Mitochondria-associated protein LRPPRC exerts cardioprotective effects against doxorubicin-induced toxicity, potentially via inhibition of ROS accumulation

Abstract

Doxorubicin (DOX) has been widely employed to treat cancer, particularly solid tumors and hematological malignancies, owing to its high efficacy; however, chemotherapy has been indicated to be cardiotoxic and induce adverse effects, including mitochondrial dysfunction and DNA damage, which limits its application. The mitochondria-associated protein leucine-rich pentatricopeptide repeat-containing (LRPPRC) has been reported to serve critical regulatory roles in physiological processes via regulating mitochondrial function. The aim of the present study was to investigate the possible protective effects of LRPPRC against DOX-induced cardiac injury. In a DOX-induced cardiotoxicity model in H9C2 cells, LRPPRC was indicated to be transcriptionally upregulated and stabilize Bcl-2 and Bax. LRPPRC overexpression exhibited protective effects against proliferation and both apoptotic and non-apoptotic cell death following DOX treatment, but not under normal conditions. It was additionally observed that overexpressed LRPPRC reversed the decreases in ATP synthesis, mitochondrial mass and transcriptional activity, which were induced by DOX exposure. Overexpressed LRPPRC also decreased the accumulation of reactive oxygen species (ROS) under DOX treatment and inhibited cell death to a similar extent as N-acetyl-L-cysteine, which is a known ROS scavenger, indicating that LRPPRC potentially exerts protective effects via inhibiting ROS accumulation. Moreover, LRPPRC overexpression protected H9C2 cells against oxidative stress induced by H₂O₂, which also indicated its ROS-scavenging function. The present study demonstrated for the first time, to the best of our knowledge, that DOX-induced LRPPRC may exert cardioprotective effects via inhibiting ROS accumulation, thereby maintaining mitochondrial function.

Keywords: H9C2 cells; cardiotoxicity; leucine-rich pentatricopeptide repeat-containing; mitochondrial function; oxidative stress; reactive oxygen species.

Figure 1

DOX treatment upregulates LRPPRC at the transcriptional level. (A) Cell Counting Kit-8 assay was performed to detect DOX cytotoxicity in H9C2 cells. (B) Following DOX treatment at IC₃₀ or IC₅₀ for 0, 24, 48, 72 and 96 h, the mRNA levels of LRPPRC, Bcl-2 and Bax were detected via reverse transcription-quantitative PCR. (C) Protein levels of LRPPRC, Bcl-2 and Bax were detected via western blot (left panel), and quantitatively analyzed via ImageJ software (right panel). (D) Via immunofluorescence staining, the expression and localization of LRPPRC (red) and nucleus (blue) were visualized. ^*P<0.05 and ^**P<0.01 vs. 0 h DOX exposure group. LRPPRC, leucine-rich pentatricopeptide repeat-containing; DOX, doxorubicin.

Figure 2

LRPPRC overexpression exerts protective effects against DOX-induced cell injury. (A) A total of 48 h post-transfection with siLRPPRC and the coding sequence of LRPPRC, the protein levels of LRPPRC were detected via western blotting. (B) Following LRPPRC overexpression, the cytotoxicity of DOX in H9C2 cells was measured. (C) Cell Counting Kit-8 assay was performed to detect the effect of LRPRC overexpression on cell proliferation under DOX treatment. (D) DOX at IC₃₀ was utilized for cell treatment for 24 h, followed by PI staining and flow cytometric analysis to detect the cell cycle phases. (E) DOX at IC₅₀ was employed for cell treatment for 24 h, followed by Annexin V-FITC/PI double staining and flow cytometric analysis to detect apoptotic and non-apoptotic cell death. ^*P<0.05 vs. vector group. LRPPRC, leucine-rich pentatricopeptide repeat-containing; DOX, doxorubicin; si, small interfering; PI, propidium iodide; OD, optical density.

Figure 3

DOX-induced LRPPRC exerts protective effects against DOX exposure potentially via scavenging ROS. (A) Following DOX exposure, the effect of LRPPRC overexpression on ATP synthesis was examined. (B) Mitochondrial mass was measured using MitoTracker Red staining. (C) To evaluate the effect of LRPPRC on mitochondrial transcriptional activity, the expression levels of COX 1, COX 3, ND1 and Cyb were detected via reverse transcription-quantitative PCR. (D) ROS accumulation was detected following DOX exposure at IC₃₀ for 24 h. (E) Annexin V-FITC/PI double staining followed by flow cytometric analysis was performed to detect apoptotic and non-apoptotic cell death. ^*P<0.05 vs. vector group; ^#P<0.05 vs. LRPPRC group. LRPPRC, leucine-rich pentatricopeptide repeat-containing; DOX, doxorubicin; PI, propidium iodide; COX, cytochrome c oxidase subunit; ND1, NADH dehydrogenase subunit 1; Cyb, cytochrome b; NAC, N-acetyl-L-cysteine; ROS, reactive oxygen species.

Figure 4

LRPPRC exerts protective effects against reactive oxygen species-induced cell injury in H9C2 cells. (A) Following treatment with 200 mM H₂O₂, the mRNA and protein expression levels of LRPPRC, Bcl-2 and Bax were detected. ^*P<0.05 vs. Mock group; ^#P<0.05 vs. 200 mM H₂O₂ group. Following H₂O₂ exposure with or without NAC treatment, the (B) cell cycle phase distribution and (C) cell death rate were measured. ^*P<0.05 vs. Mock group; ^#P<0.05 vs. 200 mM H₂O₂ group. LRPPRC, leucine-rich pentatricopeptide repeat-containing; DOX, doxorubicin; NAC, N-acetyl-L-cysteine; PI, propidium iodide.