Lapatinib induces mitochondrial dysfunction to enhance oxidative stress and ferroptosis in doxorubicin-induced cardiomyocytes via inhibition of PI3K/AKT signaling pathway

Abstract

Lapatinib (LAP) is an important anti-cancer drug and is frequently alongside doxorubicin (DOX) as a combination therapy for better anti-cancer efficacy. However, many studies have reported that LAP in combination with DOX may induce highly cardiotoxicity. Accordingly, we aimed to explore the potential mechanism involved in the synergistic effect of LAP in DOX-induced cardiotoxicity. Here, cell counting kit-8 was used to detect cell viability and lactate dehydrogenase measurement was performed to assess cell injury. Cell apoptosis was evaluated by TUNEL assay and western blot assay. Mitochondrial dysfunction was identified by JC-1 assay, adenosine triphosphate (ATP) and Cytochrome C. Moreover, the activity of ROS, SOD, CAT and GSH were measured to elucidate oxidative stress level. Ferroptosis was examined by levels of Fe²⁺, GPX4 and ASCL4. Expressions of PI3K/AKT signaling were identified by western blot assay. The results revealed that LAP inhibited the cell viability and exacerbated cell injury induced by Dox, as well as increased cell apoptosis. LAP aggravated DOX-induced mitochondria damage by changed mitochondrial membrane potential, decreased ATP and increased level of Cytochrome C. In addition, the combination of LAP and DOX induced oxidative stress and ferroptosis in H9c2 cells. The activation of PI3K/AKT signaling reversed the detrimental effects of LAP on DOX-induced H9c2 cells. The data in this study showed for the first time that LAP aggravated Dox-induced cardiotoxicity by promoting oxidative stress and ferroptosis in cardiomyocytes via PI3K/AKT-mediated mitochondrial dysfunction, suggesting that PI3K/AKT activation is a promising cardioprotective strategy for DOX /LAX combination therapies.

Keywords: Lapatinib; PI3K/AKT signaling pathway; cardiomyocytes; doxorubicin; ferroptosis; mitochondrial dysfunction.

Figure 1.

Effect of LAP on DOX-induced cytotoxicity in H9c2 cells. H9c2 cells were treated with 1 μM DOX with or without 1–3 μM LAP for 24 h. A, Chemical structure of Lapatinib. B, Cytotoxicity effect of LAP on H9c2 cells. C, LDH level in DOX-induced cells with or without different doses of LAP. D and E, Cell apoptosis was detected by TUNEL assay. Original magnification: x200. F, Protein levels of Bcl-2 and Bax were measured by western blot assay. LAP: Lapatinib; DOX: Doxorubicin; LDH: Lactate dehydrogenase. Data are expressed as mean ± SD calculated from three independent experiments. *P < 0.05, ***P < 0.001 versus control. ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 versus DOX

Figure 2.

Effect of LAP on DOX-induced mitochondria damage in H9c2 cells. H9c2 cells were treated with 1 μM DOXwith or without 1–3 μM LAP for 24 h. A, MMP was identified by JC-1 staining. Red fluorescence represents the mitochondrial aggregate form of JC-1, indicating intact mitochondrial membrane potential. Green fluorescence represents the monomeric form of JC-1, indicating dissipation of ΔΨm. Original magnification: x200. B, ATP level in DOX-induced cells with or without different doses of LAP. C, Protein level of Cytochrome C was measured by western blot assay. LAP: Lapatinib; DOX: Doxorubicin; MMP: mitochondrial membrane potential. Data are expressed as mean ± SD calculated from three independent experiments. ***P < 0.001 versus control. ^#P < 0.05, ^###P < 0.001 versus DOX

Figure 3.

Effect of LAP on DOX-induced oxidative stress in H9c2 cells. H9c2 cells were treated with 1 μM DOXwith or without 1–3 μM LAP for 24 h. A, ROS level was detected by DCFH-DA staining. Original magnification: x200. The activity of SOD (b), CAT (c) and GSH (d) was assessed in DOX-induced cells with or without different doses of LAP. LAP: Lapatinib; DOX: Doxorubicin; ROS: Reactive oxygen species; SOD: superoxide dismutase; CAT: Catalase; GSH: Glutathione. Data are expressed as mean ± SD calculated from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 versus control. ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 versus DOX

Figure 4.

Effect of LAP on DOX-induced ferroptosis in H9c2 cells. H9c2 cells were treated with 1 μM DOXwith or without 1–3 μM LAP for 24 h. A, The level of Fe²⁺ in DOX-induced cells with or without different doses of LAP. B, Protein levels of GPX4 and ASCL4 were evaluated by western blot assay. LAP: Lapatinib; DOX: Doxorubicin; GPX4: Glutathione peroxidase 4; ASCL4: acyl-CoA synthetase long-chain family member 4. Data are expressed as mean ± SD calculated from three independent experiments. *P < 0.05, **P < 0.01 versus control. ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 versus DOX

Figure 5.

Effect of LAP on PI3K/AKT signaling pathway in DOX-induced H9c2 cells. H9c2 cells were treated with 1 μM DOXwith or without 1–3 μM LAP for 24 h. Protein levels of p-PI3K, PI3K, p-AKT and AKT were detected by western blot analysis. Data are expressed as mean ± SD calculated from three independent experiments. ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 versus DOX

Figure 6.

Effects of the activation of PI3K/AKT pathway on mitochondria damage in H9c2 cells with combined treatment of LAP-plus-DOX. 30 μM of 740Y-P was added to H9c2 cells with combined treatment of LAP-plus-DOX. A, MMP was identified by JC-1 staining. Original magnification: x200. B, ATP level in DOX/LAP-induced cells with or without 740Y-P. C, Protein level of Cytochrome C was measured by western blot assay. LAP: Lapatinib; DOX: Doxorubicin; MMP: mitochondrial membrane potential. Data are expressed as mean ± SD calculated from three independent experiments. **P < 0.01 versus control. ^##P < 0.01, ^###P < 0.001 versus DOX. ^ΔP < 0.05 versus DOX+Lapatinib (3 μM)

Figure 7.

Effects of the activation of PI3K/AKT pathway on oxidative stress in H9c2 cells with combined treatment of LAP-plus-DOX. 30 μM of 740Y-P was added to H9c2 cells with combined treatment of LAP-plus-DOX. A, ROS level was detected by DCFH-DA staining. Original magnification: x200. The activity of SOD (b), CAT (c) and GSH (d) was assessed in DOX/LAP-induced cells with or without 740Y-P. LAP: Lapatinib; DOX: Doxorubicin; ROS: Reactive oxygen species; SOD: superoxide dismutase; CAT: Catalase; GSH: Glutathione. Data are expressed as mean ± SD calculated from three independent experiments. **P < 0.01 versus control. ^###P < 0.001 versus DOX. ^ΔΔΔP < 0.001 versus DOX+Lapatinib (3 μM)

Figure 8.

Effects of the activation of PI3K/AKT pathway on ferroptosis in H9c2 cells with combined treatment of LAP-plus-DOX. 30 μM of 740Y-P was added to H9c2 cells with combined treatment of LAP-plus-DOX. A, The level of Fe²⁺ in DOX/LAP-induced cells with or without 740Y-P. B, Protein levels of GPX4 and ASCL4 were evaluated by western blot assay. LAP: Lapatinib; DOX: Doxorubicin; GPX4: Glutathione peroxidase 4; ASCL4: acyl-CoA synthetase long-chain family member 4. Data are expressed as mean ± SD calculated from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 versus control. ^###P < 0.001 versus DOX. ^ΔΔP < 0.01, ^ΔΔΔP < 0.001 versus DOX+Lapatinib (3 μM)