2018 Joint Usage and Research Report
Title of Research Project

The protective role of ANT2 in chemotherapy-induced liver
damage and its therapeutic potential in cancer chemotherapy(2nd year)

ApplicantVisiting

Institution

Harbin Institute of Technology, China

Job title

Postdocand name

Kai LiName

Kai Liresearcher
Purpose of the Research Project(approx. 250 words)

Chemotherapeuticagentsaremetabolizedandeliminated mainly in the liver, and most are capable ofcausing liver damage including elevation of inflammation.
Our study has also shown that mice were injected
intraperitoneally (i.p.) with doxorubicin can significantly
induce the expression of inflammatory cytokines (IL-6, IL-1β,TNF-α) in liver. Reduction of the inflammation may protect
the liver from chemotherapy-induced damage and improve
the therapeutic effect.
ANT2(AdenineNucleotide

Translocase-2)

isa

mitochondrial protein that facilitates the exchange of ADP
and ATP across the mitochondrial membrane. Previous
studies have indicated that knockdown of ANT2 sensitizedcancer cells to anticancer drugs. However, the role of ANT2
in chemotherapy-induced liver damage and its therapeuticpotential in cancer chemotherapy are still poorly understood.
Our ongoing project has indicated that the expression ofANT2 and several cytokines (IFN-β, IL-6, TNF-α and IL-1β)
are induced by chemotherapy drugs (Doxorubicin, Cisplatinand Etoposide), and suppression of ANT2 results in anelevated expression of cytokines upon these drugs treatment.
Moreover, we found that the production of cytokines isdependent on the activation of cGAS-STING signaling
pathway via sensing of tumor cell-derived mtDNA, andANT2 has an inhibitory function on cGAS-STING signaling
by repressing the cGAS reaction product-cGAMP binding toSTING due to its competitive cGAMP binding activity.
Therefore, we hypothesize that ANT2 may function as aprotective factor in drugs-induced liver damage by inhibition

of STING-mediated inflammation in liver, and this research
project wants to clarify the protective role of ANT2 on
chemotherapeutic drugs-induced liver damage and elucidate
its therapeutic potential in cancer chemotherapy by using invivo mouse model.

Development of the Research

Research Project

Project and Results

Due to the whole-body knockout of Ant2 is lethal, and(approx.. 850 words)liver specific knockout of Ant2 in mouse has been shown anormally growth as wild-type mouse and did not show any
dysfunction of mitochondrial in liver, therefore, we will use
Cre-mediated liver-specific deletion of Ant2 mice (liver-ANT2cKO) in this study to evaluate the protective role of Ant2 inchemotherapeutic

drugs-induced

liverdamage.

To

understand weather this protective role of Ant2 is caused byinhibition of STING-mediated cytokines production, we also
will use liver-STING cKO and liver-ANT2/STING doublecKO mice in this study.
In detail, in this research, we will use four types of mice
(WT, liver-ANT2 cKO, -STING cKO, -ANT2/STING doublecKO mice). After the mice injected intraperitoneally (i.p.)with different dose of doxorubicin for 24, 48 and 72 h, we will
analysis: (1) liver damage by detection cell apoptosis using
TUNEL

assay

andwestern-blotting

todetect

apoptosis biomarkers caspase 3 and PARP; (2) liver damage
by detection AST and ALT level in serum using ELISA; (3)Liver damage by using mice liver section HE stain; (4)Inflammatory cytokines production by qRT-PCR detection
the mRNA level in liver and ELISA detection protein level inserum; (5) Mice survival after doxorubicin injection; (6)Macrophage infiltration in liver; (7) ANT2 induction in miceliver by using qRT-PCR and western-blotting; (8) Activation
states of STING signaling pathway by using western-blotting
to detect pIRF3 and STING dimerization.
Progress and results
1. By using ANT2 liver KO-mice, we found that (1) ANT2
liver KO-mice has a normal growth compare with WT mice; (2)after intraperitoneally(i.p.) injection with doxorubicin, theANT2 liver KO mice shown a decreased survival; (3)ANT2liver KO mice had higher inflammatory cytokines (IL-6,TNF-α andIL-1β)

andaminotransferase(AST,ALT)

production after i.p. injection with doxorubicin; (4) HE stain
and TUNEL stain detection also found that the liver damage
was more serious than WT mice after doxorubicin treatment;(5) ANT2 protein expression was induced by doxorubicin
treatment in WT mice liver. All the above in vivo results weobserved suggested that ANT2 can function as a protective
factor in drugs-induced liver damage.
2. A high impact journal manuscript “ANT2 functions asa negative regulator in the DNA sensing pathway via STING
and protects hepatocytes from chemotherapy-driven liverdamage” Kai Li, Seiichi Sato, Kozo Ishikawa, Chikashi Obuse,
Naohiro Terada, Akinori Takaoka et al., 2019 is in preparation.
Remaining Work and Expected Results
1. We still do not obtain STING and ANT2 double liver
KO-mice after crossing the ANT2 liver KO-mice and STING
whole body KO-mice, so we still do not understand theprotective function of ANT2 in drugs-induced liver damage invivo is via inhibition of STING signaling pathway or not, butfrom our cellular experiments, we expected that this protectiverole is via STING signaling pathway.
2. The protein level of ANT2 in mice liver was induced
after

intraperitoneally(i.p.)

injection

withdoxorubicin,

however, we did not identify the different phosphorylationsite(s) in ANT2 by using LC-MS/MS analysis.

Publication【Conference, symposium, workshop etc.】*Enter the names of conferenceor journal and its vol. No. where
the above work was presented.
【Journals】