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DDX3X/MAVS alleviates doxorubicin‑induced cardiotoxicity by regulating stress granules

  • Authors:
    • Kaixiang Zhao
    • Shaochen Wang
    • Dandan Feng
    • Dongwei Wang
    • Guang Yang
    • Fangfang Lang
  • View Affiliations / Copyright

    Affiliations: Department of Cardiology, The First Affiliated Hospital of Shandong First Medical University (Shandong Provincial Qianfoshan Hospital), Jinan, Shandong 250014, P.R China, Department of Cardiology, Shandong First Medical University, Jinan, Shandong 250017, P.R. China, Department of Obstetrics and Gynecology, Maternal and Child Health Hospital of Shandong Province, Jinan, Shandong 250013, P.R. China
    Copyright: © Zhao et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 237
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    Published online on: June 24, 2025
       https://doi.org/10.3892/mmr.2025.13602
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Abstract

The specific mechanisms of doxorubicin (Dox)‑induced cardiotoxicity (DIC) remain unclear. In the present study, H9c2 cardiomyocytes were treated with Dox, and it was revealed that DEAD‑box RNA helicase 3 X‑linked (DDX3X), mitochondrial antiviral signaling (MAVS) and stress granules (SGs) were present at lower levels in the treated H9c2 cardiomyocytes compared with those in the control cells. The present study further investigated the mechanisms through which DIC occurs. Pretreatment with arsenite, which pharmacologically accelerates SGs, alleviated the myocardial injury caused by Dox. By contrast, anisomycin, an SG inhibitor, increased cardiomyocyte apoptosis induced by Dox. In addition, both DDX3X knockdown and pretreatment with RK‑33 (a DDX3X pharmacological inhibitor) decreased SG expression, whereas DDX3X overexpression promoted SG generation. These results indicated that DDX3X mitigated DIC through the regulation of SGs. In addition, MAVS knockdown inhibited SG assembly and reduced the expression of the anti‑apoptotic inhibitor Bcl2, and MAVS was influenced by DDX3X, thereby serving as a connector between DDX3X and SGs. The results from western blotting, reverse transcription‑quantitative PCR, immunofluorescence and flow cytometry analysis demonstrated that DDX3X, MAVS, and SGs may serve as key protective factors in DIC.
View Figures

Figure 1

DDX3X and stress granules are
downregulated in Dox-induced H9c2 cells. (A) H9c2 cells were
treated with Dox (2 µmol/l) for 24 h, and then the apoptosis rate
was detected by flow cytometry. (B) Bcl2 protein levels in H9c2
cells treated with Dox (2 µmol/l) for 24 h were analyzed by western
blotting. (C) mRNA levels of DDX3X and G3BP1 in Dox (2
µmol/l)-induced H9c2 cells for 24 h were detected by reverse
transcription-quantitative PCR. (D) H9c2 cells were treated with
Dox (2 µmol/l) for 24 h, and the protein expression levels of DDX3X
and G3BP1 were detected by western blotting. Data are presented as
the mean ± SEM. Significance was determined by an unpaired
Student's t-test. *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001. Dox,
doxorubicin; DDX3X, DEAD-box RNA helicase 3 X-linked; G3BP1, ras
GTPase-activating protein-binding protein 1; NC, negative
control.

Figure 2

DDX3X alleviates Dox-induced
cardiomyocyte apoptosis and positively regulated SGs. (A) H9c2
cells were transfected with DDX3X siRNA, then treated with Dox (2
µmol/l) for 24 h, and the protein changes of DDX3X, G3BP1 and Bcl2
in each group were detected by western blotting. (B) mRNA
expression in H9c2 cells transfected with DDX3X siRNA and (C) after
DDX3X overexpression was analyzed by reverse
transcription-quantitative PCR. (D) H9c2 cells after DDX3X
overexpression were treated with Dox (2 µmol/l) for 24 h, and then
western blotting was carried out to detect G3BP1, DDX3X and Bcl2
protein expression. (E) H9c2 cells pretreated with RK-33 for 30 min
were induced with Dox (2 µmol/l) for 24 h, and the protein
expression levels of G3BP1, DDX3X and Bcl2 in each group were
detected by western blotting. All experiments were repeated at
least three times. Data are presented as the mean ± SEM.
Significance was determined by an unpaired Student's t-test (2
groups) or one-way ANOVA with Tukey's multiple comparisons test (≥3
groups), ns, P>0.05, *P≤0.05, **P≤0.01, ***P≤0.001,
****P≤0.0001. Dox, doxorubicin; DDX3X, DEAD-box RNA helicase 3
X-linked; G3BP1, ras GTPase-activating protein-binding protein 1;
siRNA, small interfering RNA.

Figure 3

SGs ameliorate Dox-induced
cardiomyocyte apoptosis. (A) H9c2 cells pretreated with An were
stimulated with Dox (2 µmol/l) for 24 h, and then western blotting
was carried out to detect G3BP1 and Bcl2 protein expression. (B)
SGs in H9c2 cells induced by Dox (2 µmol/l) for 24 h after An
pretreatment were observed by immunofluorescence. SGs were detected
using anti-G3BP1 (green) and the nuclei were stained with DAPI
(blue). (C) H9c2 cells were first treated with Dox (2 µmol/l) for
12 h, then with Ar in the continued presence of Dox for another 12
h (total Dox exposure: 24 h). Protein levels of G3BP1 and Bcl-2
were analyzed by western blotting. (D) Immunofluorescence was used
to observe SGs in H9c2 cells induced by Ar and Dox (2 µmol/l). SGs
were detected using anti-G3BP1 (green) and the nuclei were stained
with DAPI (blue). Scale bar, 50 µm. (E) Cell viability of H9c2
cells treated with Ar (10 µmol/l) for 0, 12, 24 and 48 h was
detected using the Cell Counting Kit-8 assay. All experiments were
repeated at least three times. Data are presented as the mean ±
SEM. Significance was determined by one-way ANOVA with Tukey's
multiple comparisons test. ns, P>0.05, *P≤0.05, **P≤0.01,
***P≤0.001, ****P≤0.0001. Dox, doxorubicin; DDX3X, DEAD-box RNA
helicase 3 X-linked; G3BP1, ras GTPase-activating protein-binding
protein 1; Ar, arsenite; An, anisomycin; SGs, stress granules.

Figure 4

MAVS bridges DDX3X and SGs. (A) mRNA
and protein levels of MAVS in H9c2 cells induced by Dox (2 µmol/l)
for 24 h were detected by RT-qPCR and western blotting,
respectively. (B) H9c2 cells were transfected with DDX3X siRNA,
then treated with Dox (2 µmol/l) for 24 h, and the changes in MAVS
protein expression in each group were detected by western blotting.
(C) H9c2 cells pretreated with RK-33 for 30 min were induced with
Dox (2 µmol/l) for 24 h, and then western blotting was carried out
to detect MAVS protein expression. (D) Knockdown efficiency of MAVS
was determined by RT-qPCR. (E) H9c2 cells transfected with MAVS
siRNA were induced with Dox (2 µmol/l) for 24 h, and the protein
levels of MAVS, G3BP1 and Bcl2 were determined by western blotting.
All experiments were repeated at least three times. Data are
presented as the mean ± SEM. Significance was determined by an
unpaired Student's t-test (2 groups) or one-way ANOVA with Tukey's
multiple comparisons test (≥3 groups). ns, P>0.05, *P≤0.05,
**P≤0.01, ***P≤0.001, ****P≤0.0001. Dox, doxorubicin; DDX3X,
DEAD-box RNA helicase 3 X-linked; G3BP1, ras GTPase-activating
protein-binding protein 1; RT-qPCR, reverse
transcription-quantitative PCR; SGs, stress granules; MAVs,
mitochondrial antiviral signaling; siRNA, small interfering RNA;
NC, negative control.

Figure 5

Schematic diagram of the role of
DDX3X/MAVS/SGs in Dox-induced cardiotoxicity. When Dox enters
cardiomyocytes, the expression of DDX3X is inhibited, which in turn
downregulates the expression of the mitochondrial antiviral signal
MAVS. The reduction of the DDX3X and MAVS complex inhibits the
assembly of SGs, thereby decreasing the production of
anti-apoptotic marker Bcl2. Dox, doxorubicin; DDX3X, DEAD-box RNA
helicase 3 X-linked; G3BP1, ras GTPase-activating protein-binding
protein 1; MAVs, mitochondrial antiviral signaling; SGs, stress
granules.
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Copy and paste a formatted citation
Spandidos Publications style
Zhao K, Wang S, Feng D, Wang D, Yang G and Lang F: DDX3X/MAVS alleviates doxorubicin‑induced cardiotoxicity by regulating stress granules. Mol Med Rep 32: 237, 2025.
APA
Zhao, K., Wang, S., Feng, D., Wang, D., Yang, G., & Lang, F. (2025). DDX3X/MAVS alleviates doxorubicin‑induced cardiotoxicity by regulating stress granules. Molecular Medicine Reports, 32, 237. https://doi.org/10.3892/mmr.2025.13602
MLA
Zhao, K., Wang, S., Feng, D., Wang, D., Yang, G., Lang, F."DDX3X/MAVS alleviates doxorubicin‑induced cardiotoxicity by regulating stress granules". Molecular Medicine Reports 32.3 (2025): 237.
Chicago
Zhao, K., Wang, S., Feng, D., Wang, D., Yang, G., Lang, F."DDX3X/MAVS alleviates doxorubicin‑induced cardiotoxicity by regulating stress granules". Molecular Medicine Reports 32, no. 3 (2025): 237. https://doi.org/10.3892/mmr.2025.13602
Copy and paste a formatted citation
x
Spandidos Publications style
Zhao K, Wang S, Feng D, Wang D, Yang G and Lang F: DDX3X/MAVS alleviates doxorubicin‑induced cardiotoxicity by regulating stress granules. Mol Med Rep 32: 237, 2025.
APA
Zhao, K., Wang, S., Feng, D., Wang, D., Yang, G., & Lang, F. (2025). DDX3X/MAVS alleviates doxorubicin‑induced cardiotoxicity by regulating stress granules. Molecular Medicine Reports, 32, 237. https://doi.org/10.3892/mmr.2025.13602
MLA
Zhao, K., Wang, S., Feng, D., Wang, D., Yang, G., Lang, F."DDX3X/MAVS alleviates doxorubicin‑induced cardiotoxicity by regulating stress granules". Molecular Medicine Reports 32.3 (2025): 237.
Chicago
Zhao, K., Wang, S., Feng, D., Wang, D., Yang, G., Lang, F."DDX3X/MAVS alleviates doxorubicin‑induced cardiotoxicity by regulating stress granules". Molecular Medicine Reports 32, no. 3 (2025): 237. https://doi.org/10.3892/mmr.2025.13602
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