and in a dose-dependent manner (Figure 1C and 1D). We also observed a significant increase in insoluble polyubiquinated cellular proteins in the lysates removed by high speed centrifuging before loading into the gel. This likely accounts for the reduction in ubiquitinated protein observed by Western blot when treating with higher doses of RA190 (lane 3 versus lane 2). Because RPN13 also acts to promote UCH37’s deubiquitinase function, the molecular weight of the accumulated polyubiquitinated proteins observed following exposure to RA190 was higher than seen in bortezomib-treated cells, as previously described .
RA190 binds to RPN13 in HepG2 cells
To identify RA190’s cellular target in HepG2 cells, biotin was covalently linked to RA190 via its free amine functionality (RA190B), as previously described . HepG2 cell lysate was treated with RA190B (at 0, 5, 10, or 25 µM), subjected to SDS-PAGE, and probed with streptavidin-peroxidase following protein transfer to a polyvinylidene difluoride (PVDF) membrane. The streptavidin-peroxidase bound to biotinylated cellular proteins and a new band at 42 kDa was found in treated samples (Figure 2A) that is consistent with the molecular weight of RPN13 and our previous data in other cancer cell lines . In addition, mRNA collected from HepG2 cells was treated with RA190 at either 0 or 2mM for 0, 4, 15, or 24 hr, was subjected to quantitative RT-PCR with primers specific for ADRM1, the gene encoding RPN13, and the housekeeping gene GAPDH. The ADRM1 mRNA expression was significantly increased by RA190 treatment (Figure 2B) relative to the housekeeping gene. Taken together, the results suggest that RA190 binds to and the 42 kDa RPN13 protein in HepG2 cells, triggering compensatory upregulation of ADRM1.
Rapid accumulation of polyubiquitinated proteins leads to ER stress and apoptosis
In addition to the rapid accumulation of polyubiquitinated unfolded proteins, RA190 treatment also triggered the elevation of BIP-1, ATF-4, CHOP10 and spliced XBP-1 transcript expression levels (Figures 3A-D), consistent with an ER stress response. At later time points after RA190 treatment, HepG2 cells also exhibited a significantly increased the proportion of Annexin V/PI double positive cells (Figures 4A-C), suggesting activation of apoptosis because of an unresolved ubiquitin proteasome stress response. Indeed, caspase 3 (Figure 4C) and PARP (Figure 1C) cleavage and p21 expression (Figure 1D) were also considerably increased in HepG2 cells after RA190 treatment, providing further biochemical evidence of the activation of apoptosis.
Autophagy is a potentially compensatory pathway to mitigate the impact of proteasome inhibition. Formation of the lipidated LC3-II, a biomarker of autophagy, was not elevated within 8hr after RA190 2µM treatment (Figure S1), although this was seen upon addition of 10 µM chloroquine, a positive control. Taking together, the rapid accumulation of polyubiquitinated proteins after RA190 treatment caused ER stress that could not be counteracted by the induction of autophagy, leading to apoptosis of the HepG2 cells.
RA190 blocks IκBα degradation and limits NF-κB entry to the nucleus
To examine whether RA190 blocked IκBα degradation and thereby the entry of NF-κB to the nucleus, we used immunofluorescence to visualize IκBα and NF-κB at 30 min after treating with RA190 or the 20S proteasome inhibitor MG132 (which has a similar action as bortezomib) as compared to DMSO (vehicle)-treated cells. IκBα was readily detectable in the cytoplasm of RA190 or MG132-treated cells (Figure 5 and Figure S2). In the DMSO treated cells, IκBα was almost undetectable, consistent with its rapid degradation by the proteasome. Most of the IκBα is co-located with the proteasome (Figure 5B) in RA190 or MG132-treated cells. In the DMSO-treated group, the majority NF-κB protein was nuclear. While much was still in the nucleus, the NF-κB protein was significantly increased in the cytoplasm in the RA190 treated group (Figure 5C), likely reflecting the short incubation period. The IκBα signal intensity in the cytoplasm and nuclei was quantified by Image J software and this analysis showed the fraction in the cytoplasm was significantly higher in RA190-treated group (Figure 5F). Likewise, the percentage of NF-κB signal intensity in cytoplasm was also higher in RA190-treated group (Figure5G). This result was also examined 60 min post-treatment by immunoblot of the cytoplasmic vs. nuclear cellular factions, and a similar pattern was observed (Figure 6). The NF-κB was significantly accumulated in the cytoplasm at 60 min after RA190 treatment (Figure 6A-B), and a similar finding was evident in MG132-treated HepG2 cells (Figure 6C-D). The levels of phosphorylated and ubiquitinated IκBα also built up after RA190 and bortezomib treatment (Figure S3). When considered together, these observations suggest RA190 prevents IκBα degradation by the proteasome.
RA190 treatment inhibits growth of an orthotopic HCC xenograft model
HepG2-Luc cells (5x105 cells in 20 µL) were injected into the left lobe of the liver at day 0. Once the tumor signal was detected at day 7 upon i.p. injection of luciferin and IVIS imaging, the mice were randomized into two groups (6 mice in each group). Upon randomization, treatment (RA190 20 mg/kg in the active arm, and DMSO in the control arm, intraperitoneal injection) was initiated once daily for 21 days. The tumor was visualized and bioluminescence quantified after injection of luciferin by the IVIS imaging system again at day 11, 14, 21, 28, 35 and 42. Two mice in the DMSO group were sacrificed early due to tumor burden at day 35. Surviving mice in both groups were sacrificed at day 42 and the tumor volume was smaller in the liver specimen of the RA190-treated mice (Figure 7A). Figure 7B shows the signal change in individual mice. The bioluminescence intensity in RA190 groups was significantly lower than the DMSO group (P=0.02) at day 35 time point.
RA190 and Sorafenib combination is synergistic
Synergistic combinations of multiple drug treatments targeting different pathways have proven most effective to treat cancer and stave off the emergence of resistance. Zero interaction potency (ZIP) score is an approach used to assess synergy and it captures the drug interaction relationships by comparing the change in the potency of the dose–response curves between individual drugs and their combinations . To test whether combination treatment might have a synergetic killing effect for HCC, we reduced the RA190 concentration to 1 µM and Sorafenib to 10 µM. After treating 18hr, cell viability was still around 80 % with individual drugs. However, combining RA190 and Sorafenib, significantly improved the killing effect and cell viability dropped lower than 40% (Figure 8A). The optimal combination ratios were further sought using a checkerboard analysis to assess cell viability with titrations of RA190 and sofeninib, and analyzing the data using the ZIP synergy score prediction model with the Synergy Finder application. In this experiment HepG2 cells were first treated with Sorafenib and 48 hr later with RA190 (for the final 24 hr) for a total assay time of 72 hr. A ZIP synergy score of 2.31 was achieved, which indicates a synergetic effect (Figure 8B,C) .