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Comparison of Cisplatin with Lipoplatin in Terms of Ototoxicity.

INTRODUCTION

Neuroblastoma is a common malignancy in childhood and originates from the primitive neural crest cells in the sympathetic nervous system [1-3]. The behavior of neuroblastoma varies according to the presence of the tumor and the status of metastasis [3, 4]. Neuroblastoma constitutes about 8-10% of all childhood cancers and is responsible for 15% of childhood cancer-related deaths [5]. In neuroblastoma, the patient's age, tumor histology and stage, and cytogenetic and molecular genetic markers are the most important prognostic indicators [3].

Cisplatin (CDDP) is an anti-neoplastic agent that has been used in the treatment of both pediatric and adult cancers [3, 6]. Although CDDP is used in cancer treatments, it has many side effects, such as ototoxicity, nephrotoxicity, and neurotoxicity [6, 7]. Although nephrotoxicity can be controlled and diminished after CDDP treatment, there is no prevention modality against CDDP ototoxicity in clinical practice [8, 9]. CDDP causes damage to the organ of corti in the cochlea, resulting hearing loss, which can lead to social development deficiencies, such as learning problems, in children. Therefore, ototoxicity is an extremely serious problem in childhood [3, 7]. In previous studies, we reported that CDDP shows toxic effects on House Ear Institute-Organ Corti 1 (HEI-OC1) cells [7, 10].

Liposomal platinum is the most promising drug formulation under clinical conditions [11]. Lipoplatin (LIPO) is a liposomally encapsulated form of CDDP [12]. LIPO is a nanomolecule of 110 nm in diameter, which is composed of lipids and CDDP [13]. LIPO has been shown to have toxicity lower than CDDP and more drug accumulation in the tumor [12, 14].

In the present study, we aimed to compare the anti-tumor response of LIPO and CDDP in neuroblastoma and the toxic effects in HEI-OC1 cells.

MATERIALS and METHODS

This study was in vitro study. This study was performed in accordance with the principles of the Declaration of Helsinki. Informed consent is not required for the current in vitro study.

Cell Culture and Reagents

HEI-OC1 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% (w/v) L-glutamine. The cells were incubated at 33[degrees]C in a 10% CO2 incubator [6]. KELLY (worse prognostic, MYC-N amplified) and SH-SY5Y (good prognostic, non-MYC-N amplified) neuroblastoma cells (DSMZ) were maintained in DMEM (Gibco[TM]) and Rosewell Park Memorial Institute (RPMI)-1640 (Gibco[TM]) containing 10% FBS, 1% penicillin/streptomycin, and 1% L-glutamine. The cells were incubated at 37[degrees] C and 5% CO2 conditions [5, 15, 16]. All reagents were freshly prepared with mediums before all experiments. The cell viability was determined using an automatic cell counter with a trypan blue exclusion test.

Detection of Cell Proliferation

The WST-1 assay used to determine cell proliferation is based on the basic principle the conversion of the pink tetrazolium salt (WST-1) to a dark red formazan dye via the succinate-tetrazolium reductase enzyme, which is active only in living cells in the mitochondrial respiratory chain [7]. Mitochondrial dehydrogenase enzyme activity is increased as the number of viable cells increases. As enzyme activity increases, formazan dye production also increases. Therefore, the formazan dye increases in direct proportion to the number of metabolically active cells in a cell culture. Cells were counted and 104 cells/well were seeded with least 6 replicates in a 96-well plate by overnight incubation. The cells were then treated with different concentrations of LIPO and CDDP (10 [micro]M-1000 [micro]M) for 24, 48, and 72 hours. Following the incubation periods, the WST-1 assay was performed by adding 10 [micro]L of WST reagents (Roche) to each well, and HEI-OC1 and KELLY and SH-SY5Y neuroblastoma cells were further incubated at 33[degrees]C and 37[degrees]C, respectively, for 2 hours. After incubation periods, the absorbances of the cells was measured test wavelength at 450 nm and the reference wavelength at 630 nm was measured at 630 nm on an ELISA reader (Thermo Scientific). The half-maximal inhibitory or lethal concentrations (IC50 or LD50) of the agents were calculated according to control cells viability.

Detection of Apoptotic Cells

Apoptotic cell death was determined using annexin-V-fluorescein isothiocyanate (FITC)/7-amino-actinomycin D (7-AAD; BD Biosciences) and Cycletest[TM] Plus DNA Reagent Kit (BD Biosciences) in all cells. In the normal cells, phosphatidylserine molecules on the cytoplasmic surface of the cell membrane migrate to the outer surface of the cell membrane in the event of cell apoptosis. Annexin-V can be labeled with FITC, a fluorescent substance, which can bind to the phosphatidylserine on the outer surface of the cell [17]. Thus, the apoptotic cells to which FITC is conjugated with annexin-V can be made visible. The principle of the method is based on of the extent apoptosis determination using flow cytometry of cells stained with annexin-V-FITC and 7-AAD, a non-vital dye.

The auditory cells and tumor cells were incubated for 48 hours in the presence of LIPO or CDDP. In KELLY neuroblastoma cells, LIPO was used at 750 [micro]M and CDDP was used at 20 [micro]M concentrations determined from cell proliferation assays. In SH-SY5Y cells, LIPO was used at 750 [micro]M and CDDP used at 20 [micro]M. In the auditory cells, cells were treated with 750 [micro]m LIPO and 20 [micro]M CDDP. After incubation, cells were collected in a 1.5 mL centrifuge tube and centrifuged at 500 x g for 10 minutes. Each cell pellet was re-suspended in 100 [micro]L phosphate-buffered saline (PBS), and subsequently 5 [micro]L of annexin-V and 5 [micro]L 7-AAD were added on the cells. The cells were incubated in the dark at 4[degrees]C for 15 minutes according to protocol. After incubation, 400 [micro]L annexin-V binding buffer was added in each tube. Analyses were carried out using a flow cytometer (Accuri, BD) at 488 nm excitation and 530 nm emission wavelengths for annexin-V and 488 nm excitation and 647 nm emission wavelengths for 7-AAD, and evaluated using the BD Accuri C6 Software (BD Biosciences) [6].

Cells at different stages of the cell cycle were determined using Cycletest[TM] Plus DNA Reagent Kit (BD). After the agent applications, the cells were washed thrice with PBS and re-suspended in the medium, incubated for 15 minutes at room temperature with propidium Iodide, and then analyzed using flow cytometry [18, 19].

Statistical Analysis

The Statistical Package for Social Sciences software for Windows, version 15.0, released 2007 (SPSS Inc.; Chicago IL, USA) was used for statistical evaluation of the data. Findings with a p value <0.05 for statistical significance were accepted. Non-parametric Mann-Whitney U test was also used. Each experiment was repeated at least in triplicate for statistical evaluation.

RESULTS

Cell Proliferation Results

The cell viability of KELLY human MYC-N amplified and SH-SY5Y human MYC-N non-amplified neuroblastoma cells treated with different doses of LIPO (10-1000 [micro]M) and CDDP (10-1000 [micro]M) was determined using the WST-1 assay. LIPO and CDDP inhibited neuroblastoma cell proliferations in a dose- and time-dependent manner (Figure 1, 2).

The doses of CDDP and LIPO applied for KELLY and SHSY cells were also applied for HEI-OC1 cell and cell viability was determined by the WST-1 assay. Also, proliferations of HEI-OC-1 cells were decreased with lower doses of CDDP but a higher dose of LIPO treatments.

In KELLY MYC-N amplified human neuroblastoma cells, the cell viability decreased at a rate of 41% and 75% with the treatment of 1000 [micro]M CDDP and LIPO, respectively, after 24 hours of incubation(p<0.05; Figure 1).

Cisplatin inhibited 50% cell viability at 20 [micro]M doses, while 750 [micro]M LIPO inhibited 50% cell viability in KELLY cells after 48 hours of incubation (p<0.05; Figure 1).

Also, 20 [micro]M CDDP decreased KELLY cell viability at 33%, but 750 [micro]M LIPO inhibited cell viability at 37% after 72 hours of incubation (p<0.05; Figure 1).

Also, 20 [micro]M CDDP decreased KELLY cell viability at 33%, but 750 [micro]M LIPO inhibited cell viability at 37% after 72 hours of incubation (p<0.05; Figure 1).

In the SH-SY5Y MYC-N non-amplified human neuroblastoma cells, cell viability decreased at a rate of 28% and 36% with the treatment of 1000 [micro]M CDDP and LIPO, respectively, after 24 hours of incubation (p<0.05; Figure 2).

Cisplatin inhibited 50% cell viability at 10 [micro]M doses, while LIPO inhibited cell viability at 750 [micro]M in SH-SY5Y cells after 48 hours incubation (p<0.05; Figure 2).

Also, 10 [micro]M CDDP decreased SH-SY5Y cell viability at 45%, but 750 [micro]M LIPO inhibited cell viability at 42% after 72 hours of incubation (p<0.05; Figure 2).

In the HEI-OC1 cells, cell viability decreased at 43% and 87% with the treatment of 1000 [micro]M CDDP and LIPO, respectively, after 24 hours of incubation (p<0.05; Figure 3).

Cisplatin inhibited 50% cell viability at 50 [micro]M doses, while LIPO inhibited cell viability at 750 [micro]M in SH-SY5Y cells after 48 hours of incubation (p<0.05; Figure 3).

Also, 20 [micro]M CDDP decreased SH-SY5Y cell viability at 23%, but 500 [micro]M LIPO inhibited cell viability at 58% after 72 hours of incubation (p<0.05; Figure 3).

In this study, the in vitro cytotoxic effects of LIPO and CDDP on HEIOC1 cells with neuroblastoma cells were also compared. Firstly, HEIOC1 cells were treated with the same doses of both CDDP and LIPO and the IC50 doses were determined from these experiments.

Moreover, we tested the apoptotic effects of the agents in these cells using the annexin-V and cell cycle analysis.

HEI-OC1 cells treated with 750 [micro]M LIPO for 48 hours had an 82% viability. HEI-OC1 cells treated with 20 [micro]M CDDP for 48 hours had a 65% viability.

Therefore, 20 [micro]M CDDP and 750 [micro]M LIPO concentration applied for 48 hours was designated as the IC50. Treatment with LIPO or CDDP induced a dose-dependent (CDDP, IC50=20 and 50 [micro]M; LIPO IC50=750 and 1000 [micro]M) and time-dependent (48 h) inhibition of cell proliferation.

In the SH-SY5Y cells, the cell viability was 57% for LD50 doses of LIPO 750 [micro]M, the lowest dose of CDDP is 50% of deaths from 10 [micro]M achieved, and the dose increases with decreased cell viability. The cell viability was 53% for LD50 doses of LIPO 750 [micro]M and 53% for the 20 [micro]M CDDP in KELLY cells. In the HEI-OC1 cells, 1000 [micro]M LIPO led to 66% cell viability and 50 [micro]M of CDDP decreased the cell viability below 50% (44%).

Apoptosis Results of the Cells

We also compared apoptosis level at the lC50 concentration of LIPO and CDDP on KELLY, SH-SY5Y, and HEI-OC1 cells. To compare the apoptosis level of LIPO and CDDP, flow cytometry annexin-V/7-AAD and cell cycle assays were used.

LD50 doses of CDDP caused 62.3-77.3% and LIPO caused 38.85-45.6% apoptotic cell death in KELLY cells. In SH-SY5Y, LD50 doses of CDDP caused 75.8-86.9% and LIPO induced 25.3-56.3% apoptosis (Figure 4). Cell cycle findings showed similar results in apoptosis (data not shown).

DISCUSSION

An ideal chemotherapeutic agent should be effective on cancer cells, and it should not affect normal cells. CDDP is a highly effective chemotherapeutic agent widely used for treating various adult and pediatric cancers; however, it has dose-limiting serious adverse effects, including nephrotoxicity, neurotoxicity, and ototoxicity [1]. It has been shown that CDDP has ototoxic effects in many studies [3,4]. LIPO, a nanomolecule 110 nM diameter, is a liposomal formulation of CDDP and is composed of lipids and CDDP. LIPO was developed to reduce the systemic toxicity of CDDP [12, 13].

Our previous study has shown that CDDP has toxic effects in HEI-OC1 cells [20]. In the present study, we aimed to compare the potential toxic effects of LIPO and CDDP on HEI-OC1 cells. Previous in vitro and in vivo researches have shown that LIPO has less nephrotoxic effects compared to CDDP [12]. Furthermore, chemoradiotherapy with LIPO for advanced gastric cancer treatment demonstrated minor toxicity at phase 1/2 study [12]. Fantini et al. [13] showed that LIPO therapy has lesser renal toxicity than CDDP in lung and breast cancer treatment in clinical studies.

Moreover, LIPO showed anti-tumor effect in CDDP-sensitive- and -resistant ovarian cancer cells since apoptosis was induced by caspases 3, 8, and 9 activation; Bax upregulation; and Bcl-2 downregulation [11]. LIPO caused a synergistic effect combination with doxorubicin and the albumin-bound paclitaxel abraxane. Also, LIPO inhibited ovarian xenograft tumor growth with minimum systemic toxicity in that study.

In an animal study to evaluate the renal toxicities of LIPO and CDDP, it has been shown that LIPO induces less structural and functional damage to the kidneys in mice than CDDP [21]. It was observed that intraperitoneal bolus injection of CDDP and LIPO in rat kidneys resulted in less platinum accumulation with LIPO, although CDDP and LIPO reached the same level of platinum.

CONCLUSION

In this study, the anti-tumor and apoptotic effect of LIPO was determined in neuroblastoma cells at a higher dose than CDDP and at later time periods. LIPO caused less apoptotic cell death on cochlear cells than CDDP at anti-tumoral doses, suggesting a lower toxicity in cochlear cells compared to CDDP. Further in-vivo comparative studies are needed for understanding the mechanism of ototoxic effects of LIPO versus CDDP [22].

Ethics Committee Approval: Authors declared that the research was conducted according to the principles of the World Medical Association Declaration of Helsinki "Ethical Principles for Medical Research Involving Human Subjects", (amended in October 2013)..

Informed Consent: N/A.

Peer-review: Externally peer-reviewed.

Author Contributions: Concept - Concept - N.O., S.A., Z.A., E.S.; Design - E.S., Z.A.; Supervision - N.O., S.A., Z.A.; Resource - N.O., Z.A.; Materials - Z.A, S.A., E.S.; Data Collection and/or Processing - E.S.; Analysis and/or Interpretation - E.S.; Literature Search - E.S.; Writing - E.S., Z.A.; Critical Reviews - Z.A., S.A., N.O., E.C.

Acknowledgements: The authors would like to thank Prof. F. Kalinec for ensuring HEI-OC1 cells.

Conflict of Interest: No conflict of interest was declared by the authors.

Financial Disclosure: The authors declared that this study has received no financial support.

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Efe Serinan [iD], Zekiye Altun [iD], Safiye Aktas [iD], Emre Cecen [iD], Nur Olgun [iD]

Department of Basic Oncology, Dokuz Eylul University Institute of Oncology, Izmir, Turkey (ES, ZA, SA)

Department of Pediatric Oncology, Dokuz Eylul University Institute of Oncology, Izmir, Turkey (NO)

Department of Pediatric Hematology and Oncology, Harran University School of Medicine, Sanliurfa, Turkey (EC)

ORCID IDs of the authors: E.S. 0000-0002-3682-7590; Z.A. 0000-0002-1558-4534; S.A. 0000-0002-7658-5565; N.O. 0000-0001-9591-0207; E.C. 0000-0003-0330-4715

Cite this article as: Serinan E, Altun Z, Aktas S, Cecen E, Olgun N. Comparison of Cisplatin with Lipoplatin in Terms of Ototoxicity. J Int Adv Otol 2018; 14(2): 211-5.

This study was presented at the 48th Congress of the International Pediatric Oncology (SIOP 2016 Congress), 19-22 October, Dublin, Ireland.

Corresponding Address: Zekiye Altun E-mail: zekiyesaltun@gmail.com

Submitted: 14.06.2017 * Revision Received: 03.08.2017 * Accepted: 20.11.2017 * Available Online Date: 19.02.2018

DOI: 10.5152/iao.2018.4097
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Title Annotation:Original Article
Author:Serinan, Efe; Altun, Zekiye; Aktas, Safiye; Cecen, Emre; Olgun, Nur
Publication:The Journal of the International Advanced Otology
Date:Aug 1, 2018
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