Lycopene protects human SH‑SY5Y neuroblastoma cells against hydrogen peroxide‑induced death via inhibition of oxidative stress and mitochondria‑associated apoptotic pathways

Abstract

Oxidative stress, which is characterized by excessive production of reactive oxygen species (ROS), is a common pathway that results in neuronal injury or death due to various types of pathological stress. Although lycopene has been identified as a potent antioxidant, its effect on hydrogen peroxide (H2O2)‑induced neuronal damage remains unclear. In the present study, pretreatment with lycopene was observed to protect SH‑SY5Y neuroblastoma cells against H2O2‑induced death via inhibition of apoptosis resulting from activation of caspase‑3 and translocation of apoptosis inducing factor (AIF) to the nucleus. Furthermore, the over‑produced ROS, as well as the reduced activities of anti‑oxidative enzymes, superoxide dismutase and catalase, were demonstrated to be alleviated by lycopene. Additionally, lycopene counteracted H2O2‑induced mitochondrial dysfunction, which was evidenced by suppression of mitochondrial permeability transition pore opening, attenuation of the decline of the mitochondrial membrane potential, and inhibition of the increase of Bax and decrease of Bcl‑2 levels within the mitochondria. The release of cytochrome c and AIF from the mitochondria was also reduced. These results indicate that lycopene is a potent neuroprotectant against apoptosis, oxidative stress and mitochondrial dysfunction, and could be administered to prevent neuronal injury or death.

Figure 1

Lycopene inhibited H₂O₂-induced cell death in SH-SY5Y cells. (A) MTT assay of the viability of SH-SY5Y cells exposed to H₂O₂ for 24 h at the indicated concentrations. (B) MTT assay of the changes in the viability of SH-SY5Y cells exposed to H₂O₂ as a result of pretreatment with lycopene. (C) LDH release assay of H₂O₂-induced toxicity in SH-SY5Y cells pretreated with or without lycopene. ^*P<0.01 vs. control group; ^#P<0.01 vs. H₂O₂ group. H₂O₂, hydrogen peroxide; LDH, lactate dehydrogenase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.

Figure 2

Lycopene inhibited H₂O₂-induced apoptosis in SH-SY5Y cells. (A) Representative images of nuclei stained with Hoechst 33342. (B) Quantification of the nuclei demonstrating apoptotic features observed under a fluorescence microscope. (C) Representative images of the control and apoptotic cells observed under a transmission electronic microscope. The cells treated with H₂O₂ had membrane blebbing, chromatin accumulation beneath the nuclear membrane and nuclear condensation. (D) Flow cytometric analysis of the effect of lycopene on H₂O₂-induced apoptosis in SH-SY5Y cells. ^*P<0.01 vs. control group; ^#P<0.01 vs. H₂O₂ group. H₂O₂, hydrogen peroxide.

Figure 3

Lycopene mitigated H₂O₂-induced oxidative stress in SH-SY5Y cells. (A) Representative fluorescence microscopy images of the cells stained with DCFH-DA. (B) Quantification of fluorescence density of the cells stained with DCFH-DA. (C) Activity assay of SOD. (D) Activity assay of catalase. ^*P<0.01 vs. control group. ^#P<0.01 vs. H₂O₂ group. DCFH-DA, dichloro-dihydro-fluorescein diacetate; DCF, 2′,7′-dichlorofluorescein; SOD, superoxide dismutase; H₂O₂, hydrogen peroxide.

Figure 4

Lycopene counteracted mitochondrial dysfunction caused by H₂O₂. (A) Measurement of mitochondrial permeability transition pore opening. (B) Analysis of mitochondrial membrane potentials by flow cytometry. (C) Western blot analysis of Bax and Bcl-2 protein levels within mitochondria. (D) Quantification of Bax and Bcl-2 protein levels. ^*P<0.01 vs. control group. ^#P<0.01 vs. H₂O₂ group. NRFU, normalized relative fluorescence units; Bcl-2, B-cell lymphoma 2; Bax, Bcl-2-like protein 4; COX IV, cytochrome c oxidase subunit IV; H₂O₂, hydrogen peroxide.

Figure 5

Lycopene attenuated the release of Cyt c from the mitochondria to the cytoplasm and inhibited the activation of caspase-3. (A) Western blot analysis of translocation of Cyt c from the mitochondria (Mito) to the cytoplasm (Cyto). (B) Quantification of the Cyt c protein levels in the mitochondria and cytoplasm. (C) Western blot analysis of the expression level of cleaved caspase-3. (D) Quantification of the cleaved caspase-3 protein level. ^*P<0.01 vs. control group. ^#P<0.01 vs. H₂O₂ group. Cyt c, cytochrhome c; COX IV, cytochrome c oxidase subunit IV; H₂O₂, hydrogen peroxide.

Figure 6

Lycopene suppressed the translocation of AIF from the mitochondria to the nuclei. (A) Western blot analysis of the AIF levels within the mitochondria (Mito) and nucleus (Nuc). (B) Quantification of the AIF levels. (C) Representative fluorescence microscopy images of the distribution of AIF. ^*P<0.01 vs. control group. ^#P<0.01 vs. H₂O₂ group. H₂O₂, hydrogen peroxide; AIF, apoptosis inducing factor; COX IV, cytochrome c oxidase subunit IV.