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mitochondrial pathway
Toxicology 248 (2008) 33–38
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Toxicology journal homepage: www.elsevier.com/locate/toxicol
Adenoviral-mediated up-regulation of Otos, a novel specific cochlear gene, decreases cisplatin-induced apoptosis of cultured spiral ligament fibrocytes via MAPK/mitochondrial pathway Xian-Lu Zhuo a,1,2 , Yan Wang b,1 , Wen-Lei Zhuo c,∗,1 , Yun-Song Zhang d , Yun-Jun Wei a , Xue-Yuan Zhang a,∗ a
Department of Otolaryngology, Southwest Hospital, Third Military Medical University, Chongqing, China Institute of Respiratory Diseases, Xinqiao Hospital, Third Military Medical University, Chongqing, China c Institute of Cancer, Xinqiao Hospital, Third Military Medical University, Chongqing, China d Department of Thoracic Surgery, Daping Hospital, Third Military Medical University, Chongqing, China b
a r t i c l e
i n f o
Article history: Received 7 February 2008 Received in revised form 6 March 2008 Accepted 6 March 2008 Available online 20 March 2008 Keywords: Otos Spiral ligament fibrocytes Cisplatin Recombinant Adenovirus Up-regulation
a b s t r a c t Previous reports have implicated Otos, a novel specific gene expressed by spiral ligament fibrocytes (SLFs) with unclear functions, as a protective gene for cochlea. However, whether Otos gene could protect SLFs against cisplatin (DDP)-induced apoptosis remains largely unknown. In the present study, we utilized Adenoviral-mediated gene transfection to up-regulate Otos expression in cultured SLFs and further assessed the cell viability and apoptosis as well as possible MAPK and mitochondrial pathways. As expected, the data showed that Otos up-regulation significantly decreased apoptosis of SLFs induced by DDP possibly through activation of ERK and partial inhibition of JNK and mitochondrial pathway but not p-38 pathway, suggesting Otos as a potential protective gene for cochlea and raising the possibility of Otos up-regulation as a promising approach to DDP-induced deafness therapy. © 2008 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Cisplatin (DDP), a DNA damaging agent, has been widely used in the treatment of a variety of solid cancers such as lung cancer and head and neck cancers. Its use in these and other types of tumors is limited by onset of chemoresistance and severe undesired side effects, such as nephro- and ototoxicity, whose mechanisms of action are not fully understood. The complications such as sensorineural deafness often result in poor quality of life in patients. Previous studies have conducted on the possible approaches to DDP-induced deafness prevention and therapy. Liu et al. (1998) and Wang et al. (2004) tried to prevent inner ear cells from DDP-
Abbreviations: DMEM, Dulbecco’s Modified Eagle’s Medium; DMSO, dimethyl sulfoxide; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; PBS, phosphate-buffered saline; SLFs, spiral ligament fibrocytes. ∗ Corresponding authors. E-mail addresses: [email protected] (W.-L. Zhuo), [email protected] (X.-Y. Zhang). 1 These authors contributed equally to this work. 2 The author works at Guiyang Medical College. 0300-483X/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.tox.2008.03.004
induced damage through utilization of caspase inhibitors, resulting in a decrease in cochlear cell apoptosis. Also, application of cannabinoid receptor 2 agonist (Jeong et al., 2007) and T-type calcium channel blocker (So et al., 2005) have been shown to exert preventive effects on DDP-damaged inner ear cells. Moreover, Chinese herbal medicine was suggested to play a preventive role in DDPinduced hearing loss (Lu et al., 2004). However, the therapeutic effects of these agents were weakened because of the presence of blood–labyrinth barrier that hampers entrance of the drugs into cochlea. Therefore, to find new methods for prevention of DDPinduced deafness is required. Gene therapy might be a promising approach. Recent studies have shown that adeno-associated virusmediated up-regulation of some genes such as XIAP inhibitor (Cooper et al., 2006), X-linked inhibitor (Chan et al., 2007) and neurotrophin-3 (Bowers et al., 2002) significantly rescued inner ear cells from DDP-induced apoptosis. However, little is known about the roles and mechanisms of other genes expressed in cochlea. Spiral ligament fibrocytes (SLFs) are thought to play an essential role in cochlear fluid and ion homeostasis. DDP-induced SLF apoptosis has been shown to be a cause of hearing loss. A number of genes have been detected in SLFs. Mutation or loss of these
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genes such as Cx26, Cx29, and Cx43 (Yang et al., 2005) as well as COCH (Collin et al., 2006), a novel suspected Meniere’s Diseaseassociated gene, have been identified to correlate with hearing loss. DDP application can cause functional disruption of SLFs, which may result in decreased endolymphatic potassium concentration and loss of endolymphatic potential, thereby leading to structural or functional deterioration of hear cells and spiral ganglion neurons. Therefore, preventing SLFs from DDP-induced damage might be a potential strategy for DDP-associated deafness prevention. Otos is a novel gene expressed specifically by SLFs, encoding a 6.4 kDa protein called Otospiralin that has been found to be an essential protein in the maintenance of normal hearing with unknown functions. Delprat et al. (2002) suppressed Otos gene expression via cochlear perfusion of antisense oligonucleotides, leading to irreversible deafness in guinea pigs, accompanied by inner ear cell apoptosis. Likewise, similar results were observed via Otos gene knockout in their further in vivo study (Delprat et al., 2005). According to Caravelli et al. (2004), noise stimulation might cause hearing loss through down-regulation of Otos gene. Accordingly, Otos gene seems to provide a survival signal for SLFs. However, to the best of our knowledge, whether Otos gene has a preventive effect on DDP-induced SLF damage has not virtually been reported in the open literature. Therefore, we hypothesized that up-regulation of Otos gene might attenuate DDP-induced inner ear cell apoptosis, thus contributing to prevention of hearing loss. In the present study, we aimed to determine whether upregulation of Otos gene in SLFs could decrease DDP-induced cell apoptosis. Recombinant Adenoviral-associated transfection was employed to up-regulate Otos gene expression in cultured SLFs. The cell viabilities and apoptosis as well as possible involvement of signaling pathways in response to DDP were further assessed.
CCCACCCGCACCCACCACT-3 ; for GAPDH: F: 5 -CAGTGCCAGCCTCGTCTCAT-3 and R: 5 -AGGGGCCATCCACAGTCTTC-3 . Thermal cycler parameters included 1 cycle at 94 ◦ C for 0.5 min, and 30 cycles involving denaturation at 94 ◦ C for 30 s annealing at 58 ◦ C for 45 s and extension at 72 ◦ C for 60 s. Extension was performed for an additional 10 min after the completion of the indicated cycles. The bands were quantified by densitometric scanning of band intensities and normalized to the levels of GAPDH using Image-Pro Plus 5.0 software (Media Cybernetics, Silver Spring, MD, USA).
2.4. Recombinant Adenoviral vectors construction and cell transfection The full length of Otos gene was obtained from RNA of the cultured SLFs by using RT-PCR as mentioned above. The cloned Otos cDNA fragment was ligated into the pAd-Track-GFP vector expressing GFP to form pAdTrack-GFP/Otos expressing both GFP and Otos. The recombinant pAdeasy-1 backbone vector and pAdTrack-GFP/Otos or pAdTrack-GFP vector, DNA linealized with PmeI digestion, were further cotransfected into the bacteria BJ5183 cells. The resultant pAdGFP/Otos and pAdGFP plasmid vectors were purified from the above transfected BJ5183 cells, then linealized by PacI digestion, and then transfected into the human embryonic kidney 293 (QBI-293A) cells by lipofectamine (Sigma; St. Louis, MO), leading to the formation of the recombinant adenoviruses Ad-GFP expressing GFP and Ad-Otos expressing both GFP and Otos. The Ad-viruses were amplified in QBI-293A cells, purified by cesium chloride ultracentrifugation, and stored at −80 ◦ C before use. For assessment of the optimal multiples of infection (MOIs) for maximal transgene expression, SLFs were infected with Ad-Otos or Ad-GFP at various MOIs and examined by fluorescence microscopy.
2.5. Cell viability assay For quantitative viability assays, cells were plated in 96-well plates (1 × 104 cells/well). MTT assays were used to assess cell viability. 200 l sterile MTT dye (5 mg/ml, sigma, USA) was added. After 4 h incubation at 37 ◦ C in 5% CO2 , MTT medium mixture was removed and 200 l of dimethyl sulfoxide (DMSO) was added to each well. Absorbance was measured at 490 nm using a multi-well spectrophotometer (Thermo Electron, Andover, USA).
2. Materials and methods 2.6. TUNEL staining 2.1. Reagents DDP, SP600125, PD98059 and SB203580 were purchased from Sigma Company (USA). Antibodies to Caldesmon, Na–K-ATPase, S-100, cytokeratin, GAPDH, p-JNK, JNK, ERK, p-ERK, p-38, p-p38, Bax, Bcl-2, and caspase-3 (cleaved) were purchased from Zhongshan Biotech Company (China). DDP was diluted to various concentrations with modified Eagle’s medium (DMEM). 2.2. Cell culture and immunofluorescence analysis Culture of rat SLFs was carried out as described previously (Moon et al., 2006). In brief, 7-day-old SD rats were obtained from the central laboratories of Third Military Medical University. The rats were anesthetized using ketamine (5 mg/100 g) and then decapitated. In sterile phosphate-buffered saline (PBS), the cochlea were removed from the temporal bone and then dissected free from its bony shell. The basal turn was cut from the cochlea, and transferred to a 35 mm cell culture dish. Sections of spiral ligament only located beneath the stria vascularis were carefully obtained. Explants were plated onto collagen-coated Petri dished in DMEM and 20% fetal bovine serum (FBS). They were kept in an incubator at 37 ◦ C in an atmosphere of 5% CO2 . After approximately 2 weeks in primary culture, nearly confluent monolayer cells were subcultured. For determining the types of cultured SLFs, cells seeded on slides were harvested and fixed in 0.5 ml of 4% paraformaldehyde, washed twice with PBS, and incubated with primary antibodies for 1 h at 37 ◦ C. The sources of antibodies were as follows: Caldesmon, Na–K-ATPase, S-100, cytokeratin goat polyclonal (1:500). The antibodies are specific and do not cross-react with other proteins. The cells were then washed and stained with FITC-conjugated rabbit anti-goat antibody (Zhongshan Biotech Company, China) and left for incubation in the dark. After 15 min, the cells were subsequently washed twice by double distilled H2 O (ddH2 O) and observed by a fluorescent microscopy (Leica DMRXA2, Germany) with excitation and emission settings at 488 and 530 nm, respectively.
Cells were plated on polylysine-coated slides, fixed with 4% paraformaldehyde in 0.1 M PBS for 1 h at 25 ◦ C, rinsed with 0.1 M PBS, pH 7.4, and permeabilized with 1% Triton X-100 in 0.01 M citrate buffer, pH 6.0. DNA fragmentation was detected using TUNEL detection kit (Roche Clinical Laboratories, Indianapolis, IN), which specifically labeled 3 -hydroxyl termini of DNA strand breaks using tetramethylrhodamine isothiocyanate (TRITC)-conjugated dUTP. DNA was also labeled with TRITC DNAbinding dye for 5 min. TRITC labels were observed with a fluorescence microscope. The percentage of apoptotic cells was calculated as the number of apoptotic cells per number of total cells × 100%.
2.7. Western blot assay Cells were harvested, pelleted by centrifugation, washed with ice-cold PBS, and lysed with RIPA buffer (150 mM NaCl, 50 mM Tris base pH 8.0, 1 mM EDTA, 0.5% sodium deoxycholate, 1% NP-40, 0.1% sodium dodecyl sulfate, 1 mM DTT, 1 mM PMSF, and 1 mM Na3 VO4 ) supplemented with protease inhibitor. Proteins were separated by 10% SDS-polyacrylamide gel electrophoresis and transferred onto a nitrocellulose membrane (Life Technologies, Gaithersburg, MD). Blots were then incubated in fresh blocking solution with an appropriate dilution of primary antibody at 4 ◦ C for 24 h. The sources of antibodies were as follows: GAPDH, p-JNK, JNK, ERK, p-ERK goat polyclonal; p-38, p-p38, Bax, Bcl-2, and caspase-3 (cleaved) rabbit polyclonal. After extensive washing, the membranes were incubated with horseradish peroxidase (HRP)-coupled secondary antibody (1:2000, Zhongshan Biotech Company, China) at 25 ◦ C for 1 h. The bands were visualized and quantified using Image-Pro Plus 5.0 software (Media Cybernetics, Silver Spring, MD, USA). The p-JNK, p-ERK, p-p38 band intensities were normalized to JNK, ERK, p-38 band intensities, respectively and the intensities of Bax, Bcl-2, and caspase-3 were adjusted by the GAPDH band intensity.
2.3. Semi-quantitative RT-PCR assay
2.8. Statistical analysis
Total RNA was isolated with TRIzol Reagent (Invitrogen) and first strand cDNA was synthesized from 1 g total RNA using Oligo d(T) primer (Invitrogen) and ReveTra Ace (TOYOBO, Osaka, Japan). PCR was done on the cDNA product using the following primers for Otos: F: 5 -TACGGCTGCGAGAAGACGACA-3 and R: 5 -
Data were expressed as mean value ± S.D. Differences between groups were analyzed with analysis of variances (ANOVA) or a t-test. These analyses were performed by utilizing SPSS for Windows Version 13.0 (SPSS Inc., Chicago, IL). P value less than 0.05 was considered statistically significant.
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To assess the optimal MOI for a maximal transgene expression, we transfected SLFs with Ad-Otos or Ad-GFP at various MOIs (12.5, 25, 50, 75, 100) and for varying time length (24, 48, 72 h), respectively. The results showed that more than 90% of GFP expression was found in SLFs transfected with Ad-viruses at a MOI of 50 and above for 48 h and above. Therefore, we selected a MOI of 50 with a time length of 48 h as an optimal dose and time for the transfection of the SLFs. We then transfected cells with Ad-Otos (SLFs-AdOtos) and AdGFP (SLFs-AdGFP) at a MOI of 50 for 48 h. Afterwards, RNA extracted from transfected SLFs was subjected to a RT-PCR analysis. As shown in Fig. 1B, a significant increase in Otos mRNA expression was found in SLFs-AdOtos but not in SLFs-AdGFP and SLFs-nontransfection, suggesting that vector-mediated transfection could effectively elevate Otos expression in cultured SLFs. 3.2. Otos gene elevation decreased apoptosis of SLFs in response to DDP
Fig. 1. (A) Cultured SLFs (400×) and (B) expression of Otos mRNA in SLFs-AdOtos were significantly higher than those in SLFs-AdGFP or SLFs-nontransfection, respectively (*P < 0.01 vs. 1 or 3). (1) SLFs-AdGFP; (2) SLFs-AdOtos; (3) SLFs-nontransfection.
3. Results and discussions 3.1. Vector-mediated transfection led to up-regulation of Otos The structural characteristics of the cultured cells were consistent with those of fibrocytes (Fig. 1A). The cells were immunopositive for caldesmon and S-100 but negative for Na–KATPase and cytokeratin. The results supported the notion that the cultured cells can be classified as type I fibrocytes, in line with previous report (Maeda et al., 2005).
To investigate the possible roles of Otos in DDP-induced SLFs apoptosis, we treated the cells with various concentrations (0, 5, 10, 20 M) of DDP for 12 h and 20 M DDP for various time length (0, 6, 12, 18 h), respectively. The results showed that elevation of Otos expression resulted in a significant increase in cell viability and a decrease in apoptosis of SLFs in response to DDP in both dose- and time-dependent manners compared with those of SLFs-GFP or SLFs-nontransfection cells (Fig. 2). The data indicate that Otos up-regulation may markedly enhance chemoresistance of SLFs to DDP. Notably, with low concentrations of DDP (0 and 5 M), the results of MTT and TUNEL showed little difference among the three types of cells, suggesting that sole up-regulation of Otos gene exerts slight effects on spontaneous apoptosis of SLFs. 3.3. Up-regulation of Otos decreased DDP-induced apoptosis of SLFs possibly through activation of MAPK/mitochondrial pathway The underlying mechanisms by which Otos up-regulation enhances chemoresistance of SLFs to DDP are not fully understood. A growing body of evidence suggests the possible involvement of some signaling pathways in DDP-induced cell apoptosis.
Fig. 2. (A) Effects of various concentration (0, 5, 10, 20 M) of DDP on the three types of cells for 12 h assessed by MTT and TUNEL assays, respectively (*P < 0.05 vs. 1 or 3, n = 6). (B) Effects of 20 M DDP on the three types of cells for varied time length (0, 6, 12, 18 h) assessed by MTT and TUNEL assays, respectively (*P < 0.05 vs. 1 or 3, n = 6). (1) SLFs-AdGFP; (2) SLFs-AdOtos; (3) SLFs-nontransfection.
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MAPKs, mainly comprising ERK, p38, and c-Jun N-terminal kinase (JNK), are suggested to be associated with apoptosis media´ tion of various cells in response to DDP (Sanchez-Perez et al., 1998; Wang et al., 2006; Winograd-Katz and Levitzki (2006)). Previati et al. (2007) reported that ERK activation might play a role in DDP-induced cultured inner cell apoptosis. According to Choi et al. (2007), JNK pathway activation might protect cochlear cells against DDP-induced apoptosis. However, the precise pathways by which vector-mediated up-regulation of Otos decreases sensitivity of SLFs to DDP are largely unclear. Two major apoptosis pathways have previously been described (Ashkenazi and Dixit, 1998). One pathway is mediated by death receptors, such as CD95 (Fas) and tumor necrosis factor receptors, and is casepase-8 dependent. The other is the mitochondrial pathway that involves Bcl-2 and Bax proteins. The Bcl-2 family consists of both pro-apoptotic and anti-apoptotic members that elicit opposing effects on mitochondria. The balance between proapoptotic and anti-apoptotic members determines apoptosis or survival. Bax can promote the release of cytochrome C into the cytosol from mitochondria, which in turn activates caspase-3, one of the key executioners of apoptosis. Evidence suggests that MAPKs might regulate mitochondrial pathway activation in cell apoptosis (Hong and Kim, 2007; Goel
et al., 2007; Park et al., 2007). To better understand the precise mechanisms underlying DDP-induced SLFs-AdOtos apoptosis, we hypothesized the involvement of MAPKs as well as mitochondrial pathway in this process. To test this, we conducted further experiments. Cells were divided into two major groups, both of which contained three types of cells, respectively, namely, SLFs-AdGFP, SLFs-AdOtos, and SLFs-nontransfection. The two groups were treated with DMEM or 20 M DDP for 12 h, respectively. Then the cell viability and apoptosis were analyzed by MTT and TUNEL assays. Moreover, JNK, p-JNK, ERK, p-ERK, p-38, p-p38, Bcl-2, Bax, and caspase-3 were further assessed by immunoblotting. The results (Fig. 3) showed a marked increase in p-ERK expression and a significant decrease in p-JNK expression in SLFs-AdOtos cells treated with DDP compared with those of SLFs-AdGFP or SLFsnontransfection cells in the same group, accordingly accompanied by decreased cell apoptosis and reduced mitochondrial pathway activation. Nevertheless, in DMEM group, the three MAPK pathways as well as mitochondrial pathway are unlikely to be activated among the three types of cells. The data suggest that Otos up-regulation might rescue SLFs from DDP-induced apoptosis through activation of ERK, blockade of JNK as well as inactivation of mitochondrial pathway.
Fig. 3. Analysis of the results of SLFs-AdGFP, SLFs-AdOtos and SLFs-nontransfection treated with DMEM or DDP. (A and B) The cell viability and apoptosis assessed by MTT (A) and TUNEL (B), respectively (*P < 0.05 vs. 1 or 3, n = 6). (C) Expression of the MAPK and mitochondrial pathway-related proteins assessed by immunoblotting. (1) SLFs-AdGFP; (2) SLFs-AdOtos; (3) SLFs-nontransfection.
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Fig. 4. Analysis of the results of SLFs-Otos treated with DMEM (I), DDP (II), DDP + SP600125 (III), DDP + PD98059 (IV) and DDP + SB203580 (V). The cell viability and apoptosis were assessed by MTT (A) and TUNEL (B), respectively (*P < 0.05 vs. I or II; †P < 0.05 vs. II; ‡P > 0.05 vs. II, n = 6). Further, the proteins (C) of caspase-3, Bax, Bcl-2 and GAPDH were assessed by immunoblotting, respectively.
3.4. Activation of mitochondrial pathway might be regulated by JNK and ERK signaling pathway In order to further test the possible roles of MAPK pathways, we divided SLFs-Otos cells into five groups marked I–V. Cells in group I was treated with DMEM as a control whereas cells in the remaining four groups were treated with 20 M DDP for 12 h. Nevertheless, cells in group III were pre-treated with 10 M SP600125 (a specific JNK inhibitor), in group IV with 10 M PD98059 (a specific ERK inhibitor), in group V with 10 M SB203580 (a specific p-38 inhibitor) for 1 h prior to treatment with DDP. Then the cell viability and apoptosis were assessed by MTT and TUNEL assays. As expected, pretreatment with SP600125 seemed to decrease SLFs-Otos cell apoptosis induced by DDP, whereas pretreatment with PD98059 significantly increased DDP-induced SLFs-Otos cell apoptosis, confirming the involvement of activation of ERK and partial inhibition of JNK pathway as one of the underlying mechanisms (Fig. 4). In this process, JNK activation seemed to provide an apoptotic signal, whereas ERK activation provided a survival signal, in line with previous studies (Tsuruta et al., 2004; Xia et al., 1995). In addition, western blot further confirmed the mitochondrial pathway activation in DDP-induced SLFs cell apoptosis. Interestingly, blockade of JNK markedly attenuated the mitochondrial pathway activation, whereas inhibition of ERK significantly promoted its activation, suggesting the possibility of both JNK and ERK mediating mitochondrial pathway activation, in accordance with previous reports (Hong and Kim, 2007; Goel et al., 2007; Park et al., 2007). However, whether mitochondrial pathway factors are downstream targets of MAPKs remains to be elucidated in further investigations. In the present study, to our knowledge, we provided the first insight into the roles and possible mechanisms by which Otos up-regulation protects SLFs against DDP-induced apoptosis. Since DDP-associated impairments of cochlear cells are thought to have a correlation with oxidation of free radical that leads to cell apoptosis, Otos gene might function as a factor playing a role in anti-oxidative stress. Also, Otos might act as a transcriptional factor that mediates other protective gene expressions in cochlea (Delprat et al., 2002). Notably, the protective effects that Otos up-regulation exerted on DDP-damaged SLFs were limited, though the effects were evident.
Moreover, activation of JNK was likely to provide an apoptotic signal, and nevertheless, blockade of JNK could incompletely inhibit DDP-induced SLFs-Otos cell apoptosis, suggesting that in addition to MAPK pathways, other signaling pathways might play roles in this process. However, the precise mechanisms are unclear and remain to be clarified in further studies. There were several limitations in this study. First, since antibodies to Otospiralin could not be commercially purchased and the specificity of the self-made antibodies in the literature (Delprat et al., 2002) was uncertain, we did not detect the expressions of Otospiralin protein. Nevertheless, the premiers of Otos gene for RT-PCR assay had a high specificity to Otos mRNA. The results confirmed that the Otos mRNA were markedly up-regulated in SLFs after AdOtos transfection. Second, this study had focused on the roles as well as possible mechanisms of Otos gene up-regulation in vitro. Further in vivo studies are needed to clarify the precise underlying mechanisms and to estimate the feasibility of Otos gene therapy. However, the results of this study implicate Otos as a preventive gene for SLFs and suggest Otos up-regulation as a potential strategy for DDP-induced deafness therapy. In conclusion, we successfully up-regulated Otos expression by using Adenoviral-mediated transfection in SLFs, which resulted in a marked decrease in DDP-induced SLF cell apoptosis, suggesting that Otos up-regulation in SLFs might protect the cells against DDP-induced apoptosis and raising the possibility that Otos up-regulation might be a promising approach to DDP-induced deafness therapy. Acknowledgements This work was partially supported by the specific funds of Third Military Medical University for postgraduates (2005256). We take this opportunity to specifically thank the reviewers and editors for their kind instructions that may be helpful for our further studies. References Ashkenazi, A., Dixit, V.M., 1998. Death receptors: signaling and modulation. Science 281, 1305–1308.
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Toxicology journal homepage: www.elsevier.com/locate/toxicol
Adenoviral-mediated up-regulation of Otos, a novel specific cochlear gene, decreases cisplatin-induced apoptosis of cultured spiral ligament fibrocytes via MAPK/mitochondrial pathway Xian-Lu Zhuo a,1,2 , Yan Wang b,1 , Wen-Lei Zhuo c,∗,1 , Yun-Song Zhang d , Yun-Jun Wei a , Xue-Yuan Zhang a,∗ a
Department of Otolaryngology, Southwest Hospital, Third Military Medical University, Chongqing, China Institute of Respiratory Diseases, Xinqiao Hospital, Third Military Medical University, Chongqing, China c Institute of Cancer, Xinqiao Hospital, Third Military Medical University, Chongqing, China d Department of Thoracic Surgery, Daping Hospital, Third Military Medical University, Chongqing, China b
a r t i c l e
i n f o
Article history: Received 7 February 2008 Received in revised form 6 March 2008 Accepted 6 March 2008 Available online 20 March 2008 Keywords: Otos Spiral ligament fibrocytes Cisplatin Recombinant Adenovirus Up-regulation
a b s t r a c t Previous reports have implicated Otos, a novel specific gene expressed by spiral ligament fibrocytes (SLFs) with unclear functions, as a protective gene for cochlea. However, whether Otos gene could protect SLFs against cisplatin (DDP)-induced apoptosis remains largely unknown. In the present study, we utilized Adenoviral-mediated gene transfection to up-regulate Otos expression in cultured SLFs and further assessed the cell viability and apoptosis as well as possible MAPK and mitochondrial pathways. As expected, the data showed that Otos up-regulation significantly decreased apoptosis of SLFs induced by DDP possibly through activation of ERK and partial inhibition of JNK and mitochondrial pathway but not p-38 pathway, suggesting Otos as a potential protective gene for cochlea and raising the possibility of Otos up-regulation as a promising approach to DDP-induced deafness therapy. © 2008 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Cisplatin (DDP), a DNA damaging agent, has been widely used in the treatment of a variety of solid cancers such as lung cancer and head and neck cancers. Its use in these and other types of tumors is limited by onset of chemoresistance and severe undesired side effects, such as nephro- and ototoxicity, whose mechanisms of action are not fully understood. The complications such as sensorineural deafness often result in poor quality of life in patients. Previous studies have conducted on the possible approaches to DDP-induced deafness prevention and therapy. Liu et al. (1998) and Wang et al. (2004) tried to prevent inner ear cells from DDP-
Abbreviations: DMEM, Dulbecco’s Modified Eagle’s Medium; DMSO, dimethyl sulfoxide; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; PBS, phosphate-buffered saline; SLFs, spiral ligament fibrocytes. ∗ Corresponding authors. E-mail addresses: [email protected] (W.-L. Zhuo), [email protected] (X.-Y. Zhang). 1 These authors contributed equally to this work. 2 The author works at Guiyang Medical College. 0300-483X/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.tox.2008.03.004
induced damage through utilization of caspase inhibitors, resulting in a decrease in cochlear cell apoptosis. Also, application of cannabinoid receptor 2 agonist (Jeong et al., 2007) and T-type calcium channel blocker (So et al., 2005) have been shown to exert preventive effects on DDP-damaged inner ear cells. Moreover, Chinese herbal medicine was suggested to play a preventive role in DDPinduced hearing loss (Lu et al., 2004). However, the therapeutic effects of these agents were weakened because of the presence of blood–labyrinth barrier that hampers entrance of the drugs into cochlea. Therefore, to find new methods for prevention of DDPinduced deafness is required. Gene therapy might be a promising approach. Recent studies have shown that adeno-associated virusmediated up-regulation of some genes such as XIAP inhibitor (Cooper et al., 2006), X-linked inhibitor (Chan et al., 2007) and neurotrophin-3 (Bowers et al., 2002) significantly rescued inner ear cells from DDP-induced apoptosis. However, little is known about the roles and mechanisms of other genes expressed in cochlea. Spiral ligament fibrocytes (SLFs) are thought to play an essential role in cochlear fluid and ion homeostasis. DDP-induced SLF apoptosis has been shown to be a cause of hearing loss. A number of genes have been detected in SLFs. Mutation or loss of these
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genes such as Cx26, Cx29, and Cx43 (Yang et al., 2005) as well as COCH (Collin et al., 2006), a novel suspected Meniere’s Diseaseassociated gene, have been identified to correlate with hearing loss. DDP application can cause functional disruption of SLFs, which may result in decreased endolymphatic potassium concentration and loss of endolymphatic potential, thereby leading to structural or functional deterioration of hear cells and spiral ganglion neurons. Therefore, preventing SLFs from DDP-induced damage might be a potential strategy for DDP-associated deafness prevention. Otos is a novel gene expressed specifically by SLFs, encoding a 6.4 kDa protein called Otospiralin that has been found to be an essential protein in the maintenance of normal hearing with unknown functions. Delprat et al. (2002) suppressed Otos gene expression via cochlear perfusion of antisense oligonucleotides, leading to irreversible deafness in guinea pigs, accompanied by inner ear cell apoptosis. Likewise, similar results were observed via Otos gene knockout in their further in vivo study (Delprat et al., 2005). According to Caravelli et al. (2004), noise stimulation might cause hearing loss through down-regulation of Otos gene. Accordingly, Otos gene seems to provide a survival signal for SLFs. However, to the best of our knowledge, whether Otos gene has a preventive effect on DDP-induced SLF damage has not virtually been reported in the open literature. Therefore, we hypothesized that up-regulation of Otos gene might attenuate DDP-induced inner ear cell apoptosis, thus contributing to prevention of hearing loss. In the present study, we aimed to determine whether upregulation of Otos gene in SLFs could decrease DDP-induced cell apoptosis. Recombinant Adenoviral-associated transfection was employed to up-regulate Otos gene expression in cultured SLFs. The cell viabilities and apoptosis as well as possible involvement of signaling pathways in response to DDP were further assessed.
CCCACCCGCACCCACCACT-3 ; for GAPDH: F: 5 -CAGTGCCAGCCTCGTCTCAT-3 and R: 5 -AGGGGCCATCCACAGTCTTC-3 . Thermal cycler parameters included 1 cycle at 94 ◦ C for 0.5 min, and 30 cycles involving denaturation at 94 ◦ C for 30 s annealing at 58 ◦ C for 45 s and extension at 72 ◦ C for 60 s. Extension was performed for an additional 10 min after the completion of the indicated cycles. The bands were quantified by densitometric scanning of band intensities and normalized to the levels of GAPDH using Image-Pro Plus 5.0 software (Media Cybernetics, Silver Spring, MD, USA).
2.4. Recombinant Adenoviral vectors construction and cell transfection The full length of Otos gene was obtained from RNA of the cultured SLFs by using RT-PCR as mentioned above. The cloned Otos cDNA fragment was ligated into the pAd-Track-GFP vector expressing GFP to form pAdTrack-GFP/Otos expressing both GFP and Otos. The recombinant pAdeasy-1 backbone vector and pAdTrack-GFP/Otos or pAdTrack-GFP vector, DNA linealized with PmeI digestion, were further cotransfected into the bacteria BJ5183 cells. The resultant pAdGFP/Otos and pAdGFP plasmid vectors were purified from the above transfected BJ5183 cells, then linealized by PacI digestion, and then transfected into the human embryonic kidney 293 (QBI-293A) cells by lipofectamine (Sigma; St. Louis, MO), leading to the formation of the recombinant adenoviruses Ad-GFP expressing GFP and Ad-Otos expressing both GFP and Otos. The Ad-viruses were amplified in QBI-293A cells, purified by cesium chloride ultracentrifugation, and stored at −80 ◦ C before use. For assessment of the optimal multiples of infection (MOIs) for maximal transgene expression, SLFs were infected with Ad-Otos or Ad-GFP at various MOIs and examined by fluorescence microscopy.
2.5. Cell viability assay For quantitative viability assays, cells were plated in 96-well plates (1 × 104 cells/well). MTT assays were used to assess cell viability. 200 l sterile MTT dye (5 mg/ml, sigma, USA) was added. After 4 h incubation at 37 ◦ C in 5% CO2 , MTT medium mixture was removed and 200 l of dimethyl sulfoxide (DMSO) was added to each well. Absorbance was measured at 490 nm using a multi-well spectrophotometer (Thermo Electron, Andover, USA).
2. Materials and methods 2.6. TUNEL staining 2.1. Reagents DDP, SP600125, PD98059 and SB203580 were purchased from Sigma Company (USA). Antibodies to Caldesmon, Na–K-ATPase, S-100, cytokeratin, GAPDH, p-JNK, JNK, ERK, p-ERK, p-38, p-p38, Bax, Bcl-2, and caspase-3 (cleaved) were purchased from Zhongshan Biotech Company (China). DDP was diluted to various concentrations with modified Eagle’s medium (DMEM). 2.2. Cell culture and immunofluorescence analysis Culture of rat SLFs was carried out as described previously (Moon et al., 2006). In brief, 7-day-old SD rats were obtained from the central laboratories of Third Military Medical University. The rats were anesthetized using ketamine (5 mg/100 g) and then decapitated. In sterile phosphate-buffered saline (PBS), the cochlea were removed from the temporal bone and then dissected free from its bony shell. The basal turn was cut from the cochlea, and transferred to a 35 mm cell culture dish. Sections of spiral ligament only located beneath the stria vascularis were carefully obtained. Explants were plated onto collagen-coated Petri dished in DMEM and 20% fetal bovine serum (FBS). They were kept in an incubator at 37 ◦ C in an atmosphere of 5% CO2 . After approximately 2 weeks in primary culture, nearly confluent monolayer cells were subcultured. For determining the types of cultured SLFs, cells seeded on slides were harvested and fixed in 0.5 ml of 4% paraformaldehyde, washed twice with PBS, and incubated with primary antibodies for 1 h at 37 ◦ C. The sources of antibodies were as follows: Caldesmon, Na–K-ATPase, S-100, cytokeratin goat polyclonal (1:500). The antibodies are specific and do not cross-react with other proteins. The cells were then washed and stained with FITC-conjugated rabbit anti-goat antibody (Zhongshan Biotech Company, China) and left for incubation in the dark. After 15 min, the cells were subsequently washed twice by double distilled H2 O (ddH2 O) and observed by a fluorescent microscopy (Leica DMRXA2, Germany) with excitation and emission settings at 488 and 530 nm, respectively.
Cells were plated on polylysine-coated slides, fixed with 4% paraformaldehyde in 0.1 M PBS for 1 h at 25 ◦ C, rinsed with 0.1 M PBS, pH 7.4, and permeabilized with 1% Triton X-100 in 0.01 M citrate buffer, pH 6.0. DNA fragmentation was detected using TUNEL detection kit (Roche Clinical Laboratories, Indianapolis, IN), which specifically labeled 3 -hydroxyl termini of DNA strand breaks using tetramethylrhodamine isothiocyanate (TRITC)-conjugated dUTP. DNA was also labeled with TRITC DNAbinding dye for 5 min. TRITC labels were observed with a fluorescence microscope. The percentage of apoptotic cells was calculated as the number of apoptotic cells per number of total cells × 100%.
2.7. Western blot assay Cells were harvested, pelleted by centrifugation, washed with ice-cold PBS, and lysed with RIPA buffer (150 mM NaCl, 50 mM Tris base pH 8.0, 1 mM EDTA, 0.5% sodium deoxycholate, 1% NP-40, 0.1% sodium dodecyl sulfate, 1 mM DTT, 1 mM PMSF, and 1 mM Na3 VO4 ) supplemented with protease inhibitor. Proteins were separated by 10% SDS-polyacrylamide gel electrophoresis and transferred onto a nitrocellulose membrane (Life Technologies, Gaithersburg, MD). Blots were then incubated in fresh blocking solution with an appropriate dilution of primary antibody at 4 ◦ C for 24 h. The sources of antibodies were as follows: GAPDH, p-JNK, JNK, ERK, p-ERK goat polyclonal; p-38, p-p38, Bax, Bcl-2, and caspase-3 (cleaved) rabbit polyclonal. After extensive washing, the membranes were incubated with horseradish peroxidase (HRP)-coupled secondary antibody (1:2000, Zhongshan Biotech Company, China) at 25 ◦ C for 1 h. The bands were visualized and quantified using Image-Pro Plus 5.0 software (Media Cybernetics, Silver Spring, MD, USA). The p-JNK, p-ERK, p-p38 band intensities were normalized to JNK, ERK, p-38 band intensities, respectively and the intensities of Bax, Bcl-2, and caspase-3 were adjusted by the GAPDH band intensity.
2.3. Semi-quantitative RT-PCR assay
2.8. Statistical analysis
Total RNA was isolated with TRIzol Reagent (Invitrogen) and first strand cDNA was synthesized from 1 g total RNA using Oligo d(T) primer (Invitrogen) and ReveTra Ace (TOYOBO, Osaka, Japan). PCR was done on the cDNA product using the following primers for Otos: F: 5 -TACGGCTGCGAGAAGACGACA-3 and R: 5 -
Data were expressed as mean value ± S.D. Differences between groups were analyzed with analysis of variances (ANOVA) or a t-test. These analyses were performed by utilizing SPSS for Windows Version 13.0 (SPSS Inc., Chicago, IL). P value less than 0.05 was considered statistically significant.
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To assess the optimal MOI for a maximal transgene expression, we transfected SLFs with Ad-Otos or Ad-GFP at various MOIs (12.5, 25, 50, 75, 100) and for varying time length (24, 48, 72 h), respectively. The results showed that more than 90% of GFP expression was found in SLFs transfected with Ad-viruses at a MOI of 50 and above for 48 h and above. Therefore, we selected a MOI of 50 with a time length of 48 h as an optimal dose and time for the transfection of the SLFs. We then transfected cells with Ad-Otos (SLFs-AdOtos) and AdGFP (SLFs-AdGFP) at a MOI of 50 for 48 h. Afterwards, RNA extracted from transfected SLFs was subjected to a RT-PCR analysis. As shown in Fig. 1B, a significant increase in Otos mRNA expression was found in SLFs-AdOtos but not in SLFs-AdGFP and SLFs-nontransfection, suggesting that vector-mediated transfection could effectively elevate Otos expression in cultured SLFs. 3.2. Otos gene elevation decreased apoptosis of SLFs in response to DDP
Fig. 1. (A) Cultured SLFs (400×) and (B) expression of Otos mRNA in SLFs-AdOtos were significantly higher than those in SLFs-AdGFP or SLFs-nontransfection, respectively (*P < 0.01 vs. 1 or 3). (1) SLFs-AdGFP; (2) SLFs-AdOtos; (3) SLFs-nontransfection.
3. Results and discussions 3.1. Vector-mediated transfection led to up-regulation of Otos The structural characteristics of the cultured cells were consistent with those of fibrocytes (Fig. 1A). The cells were immunopositive for caldesmon and S-100 but negative for Na–KATPase and cytokeratin. The results supported the notion that the cultured cells can be classified as type I fibrocytes, in line with previous report (Maeda et al., 2005).
To investigate the possible roles of Otos in DDP-induced SLFs apoptosis, we treated the cells with various concentrations (0, 5, 10, 20 M) of DDP for 12 h and 20 M DDP for various time length (0, 6, 12, 18 h), respectively. The results showed that elevation of Otos expression resulted in a significant increase in cell viability and a decrease in apoptosis of SLFs in response to DDP in both dose- and time-dependent manners compared with those of SLFs-GFP or SLFs-nontransfection cells (Fig. 2). The data indicate that Otos up-regulation may markedly enhance chemoresistance of SLFs to DDP. Notably, with low concentrations of DDP (0 and 5 M), the results of MTT and TUNEL showed little difference among the three types of cells, suggesting that sole up-regulation of Otos gene exerts slight effects on spontaneous apoptosis of SLFs. 3.3. Up-regulation of Otos decreased DDP-induced apoptosis of SLFs possibly through activation of MAPK/mitochondrial pathway The underlying mechanisms by which Otos up-regulation enhances chemoresistance of SLFs to DDP are not fully understood. A growing body of evidence suggests the possible involvement of some signaling pathways in DDP-induced cell apoptosis.
Fig. 2. (A) Effects of various concentration (0, 5, 10, 20 M) of DDP on the three types of cells for 12 h assessed by MTT and TUNEL assays, respectively (*P < 0.05 vs. 1 or 3, n = 6). (B) Effects of 20 M DDP on the three types of cells for varied time length (0, 6, 12, 18 h) assessed by MTT and TUNEL assays, respectively (*P < 0.05 vs. 1 or 3, n = 6). (1) SLFs-AdGFP; (2) SLFs-AdOtos; (3) SLFs-nontransfection.
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MAPKs, mainly comprising ERK, p38, and c-Jun N-terminal kinase (JNK), are suggested to be associated with apoptosis media´ tion of various cells in response to DDP (Sanchez-Perez et al., 1998; Wang et al., 2006; Winograd-Katz and Levitzki (2006)). Previati et al. (2007) reported that ERK activation might play a role in DDP-induced cultured inner cell apoptosis. According to Choi et al. (2007), JNK pathway activation might protect cochlear cells against DDP-induced apoptosis. However, the precise pathways by which vector-mediated up-regulation of Otos decreases sensitivity of SLFs to DDP are largely unclear. Two major apoptosis pathways have previously been described (Ashkenazi and Dixit, 1998). One pathway is mediated by death receptors, such as CD95 (Fas) and tumor necrosis factor receptors, and is casepase-8 dependent. The other is the mitochondrial pathway that involves Bcl-2 and Bax proteins. The Bcl-2 family consists of both pro-apoptotic and anti-apoptotic members that elicit opposing effects on mitochondria. The balance between proapoptotic and anti-apoptotic members determines apoptosis or survival. Bax can promote the release of cytochrome C into the cytosol from mitochondria, which in turn activates caspase-3, one of the key executioners of apoptosis. Evidence suggests that MAPKs might regulate mitochondrial pathway activation in cell apoptosis (Hong and Kim, 2007; Goel
et al., 2007; Park et al., 2007). To better understand the precise mechanisms underlying DDP-induced SLFs-AdOtos apoptosis, we hypothesized the involvement of MAPKs as well as mitochondrial pathway in this process. To test this, we conducted further experiments. Cells were divided into two major groups, both of which contained three types of cells, respectively, namely, SLFs-AdGFP, SLFs-AdOtos, and SLFs-nontransfection. The two groups were treated with DMEM or 20 M DDP for 12 h, respectively. Then the cell viability and apoptosis were analyzed by MTT and TUNEL assays. Moreover, JNK, p-JNK, ERK, p-ERK, p-38, p-p38, Bcl-2, Bax, and caspase-3 were further assessed by immunoblotting. The results (Fig. 3) showed a marked increase in p-ERK expression and a significant decrease in p-JNK expression in SLFs-AdOtos cells treated with DDP compared with those of SLFs-AdGFP or SLFsnontransfection cells in the same group, accordingly accompanied by decreased cell apoptosis and reduced mitochondrial pathway activation. Nevertheless, in DMEM group, the three MAPK pathways as well as mitochondrial pathway are unlikely to be activated among the three types of cells. The data suggest that Otos up-regulation might rescue SLFs from DDP-induced apoptosis through activation of ERK, blockade of JNK as well as inactivation of mitochondrial pathway.
Fig. 3. Analysis of the results of SLFs-AdGFP, SLFs-AdOtos and SLFs-nontransfection treated with DMEM or DDP. (A and B) The cell viability and apoptosis assessed by MTT (A) and TUNEL (B), respectively (*P < 0.05 vs. 1 or 3, n = 6). (C) Expression of the MAPK and mitochondrial pathway-related proteins assessed by immunoblotting. (1) SLFs-AdGFP; (2) SLFs-AdOtos; (3) SLFs-nontransfection.
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Fig. 4. Analysis of the results of SLFs-Otos treated with DMEM (I), DDP (II), DDP + SP600125 (III), DDP + PD98059 (IV) and DDP + SB203580 (V). The cell viability and apoptosis were assessed by MTT (A) and TUNEL (B), respectively (*P < 0.05 vs. I or II; †P < 0.05 vs. II; ‡P > 0.05 vs. II, n = 6). Further, the proteins (C) of caspase-3, Bax, Bcl-2 and GAPDH were assessed by immunoblotting, respectively.
3.4. Activation of mitochondrial pathway might be regulated by JNK and ERK signaling pathway In order to further test the possible roles of MAPK pathways, we divided SLFs-Otos cells into five groups marked I–V. Cells in group I was treated with DMEM as a control whereas cells in the remaining four groups were treated with 20 M DDP for 12 h. Nevertheless, cells in group III were pre-treated with 10 M SP600125 (a specific JNK inhibitor), in group IV with 10 M PD98059 (a specific ERK inhibitor), in group V with 10 M SB203580 (a specific p-38 inhibitor) for 1 h prior to treatment with DDP. Then the cell viability and apoptosis were assessed by MTT and TUNEL assays. As expected, pretreatment with SP600125 seemed to decrease SLFs-Otos cell apoptosis induced by DDP, whereas pretreatment with PD98059 significantly increased DDP-induced SLFs-Otos cell apoptosis, confirming the involvement of activation of ERK and partial inhibition of JNK pathway as one of the underlying mechanisms (Fig. 4). In this process, JNK activation seemed to provide an apoptotic signal, whereas ERK activation provided a survival signal, in line with previous studies (Tsuruta et al., 2004; Xia et al., 1995). In addition, western blot further confirmed the mitochondrial pathway activation in DDP-induced SLFs cell apoptosis. Interestingly, blockade of JNK markedly attenuated the mitochondrial pathway activation, whereas inhibition of ERK significantly promoted its activation, suggesting the possibility of both JNK and ERK mediating mitochondrial pathway activation, in accordance with previous reports (Hong and Kim, 2007; Goel et al., 2007; Park et al., 2007). However, whether mitochondrial pathway factors are downstream targets of MAPKs remains to be elucidated in further investigations. In the present study, to our knowledge, we provided the first insight into the roles and possible mechanisms by which Otos up-regulation protects SLFs against DDP-induced apoptosis. Since DDP-associated impairments of cochlear cells are thought to have a correlation with oxidation of free radical that leads to cell apoptosis, Otos gene might function as a factor playing a role in anti-oxidative stress. Also, Otos might act as a transcriptional factor that mediates other protective gene expressions in cochlea (Delprat et al., 2002). Notably, the protective effects that Otos up-regulation exerted on DDP-damaged SLFs were limited, though the effects were evident.
Moreover, activation of JNK was likely to provide an apoptotic signal, and nevertheless, blockade of JNK could incompletely inhibit DDP-induced SLFs-Otos cell apoptosis, suggesting that in addition to MAPK pathways, other signaling pathways might play roles in this process. However, the precise mechanisms are unclear and remain to be clarified in further studies. There were several limitations in this study. First, since antibodies to Otospiralin could not be commercially purchased and the specificity of the self-made antibodies in the literature (Delprat et al., 2002) was uncertain, we did not detect the expressions of Otospiralin protein. Nevertheless, the premiers of Otos gene for RT-PCR assay had a high specificity to Otos mRNA. The results confirmed that the Otos mRNA were markedly up-regulated in SLFs after AdOtos transfection. Second, this study had focused on the roles as well as possible mechanisms of Otos gene up-regulation in vitro. Further in vivo studies are needed to clarify the precise underlying mechanisms and to estimate the feasibility of Otos gene therapy. However, the results of this study implicate Otos as a preventive gene for SLFs and suggest Otos up-regulation as a potential strategy for DDP-induced deafness therapy. In conclusion, we successfully up-regulated Otos expression by using Adenoviral-mediated transfection in SLFs, which resulted in a marked decrease in DDP-induced SLF cell apoptosis, suggesting that Otos up-regulation in SLFs might protect the cells against DDP-induced apoptosis and raising the possibility that Otos up-regulation might be a promising approach to DDP-induced deafness therapy. Acknowledgements This work was partially supported by the specific funds of Third Military Medical University for postgraduates (2005256). We take this opportunity to specifically thank the reviewers and editors for their kind instructions that may be helpful for our further studies. References Ashkenazi, A., Dixit, V.M., 1998. Death receptors: signaling and modulation. Science 281, 1305–1308.
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