RA190 had a superior effect against HepG2 cell growth compared to Sorafenib
Cells seeded at the concentration of 2,500 cells/well in 100 mL DMEM medium supplemented with 10% FBS in 96-well plate. Twenty-four hours post seeding 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 (0.15 µM) was significantly lower than for Sorafenib (9.7 µM) using 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 accumulation of polyubiquitinated proteins
Since compounds related to RA190 are proteasome inhibitors [12], we examined its impact on the levels of polyubiquitinated proteins in HepG2 cells by ubiquitin immunoblot analysis. RA190 treatment (1 µM, 2 µM) of HepG2 cells (12 hr) dramatically increased the levels of polyubiquitinated proteins and in a dose-dependent manner (Figure 1C and 1D). We also observed that a large number of ubiquitinated proteins have more insoluble in the lysates after high speed centrifuging before loading into the gel. Thus, it is possible less ubiquitinated protein observed with greater quantities 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 previous mentioned [20].
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 [12]. 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 our previous data in other cancer cell lines [12]. In addition, HepG2 cell mRNA was treated with RA190B 2mM (at 0, 4, 15, or 24 hr), subjected to quantitative RT-PCR with RPN13 (ADRM1) primer. The RPN13 (ADRM1) mRNA expression was significantly increased RA190 treatment (Figure 2B). Taking together, the results suggest that RA190 binds to the 42kDa RPN13 protein in HepG2 cells with specificity.
Rapid accumulation of polyubiquitinated proteins leads to ER stress and apoptosis
In addition, with the rapid accumulation of polyubiquitinated unfolded proteins, RA190 treatment also triggered the rapid elevation of BIP-1, ATF-4, CHOP10 and Spliced XBP-1 transcript expression levels (Figures 3A-D), consistent with an ER stress response. HepG2 cells after RA190 treatment thereafter also significantly increased the proportion of Annexin V/PI double positive cells (Figures 4A-C), suggesting activation of apoptosis by 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 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.
Impact of RA190 on NF-κB pathway
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 and 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, IκBα was almost undetected, 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 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). The IκBα signal intensity in the cytoplasm and nuclei was quantified by Image J software and showed the percentage was significantly higher in RA190 treated group (Figure 5F). 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). Furthermore, the phosphorylated/ ubiquitinated IκBα was accumulation after RA190 and bortezomib treatment (Figure S3). Taking together, we suggest RA190 blocked IκBα degradation through proteasome degradation pathway.
RA190 possess superior in vivo tumor growth inhibition
HepG2-Luc cells (1x105 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 by i.p. injection of luciferin and IVIS imaging, the mice were randomized into two groups (6 mice in each group). Upon randomization, treatment (RA190 20mg/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
Drug combinations that consist of multiple chemical agents have shown great promises to improve efficacy and overcome resistance for treating cancer. We utilized zero interaction potency (ZIP) score, which captures the drug interaction relationships by comparing the change in the potency of the dose–response curves between individual drugs and their combinations [21], to test whether combination treatment may have a synergetic killing effect 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). Under a checkerboard analysis and the ZIP synergy score prediction model using a synergy finder application we further sought the optimal combination ratios. In this experiment HepG2 cells were treated with Sorafenib (first 48 hr) then RA190 was added (last 24 hr) for a total assay time of 72 hr. It showed the ZIP synergy score of 2.31 which indicates a synergetic effect (Figure 8B,C) [21].