C-phycocyanin from Limnothrix Species KNUA002 Alleviates Cisplatin-Induced Ototoxicity by Blocking the Mitochondrial Apoptotic Pathway in Auditory Cells

Abstract

Ototoxicity, or adverse pharmacological effects on the inner ear or auditory nerve, is a common side effect of cisplatin, a platinum-based drug widely used in anticancer chemotherapy. Although the incidence of ototoxicity is high among patients that receive cisplatin therapy, there is currently no effective treatment for it. The generation of excessive reactive oxygen species (ROS) is considered to be the major cause of cisplatin-induced ototoxicity. C-phycocyanin (C-PC), a blue phycobiliprotein found in cyanobacteria and red algae, has antioxidant and anticancer activities in different experimental models in vitro and in vivo. Thus, we tested the ability of C-PC from Limnothrix sp. KNUA002 to protect auditory cells from cisplatin-induced ototoxicity in vitro. Pretreatment with C-PC from Limnothrix sp. KNUA002 inhibited apoptosis and protected mitochondrial function by preventing ROS accumulation in cisplatin-treated House Ear Institute-Organ of Corti 1 (HEI-OC1) cells, a mouse auditory cell line. Cisplatin increased the expression of Bax and reduced the expression of Bcl-2, which activate and inhibit, respectively, the mitochondrial apoptotic pathway in response to oxidative stress. Pretreatment with C-PC prior to cisplatin treatment caused the Bax and Bcl-2 levels to stay close to the levels in untreated control cells. Our results suggest that C-PC from Limnothrix sp. KNUA002 protects cells against cisplatin-induced cytotoxicity by inhibiting the mitochondrial apoptotic pathway.

Keywords: C-phycocyanin; HEI-OC1; Limnothrix; cisplatin; ototoxicity.

Figure 1

Effect of C-PC treatment on cell viability in cisplatin-treated HEI-OC1 cells. (A) Cells were cultured with 0.1, 0.5, 1, 2, 5, 10, or 20 μg/mL C-PC for 30 h. Cytotoxicity was evaluated by MTT assay. (B) Cells were treated with 0.1–20 μg/mL C-PC for 1 h and then treated with 30 μM CP for 30 h. Data represent the mean ± standard error of three separate experiments; * p < 0.05, compared with the cells treated with CP alone. C-PC, C-phycocyanin; CP, cisplatin.

Figure 2

Effect of C-PC on cell cycle arrest and apoptosis in cisplatin-treated HEI-OC1 cells. (A) Cell cycle analysis by flow cytometry and (B) comparison of the sub-G0/G1 ratio between the cells treated with CP alone and those pretreated with C-PC. (C) Western blot showing caspase-3 expression in cells treated with CP and C-PC. Data are shown as the mean ± standard deviation; * p < 0.05, compared with the cells treated with CP alone. C-PC, C-phycocyanin; CP, cisplatin. (D) TUNEL assay to detect apoptotic cells. Fragmented DNA (green) and nuclei (blue) were stained and observed under fluorescence microscopy. Scale bar represents 100 µm. The cells were pretreated with 1 μg/mL C-PC for 1 h, followed by treatment with 30 μM CP for 30 h.

Figure 3

ROS scavenging capacity of C-PC in cisplatin-treated cells. (A) Measurement of intracellular ROS levels using a fluorescent dye 2′, 7′-dichlorodihydrofluorescein diacetate (DCFH-DA) probe. The fluorescence intensity was detected by flow cytometry. (B) Relative fluorescence-activated cell sorting (FACS) fluorescence intensities. The cells were cultured with 1 μg/mL C-PC for 1 h, followed by treatment with 30 μM CP for 24 h. C-PC, C-phycocyanin; CP, cisplatin. Data are shown as the mean ± standard deviation; * p < 0.05, compared with the cells treated with CP alone.

Figure 4

Effect of C-PC on Bax-mediated apoptosis caused by cisplatin. (A) Western blot showing Bax expression, compared with β-actin as the loading control (n = 3 per lane). (B) Western blot showing Bcl-2 expression, compared with β-actin as the loading control (n = 3 per lane). (C) Relative changes in the Bax/Bcl-2 expression ratio. CP treatment upregulated Bax expression compared with that in the control cells, and pretreatment with C-PC significantly alleviated the CP-induced increase in Bax expression. CP treatment downregulated Bcl-2 expression, and C-PC pretreatment alleviated the CP-induced downregulation of Bcl-2 expression. Cells were incubated with 1 μg/mL C-PC for 1 h and/or 30 μM CP for 24 h. Data are shown as the mean ± standard deviation; * p < 0.05, compared with the cells treated with CP alone. C-PC, C-phycocyanin; CP, cisplatin.