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Page 12 of 17 Matsushita et al. Hepatoma Res 2018;4:61 I http://dx.doi.org/10.20517/2394-5079.2018.81

C

Figure 5. BK697 inhibit far upstream element-binding protein-interacting repressor Δexon2 (FIRΔexon2) that is considered as a dominant
negative of FIR. (A) From computer screening to search synthesized chemicals that mimicking the structure of the identified compound
using Namiki database (Namiki Shoji Co., Ltd., Tokyo, Japan) that was composed of commercially available chemicals after natural
product depository array at RIKEN (Japan). Affiliated chemicals were screened and indicated in the square (square). Based on the
computer screening, lots of similar chemicals were designed and seven compounds were selected for treating with HLE and HLF cells to
examine cell growth inhibition (arrows); (B) BK697 effectively suppressed hepatoblastoma cells, HLE and HLF cells. BK697 effectively
suppressed hepatoblastoma cells, HLE and HLF cells by MTS assay (see materials and methods). Small molecular weight indicated
arrows (A) were examined the cell growth suppression in HLE and HLF cells. All samples are tested in duplicate, absorbencies were
tested 3 times. Same volume of DMSO was used as negative control. Same volume of 3% H 2 O 2 was used as positive control; (C) BK697
suppressed FIR/FIRΔexon2 expression on dose-dependent manner in gastric cancer cells (left) and HeLa cells (right). Note SAP155
(SF3B1) was also suppressed by BK697 along with FIR/FIRΔexon2 expression. H2AX is a marker of DNA damage. FIR: FUBP1-interacting
repressor; HLE: hepatoblastoma cell line; MTS: 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-
tetrazolium, inner salt; DMSO: dimethyl sulfoxide

cancer therapies directed against c-Myc and splicing of SF3B inhibition may go through FIR and its splicing
variants. In this study, BK697 has been screened to target SAP155-binding FIRΔexon2 for cancer therapy
[Figure 2B]. According to recent cancer gene therapy, adenovirus-mediated (Ad) TP53 gene transfer is
frequently used, together with cis-dichloro-diammineplatinum administration or ionizing radiation [1,10] . As
for Ad-FIR or Sendai virus-FIR vector, the transduction efficiency showed that the efficacy in preclinical
trials and combination treatment with standard chemoradiation and Ad-FIR/Sendai-FIR gene therapy may
be an attractive modality in the future [10-14] .

HBV has a small (3.2 kb), partially-double stranded, relaxed-circular DNA genome that encodes four
[54]
overlapping open reading frames (ORFs) . The genomic transcripts from these overlapping four ORFs act
mRNAs for precore, core and polymerase. The genomic transcript that encodes both core and polymerase is
[54]
multifunctional and referred to as pgRNA . The core protein binds to HBV covalently closed circular DNA
[54]
(cccDNA). The cccDNA forms a minichromosome in the nucleus of the hepatocyte . Recent nucleoside
analogues and interferons treatment for HBV-positive patients do not achieve complete clearance of viral
genome cccDNA in the nucleus [Figure 6]. To our interest, PUF60 was identified as a versatile regulator of
[34]
transcriptional and post-transcriptional steps in expression of HBV 3.5 kb, precore plus pgRNA . This is
the first to identify a host cell factor (protein) involved in not only positively regulating viral gene expression
[34]
but also negative regulation of the same viral life cycle . Therefore, FIR/PUF60 is also a novel promising
target to inhibit HBV cccDNA transcription as well as interfering FBW7 function [Figure 6]. Given the FIR/
[34]
PUF60 is required for HBV cccDNA replication and novel small molecular weight chemicals including
BK697 that suppresses FIR/PUF60 expression [Figure 5C], those chemicals have advantage to eliminate
HBV cccDNA than other strategies as recent nucleoside analogues and interferons treatment. Further, the
amino terminus of FIR was necessary to repress transcription from the c-Myc promoter by suppressing
[22]
FUBP1, FUBP [Figures 2 and 6] . FUBP1/FIR(PUF60)/TFIIH system FIR suppresses endogenous c-Myc