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. 2020 May 6;20(1):386.
doi: 10.1186/s12885-020-06896-0.

Bis-benzylidine Piperidone RA190 treatment of hepatocellular carcinoma via binding RPN13 and inhibiting NF-κB signaling

Ruey-Shyang Soong  1   2   3 , Ravi K Anchoori  4 , Richard B S Roden  5 , Rou-Ling Cho  3 , Yi-Chan Chen  1 , Sheng-Chieh Tseng  3 , Yun-Li Huang  3 , Po-Cheng Liao  3 , Yu-Chiau Shyu  6   7   8
Affiliations

Bis-benzylidine Piperidone RA190 treatment of hepatocellular carcinoma via binding RPN13 and inhibiting NF-κB signaling

Ruey-Shyang Soong et al. BMC Cancer. .

Abstract

Background: According to GLOBOSCAN, hepatocellular carcinoma (HCC) claimed 782,000 lives in 2018. The tyrosine kinase inhibitor sofafenib is used to treat HCC, but new anticancer agents targeting different pathways are urgently needed to improve outcomes for patients with advanced disease. The aberrant metabolism and aggressive growth of cancer cells can render them particularly susceptible to proteasome inhibition, as demonstrated by bortezomib treatment of multiple myeloma. However, resistance does emerge, and this 20S proteasome inhibitor has not proven active against HCC. The bis-benzylidine piperidone RA190 represents a novel class of proteasome inhibitor that covalently binds to cysteine 88 of RPN13, an ubiquitin receptor subunit of the proteasome's 19S regulatory particle. RA190 treatment inhibits proteasome function, causing rapid accumulation of polyubiquitinated proteins. Considerable evidence suggests that nuclear factor κB (NF-κB) signaling, which is dependent upon the proteasome, is a major driver of inflammation-associated cancers, including HCC.

Methods: Human HCC cell lines were treated with titrations of RA190. The time course of endoplasmic reticulum stress and NF-κB-related mechanisms by which RA190 may trigger apoptosis were assessed. The therapeutic activity of RA190 was also determined in an orthotopic HCC xenograft mouse model.

Results: RA190 is toxic to HCC cells and synergizes with sofafenib. RA190 triggers rapid accumulation of polyubiquitinated proteins, unresolved endoplasmic reticulum stress, and cell death via apoptosis. RA190 blocks proteasomal degradation of IκBα and consequent release of NF-κB into the nuclei of HCC cells. Treatment of mice bearing an orthotopic HCC model with RA190 significantly reduced tumor growth.

Conclusions: RA190 has therapeutic activity in a xenograft model, and with sorafenib exhibited synergetic killing of HCC cells in vitro, suggesting further exploration of such a combination treatment of HCC is warranted.

Keywords: Apoptosis; Hepatocellular carcinoma; NF-κB; Proteasome inhibitor; RA190.

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Conflict of interest statement

The authors declare the following competing financial interest(s): Under a licensing agreement between Pontifax/PI Therapeutics and Johns Hopkins University, Drs. Anchoori and Roden are entitled to royalties on an invention (US patent application 20190175572) described in this article. This arrangement has been reviewed and approved by Johns Hopkins University in accordance with its conflict of interest policies. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Figures

Fig. 1
Fig. 1
Impact of RA190 upon HepG2 cell viability, colony formation, polyubiquitinated protein, and apoptotic markers. a HepG2 cells were plated at 96/well of a microtiter plate in the presence of titrations of RA190 or sorafinib, and after 72 h the cell viability was determined by MTT assay. b Treatment of HepG2 cells with RA190 reduced the number tumor colonies in a clonogenicity assay. c HepG2 cells after 12 h treatment with 0, 1, or 2 μM RA190 were harvested and subjected to Western blot analysis with antibody to Ubiquitin, PARP and Actin (full-length blots/gels are presented in Fig. S4). d HepG2 cells were treated with 2 μM RA190 and harvested at the indicated times. Cell lysates were subjected to Western blot analysis with antibody to Ubiquitin, p21 and Actin (full-length blots/gels are presented in Fig. S5)
Fig. 2
Fig. 2
RA190B binds to RPN13 in HepG2 cells. a HepG2 cell lysates were incubated with 0, 5, 10 or 25 μM RA190B, separated by SDA-PAGE, transferred to a membrane and probed with HRP-streptavidin. After development using chemiluminescence, a prominent band at 42 KDa was evident, a molecular weight consistent with RPN13 (full-length blots/gels are presented in Fig. S6). b RPN13 (ADRM1) mRNA expression as determined by quantitative RT-PCR was significantly increased after 4 h RA190 treatment
Fig. 3
Fig. 3
Elevation of mRNA levels of UPR genes after RA190 treatment. a, b, c, d The mRNA expression levels of ER stress proteins BIP-1, ATF-4, CHOP10, and spliced XBP-1 were determined by quantitative RT-PCR in HepG2 cells 0, 4, 15 and 24 h after 2 μM RA190 treatment
Fig. 4
Fig. 4
RA190 triggers apoptosis in HepG2 cells. a, b The percentage of PI+/Annexin V+ HepG2 cells was determined by flow cytometry after treatment with 0, 2 or 4 μM RA190 for 0, 8 or 24 h. c Active Caspase 3 level was determined by flow cytometry after treatment with 0, 2 or 4 μM RA190 for 8 h
Fig. 5
Fig. 5
RA190 causes accumulation of IκBα and NF-κB in the cytoplasm, co-localized with proteasomes. HepG2 cells were treated with DMSO, 2 μM RA190 for or 25 μM MG132 for 30 min. The cells were fixed, permeabilized and stained for IκBα a, proteasome b, NF-κB c and viewed by confocal fluorescence microscopy individually, or d merged, or e under phase contrast. a, b, c IκBα and NF-κB in RA190 and MG132-treated cells is significantly accumulated in cytoplasm, and apparently co-localized with the proteasome. The proportion of fluorescence in the cytoplasm versus the nucleus was quantified for f IκBα, or g NF-κB
Fig. 6
Fig. 6
NF-κB is significantly accumulated in the cytoplasmic fraction after RA190 treatment. HepG2 cells were treated with DMSO or 2 μM RA190 for 60 min, harvested and lysed (total fraction). Cytoplasmic and nuclear fractions were separated, and each fraction was subjected to Western blot with antibody to NF-κB, lamin a (a nuclear marker) and tubulin (a cytoplasmic marker). a NF-κB showed significant accumulation in the cytoplasmic fraction of cells when treated with RA190 for 60 min, as compared with DMSO (full-length blots/gels are presented in Fig. S7). The quantified data is presented in a bar graph b. c Similar results were obtained with MG132 treatment (full-length blots/gels are presented in Fig. S8) and the quantification is presented in a bar graph d
Fig. 7
Fig. 7
RA190 treatment inhibited the growth of orthotopic HCC tumor model. a Bioluminescence imaging showing the HepG2 tumors following orthotopic challenge with HepG2-Luc cells (5 × 105 /mouse) administered in the left lobe of liver of nude mice. Mice were treated with either DMSO or RA190 (20 mg/kg) once daily via the intra peritoneal route beginning at day 7 and ending at day 28. Imaging of bioluminescence after injection of luciferin via the intraperitoneal route was performed on an IVIS 200 imager and the data plotted b. Bioluminescence was significantly different (P < 0.02) between the groups on day 42
Fig. 8
Fig. 8
RA190 combination with Sorafenib showed synergy against HepG2 cells. a Bar graph depicting HepG2 cell viability after 48 h of each treatment regimen. To seek the conditions of optimal synergy, a checkerboard analysis was performed using titrations of RA190 and Sorafenib. b, c A surface plot of the Synergy score showed the most synergistic cell killing occurred at 0.3 μM RA190 and 0.5 mM Sorafenib
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