Protective Effects of Laminarin on Cisplatin-induced Ototoxicity in HEI- OC1 Auditory Cells

Cisplatin is an effective chemotherapeutic agent against many common types of cancers. However, one of its most severe and debilitating side effects is ototoxicity. The purpose of this study was to investigate the mechanisms of laminarin protective effects on cisplatin-induced damage in HEI-OC1 auditory cell line. In our research, HEI-OC1 cells were pretreated with laminarin and then exposed to cisplatin. Here, we examined the cell viability, cell apoptosis and the factors associated with apoptotic pathway. Compared with cisplatin injured group, pretreatment with laminarin increased cell viability and decreased the cell apoptosis and necrosis. Furthermore, treatment with laminarin reduced intracellular ROS production, bax mRNA, and cleaved caspase-3, caspase-9, caspase-8 expression, but increased the bcl-2 mRNA level. The protection was ascribed to the radical scavenging activity of laminarin, and the inhibition of both mitochondrial apoptosis pathway and extrinsic apoptotic pathway.


Introduction
Cisplatin is widely used as chemotherapeutic agent against a variety of human neoplasms, including lung, testicular, cervical, and various head and neck cancers [1,2]. However, frequent administration of high doses of cisplatin has severe adverse effects, including ototoxicity, nephrotoxicity, neurotoxicity and bone marrow toxicity [1,3]. The ototoxicity was characterized by irreversible, progressive, bilateral, high-frequency, sensorineural hearing loss and may be accompanied by tinnitus [4]. The ototoxic mechanism of cisplatin has been disclosed to be related to several factors, including mitochondrial dysfunction, increased generation of intracellular ROS [5] and reactive nitrogen species [6], which was the widely accepted mechanism. Moreover, cisplatin can bind with guanine, leading to cell apoptosis via the formation of inter-and intrastrand DNA crosslinks, aberrant genetic transcription and cell cycle arrest [7]. All of these results which decrease the protective factors, such as glutathione and antioxidant enzymes in cochlear tissues, lead to the apoptosis and death of the outer hair cells of Corti [8].
Laminarin was naturally extracted from laminaria japonica, contained mannose (3.27%), arabi-nose (8.61%), glucose (4.23%), galactose (12.12%), fucose (46.93%), widely used in traditional Chinese medicine. It was introduced as a treatment for several diseases, including anticancer [9], immunoregulation [10], anticoagulation [11]. A previous study had suggested that the main compounds of laminarin (fucose and sulphate group) extracted from laminaria japonica were active antioxidants, especially in scavenging superoxide radical and hydroxyl radical. However, whether laminarin has protective effects on cisplatin-induced hair cell damage and the related mechanisms had not been defined. Furthermore, the HEI-OC1 is appropriate cell line for effectively studying signaling pathways, and has shown a good correlation with in vivo studies. Moreover, many studies have shown the HEI-OC1 cell line is also a good model for studying cisplatininduced ototoxicity [12].
Our study aimed to investigate the protective effects of laminarin on cisplatin-induced ototoxicity and the underlying mechanisms in HEI-OC1 cell line.

Cell viability assessment
The cisplatin (Sigma, USA) and laminarin (Sigma, USA) were freshly diluted in non-serum culture medium. The HEI-OC1 cells (5000 cells/well in a 96-well plate) were incubated in varying cisplatin concentrations (0-40 μM) for 24 h to determine the half maximal inhibitory concentration (IC50), and the cell viability was measured by MTT assay (Sigma, USA), as described previously [13].
The cells were incubated in varying laminarin concentrations (1-30 μM) for 12 h, then determined the dose-dependent influence of laminarin on cell viability by MTT.
The cells were seeded and incubated overnight. Then they were pretreated with varying doses of laminarin for 12 h, discarded the culture medium, replaced with medium containing 20 μM cisplatin and incubated for 24 h. Medium with vehicle alone was used as the control. MTT assay was used to measure the effects of laminarin on cell viability.

Hoechst 33258 staining
The cells were cultured in the medium with or without laminarin treatment (10 μM) for 12 h, and then replaced with medium with or without cisplatin (20 μM) for 24 h. Medium with vehicle alone was used as the control. The cells were washed twice with phosphate buffered saline, then fixed with 4% paraformaldehyde for 10 min at room temperature. After fixation, the cells were washed twice again with PBS and stained with Hoechst 33258 (Beyotime Company of Biotechnology, China) for 5 min. After washing twice, the cells were observed under confocal fluorescent microscopy ( Leica, Germany).

Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) double staining and flow cytometry
To quantify and verify the percentage of apoptotic and necrotic cells induced by cisplatin with or without laminarin pretreatment, we used Annexin V-FITC/PI double staining and flow cytometry to detect it. The cells were treated as described above, afterwards, we collected the cells and stained them by the Annexin V-FITC/PI apoptosis detection kit (Beyotime Company of Biotechnology, China) following the manufacturer's protocol. Cell distributions were detected by flow cytometry (Beckman Coulter, USA). Ten thousand cells were examined in each sample. The data were analyzed by FlowJo 7.6.1 software.

Measurement of intracellular ROS generation
The generation of ROS was detected by 5-(and 6)-carboxyl-20, 70dichlorodihydro fluorescein diacetate (DCFDA; Sigma, USA). The cells were seeded and incubated as mentioned previously. Thereafter, the cells were treated with 10 mM DCFDA in serum-free medium for 10 min at 33°C and protected from light. The oxidative burst was measured by flow cytometry with 480 nm excitation wavelength and 530 nm emission wavelengths.

mRNA extraction and quantitative real-time PCR (q-PCR)
Total RNA was extracted from samples by Trizol Reagent (Invitrogen, USA) according to the manufacturer's protocols. With the hexamer primer, 1 mg total mRNA was reverse-transcripted into cDNA by the Revert Aid kit (Fermentas, Canada). Real-time PCR was performed with the Light Cycler Fast Start DNA SYBR Green kit and run in triplicate on Master cycler ep realplex (Eppendorf AG, Germany). The primer sequences used for PCR amplification were as follows: bax

Western blotting analysis
The cells were seeded and incubated as previously mentioned. The isolation of proteins was achieved according to the manufacture's protocol (Beyotime, China). The protein concentration was determined using BCA method 20 μg of total protein were mixed with sample buffer containing 2% mercaptoethanol. The samples were heated at 95° for 5 min, and separated by 12% Tris-HCl gels electrophoresis. Gels were then transferred onto polyvinylidene difluoride membranes and blocked with 5% non-fat dry milk for 1 h at room temperature. Next, the membranes were incubated with 3% nonfat dry milk with the primary antibodies of cleaved caspase-3 (17 kDadiluted at 1:600, goat monoclonal, Life Technologies, USA), caspase-9 (35, 37 kDa, diluted at 1:1000, rabbit monoclonal, Life Technologies, USA), caspase-8 (55 kDa, diluted at 1:1000, rabbit polyclonal, Abcam, UK), β-actin (43 kDa, diluted at 1:1000, mouse monoclonal, Santa Cruz, USA), at 4°C overnight. Then the membranes were incubated with the secondary donkey anti-goat, anti-rabbit or anti-mouse IgG antibodies (1:5000, Santa Cruz, USA) for 1 h. The membranes were developed using an ECL detection kit (Santa Cruz, USA) and exposed to X-ray film. The relative expression level was calculated with the Image J software by comparison with β-actin.

Statistical analysis
Results were obtained from triplicate determinations and three independent experiments. Statistical analyses were performed by SPSS 17.0 software package. One-way ANOVA and factorial design were applied and data were expressed as the mean ± SD. P<0.05 was considered as statistically significant.

Effects of laminarin on cisplatin-induced apoptosis and necrosis in HEI-OC1 cells
The apoptosis of HEI-OC1 cells was determined by observing the nuclear morphology using Hoechst 33258 staining (Figure 2). The nuclei of normal cells were roundness with homogeneous intensity. Laminarin (10 μM) alone had no effect on the gross morphology of the HEI-OC1 cells compared with that of the controls. However, cisplatin (20 μM) alone lead to the characteristic apoptotic morphology of heterogeneous intensity, condensation and fragmentation of the nuclei.  Those results showed that cisplatin promoted apoptotic and necrotic cells, which was significantly inhibited by laminarin pretreatment.

Effects of laminarin on ROS generation
We measured the intracellular levels of ROS generated by HEI-OC1 cells in response to cisplatin and laminarin using the fluorescent probe DCFH-DA (Figure 4). Figure 4A showed that HEI-OC1 cells treated with 20 μM cisplatin significantly increased fluorescence intensity in comparison to the control group. Pretreatment with 10 μM laminarin for 12 h markedly reduced the cisplatin-induced generation of ROS in HEI-OC1 cells ( Figure 4A). The mean probe intensity in comparison to that of the control group is shown in Figure 4B. Treatment with 20 μM cisplatin markedly increased the fluorescence intensity, whereas pretreatment with laminarin resulted in a decrease in the fluorescence intensity compared with that of cisplatin alone, and treatment with laminarin alone did not alter the fluorescence.

Effects of laminarin on the apoptosis pathways
RT-PCR analysis indicated decreased bcl-2 mRNA and increased bax mRNA levels 24 h after cisplatin treatment. However, pretreatment with laminarin for 12 h resulted in a significant decrease in the bax mRNA expression ( Figure 5A) and increase in the bcl-2 mRNA expression ( Figure 5B) in comparison to treatment with cisplatin alone.
Simultaneously, we examined the effects of laminarin on cleaved caspase-3, caspase-8 and caspase-9 activation using western blot analysis ( Figure 6). Exposure of cells to 20 μM cisplatin increased the expression of cleaved caspase-3, caspase-8 and caspase-9. Pretreatment of cells with laminarin for 12 h prior to cisplatin treatment induced a marked reduction in cleaved caspase-3, caspase-8 and caspase-9 (P<0.05), in comparison to the cisplatin-treated group.  The overexpression of ROS caused by cisplatin was recognized as a central mechanism of its ototoxicity [14], which can overwhelm the antioxidant defense mechanisms in the cochlea, leading to mitochondrial release of cytochrome c, activating the apoptosis pathway, inducing out hair cells death. Therefore, antioxidants could be a desirable method for protection against cisplatin-induced hearing loss [15]. Laminarin is a tropical plant traditionally used in Chinese medicine, which has been proven to inhibit sepsis-induced oxidative stress, lipid peroxidation in rats by inhibiting caspase-3 activity and enhancing SOD, CAT and GSH-Px activities [16]. Our results showed the laminarin decreased the cisplatin-induced ROS production, indicating its antioxidant activity.
We evaluated the caspases pathways, which are the key markers of apoptosis, and have been proven to be key factors in cisplatin-induced apoptosis of hair cells [2]. To address the apoptosis, we focused on bcl-2, bax, caspase-3, caspase-8 and caspase-9. In this study, caspase-3, an ultimate mediator in apoptosis [4], was down-regulated when the cells were pretreated with laminarin, indicating that the preventive effect of laminarin is related to the apoptosis pathway. For the apoptotic pathway, it contains mitochondrial apoptotic pathway and extrinsic apoptotic pathway. Previous findings have indicated that Bax/ Bcl-2 and caspase-9 in mitochondrial pathways have a critical role in cisplatin-induced apoptosis of auditory cells [17,18]. Our results showed that laminarin reduced the levels of bax mRNA and caspase-9, elevated the levels of bcl-2 mRNA, which indicated laminarin protect cells from cisplatin-induced auditory cell damage through inhibiting the mitochondrial apoptotic pathway. For the extrinsic apoptotic pathway, caspase-8 was thought to be the primary mediator, which can cause cellular destruction by apoptosis without mitochondria participation [19]. Therefore, we assayed caspase-8 expression, which showed a decrease when pretreated with laminarin in cell. These results demonstrated that the preventive effect of laminarin is also related to the inhibition of extrinsic apoptotic pathway.
Despite the protective effect of laminarin against cisplatin-induced ototoxicity, its clinical application in patients remains inconclusive. Additionally, it must be noted that high doses of laminarin have a cytotoxic effect instead of the beneficial effects. Future studies will concentrate on animal models to confirm the underlying protective mechanisms and to establish an efficacious laminarin dose before any clinical recommendations.
In conclusion, we have observed that low doses of laminarin could provide protective effects against cisplatin-induced ototoxicity. Moreover, the preventive effect of laminarin is related to its radical scavenging effect and inhibition of both mitochondrial apoptotic pathway and extrinsic apoptotic pathway.