Chapter 5 Discussion
5.3 The clinical implication of WP1066
IFN-α is currently approved by Food and Drug Administration for patients with
HCV infection. In our results, the combination of IFN-α and WP1066 enhances the
antiviral ability of cells, it provides a possibility that a STAT3 inhibitor like
WP1066 can enforce IFN-mediated antiviral therapy (Fig. 9 and Fig. 10). However,
there are some problems in application of WP1066. We observe that the toxic
concentration of WP1066 is very close to the effective concentration. Therefore, the
therapeutic window for WP1066 is quite narrow. However, in research of cancer
therapy, normal cell lines are much more resistant to WP1066 than cancer cell lines
(Ferrajoli et al., 2007), and that the inhibitor has been used in animal models (Kong
et al., 2008). Since WP1066 has shown efficacy in vitro, we would further examine
the effect of WP1066 on IFN-mediated antiviral ability in animal models, hoping to
gain insight into its in vivo activity.
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Figures
Figures 1. The effect of FLLL32 on ISG gene
pretreated with the indicated dose
IFN-α4 (1000 IU/ml) for 6 hours.
using primers to PKR (A), IRF-7
was normalized to β-actin level.
The effect of FLLL32 on ISG gene induction by IFN-α4. WT MEFs were
pretreated with the indicated doses of FLLL32 for 1 hour, followed by treatment of
IU/ml) for 6 hours. Total RNA was prepared for quantitative RT
7 (B), IP-10 (C), RIG-I (D) and β-actin. Relative mRNA WT MEFs were
followed by treatment of
quantitative RT-PCR
Relative mRNA
Figures 2. The effect of LLL12 on ISG gene
MEFs were pretreated with the indicated dose
treatment of IFN-α4 (1000 IU/ml) for 6 hours. Total
RT-PCR using primers to PKR (A),
Relative mRNA was normalized to
The effect of LLL12 on ISG gene induction by IFN-α4 stimulation.
MEFs were pretreated with the indicated doses of LLL12 for 1 hour, followed by
IU/ml) for 6 hours. Total RNA was prepared for quantitative
PKR (A), IRF-7 (B), IP-10 (C), RIG-I (D) and
Relative mRNA was normalized to β-actin level.
4 stimulation. WT
hour, followed by
quantitative
and β-actin.
Figures 3. The effect of Cpd188 on ISG gene
MEFs were pretreated with the in
treatment of IFN-α4 (1000 IU/ml) for 6 hours. Total
RT-PCR using primers to PKR (A),
Relative mRNA was normalized to
The effect of Cpd188 on ISG gene induction by IFN-α4 stimulation.
MEFs were pretreated with the indicated doses of Cpd188 for 1 hour, followed by
IU/ml) for 6 hours. Total RNA was prepared for quantitative
PKR (A), IRF-7 (B), IP-10 (C), RIG-I (D) and
Relative mRNA was normalized to β-actin level
.
4 stimulation. WT
hour, followed by
quantitative
and β-actin.
Figures 4. The effect of Stattic
MEFs were pretreated with the in
treatment of IFN-α4 (1000 IU/ml) for 6 hours. Total
RT-PCR using primers to PKR (A),
Relative mRNA was normalized to
Stattic on ISG gene induction by IFN-α4 stimulation.
MEFs were pretreated with the indicated doses of Stattic for 1 hour, followed by
IU/ml) for 6 hours. Total RNA was prepared for quantitative
PKR (A), IRF-7 (B), IP-10 (C), RIG-I (D) and
Relative mRNA was normalized to β-actin level
.
4 stimulation. WT
hour, followed by
quantitative
and β-actin.
Figures 5. WP1066 enhances ISG gene induction by IFN-α4 in a dose-dependent manner in WT MEFs. WT MEFs were pretreated with the indicated doses of WP1066
for 1 hour, followed by treatment with or without IFN-α4 (1000 IU/ml) for 6 hours.
Total RNA was prepared for quantitative RT-PCR using primers to PKR (A), IRF-7 (B),
IP-10 (C), RIG-I (D), RNaseL (E), MDA5 (F), and β-actin. Relative mRNA was
normalized to β-actin level.
Figures 6. Enhanced ISG gene expression by WP1066 is abrogated in STAT3KO MEFs. STAT3KO MEFs were pretreated with the indicated doses of WP1066 for 1
hour, followed by treatment with or without IFN-α4 (1000 IU/ml) for 6 hours. Total
RNA was prepared for quantitative RT-PCR using primers to PKR (A), IRF-7 (B),
IP-10 (C), RIG-I (D), RNaseL (E), MDA5 (F) and β-actin. Relative mRNA was
normalized to β-actin level.
Figures 7. WP1066 enhances ISG gene induction by IFN-α4 in a dose-dependent manner in WT BMMs. WT BMMs were pretreated with the indicated dose of
WP1066 for 1 hour, followed by treatment with or without IFN-α4 (1000 IU/ml) for 4
hours. Total RNA was prepared for quantitative RT-PCR using primers for PKR (A) ,
IRF-7 (B), RIG-I (C), IFIT1 (D), IFIT2 (E), IFIT3 (F) and β-actin. Relative mRNA was
normalized to β-actin level.
Figures 8. Enhanced IFN-α4-stimulated ISG gene expression by WP1066 is abrogated in STAT3KO BMMs. STAT3KO BMMs were pretreated with WP1066 for
1 hour, followed by stimulation with or without IFN-α4 (1000 IU/ml) for 4 hours. Total
RNA of treated cells was subjected to quantitative RT-PCR using primers for PKR (A) ,
IRF-7 (B), RIG-I (C), IFIT1 (D), IFIT2 (E) IFIT3 (F) and β-actin. Relative mRNA was
normalized to β-actin level.
Figures 9. WP1066 enhances IFN-α4-mediated antiviral response to EMCV in WT MEFs and BMMs. WT MEFs (A) and BMMs (B) were pretreated with the indicated
doses of WP1066 for 1 hour, followed by stimulation with or without IFN-α4 (1 or 10
IU/ml) for 16 hours. The treated MEFs and BMMs were infected with EMCV at an
MOI of 1 or 10 , respectively for 4 or 3 hours. Total RNA of the infected cells were
subjected to quantitative RT-PCR using primers for EMCV (2A-2B) and β-actin.
Relative mRNA was normalized to β-actin level. Inhibition percentage was cauculated
according to the following formula in (C) and (D) .
Percent inhibition = 1 – 2A-2B related mRNA+IFN±WP x 100%
2A-2B related mRNA-IFN
Figures 10. Enhancement of
WP1066 is abrogated in STAT3KO
the indicated doses of WP1066 for 1 hour, followed by s
IFN-α4 (1 IU/ml) for 16 hours. The treated cells were
of 1 for 4 hours. Total RNA of the infected cells were subjected to quantitative RT
using primers for EMCV 2A-2B (
(B) Percent inhibition was calculated
Percent inhibition = 1 – 2A-2B related mRNA 2A
Enhancement of IFN-α4-mediated antiviral response to EMCV s abrogated in STAT3KO MEFs. STAT3KO MEFs were pretreated
of WP1066 for 1 hour, followed by stimulation with or
IU/ml) for 16 hours. The treated cells were infected with EMCV at
of 1 for 4 hours. Total RNA of the infected cells were subjected to quantitative RT
2B (A). Relative mRNA was normalized to β-actin lev
calculated according to the following formula:
x 100%
2B related mRNA+IFN±WP 2A-2B related mRNA-IFN
to EMCV by
pretreated with
ion with or without
with EMCV at an MOI
of 1 for 4 hours. Total RNA of the infected cells were subjected to quantitative RT-PCR
actin level.
Figures 11. WP1066 does not alter IFN-α4-stimulated activation of STAT1, STAT2 and STAT3. WT MEFs (A) and BMMs (B) were pretreated with the indicated dose of
WP1066 for 1hr. Followed by treatment with or without IFN-α4 1000 IU/ml for 30
minutes. Total cell lysates were subjected to immunoblotting using antibodies to
pSTAT1, pSTAT2, pSTAT3, STAT1, STAT2, STAT3 and α-tubulin.
Figures 12. WP1066 does not STAT1, STAT2 and STAT3
indicated doses of WP1066 for 1 hour, followed by treatment with or without IFN
1000 IU/ml for 30 minutes. C
immunoblotting using antibodies to pSTAT1, pSTAT2, pSTAT3, STAT1, STAT2,
STAT3, α-tubulin and lamin B.
WP1066 does not affect nuclear translocation of IFN-α4 activated in WT MEFs. WT MEFs were pretreated with the
of WP1066 for 1 hour, followed by treatment with or without IFN
Cytoplasmic and nuclear extracts were subjected to
immunoblotting using antibodies to pSTAT1, pSTAT2, pSTAT3, STAT1, STAT2, activated
WT MEFs were pretreated with the
of WP1066 for 1 hour, followed by treatment with or without IFN-α4
were subjected to
immunoblotting using antibodies to pSTAT1, pSTAT2, pSTAT3, STAT1, STAT2,
Figures 13. WP1066 does not affect n STATs in WT BMMs. WT BMMs
WP1066 for 1 hour, followed by treatment with or without IFN
minutes. Cytoplasmic and nuclear extracts
antibodies to pSTAT1, pSTAT2, pSTAT3, STAT1, STAT2, STAT3,
lamin B.
WP1066 does not affect nuclear translocation of IFN-α4 activated
WT BMMs were pretreated with the indicated dose
WP1066 for 1 hour, followed by treatment with or without IFN-α4 1000 IU/ml for 30
ytoplasmic and nuclear extracts were subjected to immunoblotting
antibodies to pSTAT1, pSTAT2, pSTAT3, STAT1, STAT2, STAT3, α-tubulin and activated
were pretreated with the indicated doses of
IU/ml for 30
were subjected to immunoblotting using
tubulin and
Figures 14. WP1066 enhances the recruitment of transcription complex ISGF3 to ISRE of ISGs. WT BMMs were pretreated with the indicated doses of WP1066 for 1
hour, followed by treating with or without IFN-α4 1000 IU/ml for 15 mins. ChIP was
performed using antibody to STAT1, and QPCR using primers specific to the ISRE
region of IFIT1 (A) and ISRE or exon region of MDA5 (B). Relative abundance was
normalized to the values of enriched to that of regions of input control. (C) Gel
electrophoresis analysis of enriched chromatins were amplified by PCR with indicated
primers. (D) and (E) Quantitative analysis of PCR signals in (C). The results were
expressed as the percentage of immunoprecipitated DNA over total input DNA.
Figures 15. WP1066 reverses the suppression effect of full length and N-terminal domain of STAT3 on ISG induction. STAT3KO MEFs transfected with empty vector
(EV), wild type STAT3 (ST3-FL) and NTD of STAT3 (ST3-NTD) were pretreated with
the indicated doses of WP1066 for 1 hour, followed by treatment with or without
IFN-α4 100 IU/ml for 6 hours. Total RNA were subjected to quantitative RT-PCR using
primers for PKR (A), IRF-7 (B), IFIT1 (C), IFIT2 (D), IFIT3 (E) andβ-actin. Relative
mRNA was normalized to β-actin level.
Figures 16. WP1066 reverses domain of STAT3 on type I IFN
transfected with empty vector (EV), wild type STAT3 (ST3
(ST3-NTD) were pretreated with the indicated dose of WP1066 for 1 hour, followed by
treatment with or without IFN-α
with EMCV at an MOI of 1 for 8
to quantitative RT-PCR using primers for
normalized to β-actin level.
the repression effect of full length and N-terminal ype I IFN-inducing antiviral function. STAT3KO MEFs
vector (EV), wild type STAT3 (ST3-FL) and NTD of STAT3
NTD) were pretreated with the indicated dose of WP1066 for 1 hour, followed by
α4 1IU/ml for 16 hours. The treated cells were
MOI of 1 for 8 hours. Total RNA of the infected cells were subjected
PCR using primers for EMCV 2A-2B. Relative mRNA was terminal
STAT3KO MEFs
FL) and NTD of STAT3
NTD) were pretreated with the indicated dose of WP1066 for 1 hour, followed by
The treated cells were infected
hours. Total RNA of the infected cells were subjected
. Relative mRNA was