Knockdown of sorcin increases HEI-OC1 cell damage induced by cisplatin in vitro

Abstract

Hearing loss caused by ototoxic drugs is a kind of acquired hearing loss. Cisplatin is one of the most commonly used drugs and its main action sites are hair cells (HCs). Sorcin is a drug-resistant calcium-binding protein belonging to the small penta-EF-hand protein family. Sorcin is highly expressed in many tissues, including bone, heart, brain, lung, and skin tissues. Single-cell RNA sequencing showed that sorcin was expressed in the outer HCs of mice, but its role remained unknown. We also found that sorcin was highly expressed in the cytoplasm of cochlear HCs and HEI-OC1 cells. After cisplatin injury, the expression of sorcin in HCs and HEI-OC1 cells decreased significantly. SiRNA transfection technology was used to knock down the expression of sorcin. The results showed that the number of apoptotic cells, the expression of cleaved caspased-3, and the expression of Bax increased while the anti-apoptotic factor Bcl-2 decreased in the siRNA-Sorcin + CIS group. The observed increase in apoptosis was related to the increase of reactive oxygen species (ROS) and the destruction of the mitochondrial membrane potential (MMP). Finally, we found that the downregulated sorcin worked by activating the P-ERK1/2 signaling pathway. Overall, this study showed that sorcin can be used as a new target to prevent the ototoxicity of platinum drugs.

Introduction

Approximately one billion people worldwide have hearing loss, which is a disabling disease that is common in humans. Hearing loss can be caused by a wide variety of factors, such as aging, drugs, noise, and genetics. Platinum anticancer drugs are among the most commonly used ototoxic drugs in clinics. Cisplatin is a metal complex with a platinum atom at its center. This complex is used as an acyclic cytotoxic chemotherapeutic drug, similar to the bifunctional alkylating agent [1]. Since its FDA approval in 1978, cisplatin has gradually been applied to a wide variety of malignant tumors, including genital tract tumors, nasopharyngeal carcinoma, esophageal cancer, and malignant lymphoma. The main mechanism of cisplatin is to inhibit the DNA replication of cancer cells [2]. With regard to its effects on hearing, cisplatin mainly causes high-frequency hearing loss, and the ototoxicity induced by this drug is irreversible. The degree of hearing loss caused by cisplatin gradually progresses with the passage of time [3,4]. Previous studies have found that large accumulations of cisplatin in the cochlea are associated with the loss of outer hair cells (HCs) in the organ of Corti [5,6]. The mechanism of cisplatin-induced ototoxicity has not been fully elucidated but may be related to oxidative stress, apoptosis, and inflammation. The absorption of cisplatin involves two pathways: passive diffusion and active uptake [7]. In active uptake, copper ion transporters (Ctr1, Ctr2) and organic cation transporters (OCT2) mediate cisplatin into the cytoplasm to form water-based complexes, which cause DNA damage and cross-binding, increase the release of cytochrome C, and finally lead to caspase-3 activation and apoptosis [8]. Cisplatin produces excessive reactive oxygen species (ROS), leading to an imbalance of the antioxidant defense system and reducing the mitochondrial membrane potential (MMP), activating caspase-9 and caspase-3, and triggering mitochondrial-mediated apoptosis [9]. The Bcl-2 family includes the anti-apoptotic member, the pro-apoptotic member, and the BH3-only subfamily. Bcl-2 and Bax belong to the anti-apoptotic member and the pro-apoptotic member respectively. Bax and Bcl-2 can regulate the release of cytochrome C, which is upstream of caspase-3 [10].

Sorcin, also known as cp-22 and v19, is a calcium-binding protein associated with soluble resistance that belongs to the penta-EF-hand protein family [11]. Sorcin is expressed not only in normal tissues including kidney, brain, bones, skin, but also in cancer tissues including colorectal cancer, lung cancer, breast cancer, and ovarian cancer [[11], [12], [13], [14], [15]]. The role of sorcin is still under exploration. At present, sorcin is mainly focused on the regulation of calcium homeostasis, cancer development, multidrug resistance, apoptosis, and so on. Sorcin promotes tumor progression by enhancing tumor invasion and migration [13]. In addition, sorcin has a certain relationship with mitochondrial apoptosis, which can reduce apoptosis by interacting with TNF receptor associated protein 1 (TRAP1) [16]. Sorcin regulates apoptosis induced by Bax, Bcl-2, caspase-3, and other apoptotic proteins [17,18]. The effect of sorcin on cells is achieved by regulating different signal pathways, such as P-ERK, STAT3, and P13K/Akt [19,20].

In the present study, we found that sorcin was expressed in cochlear HCs and auditory HEI-OC-1 cells and then explored the role of sorcin in cisplatin-induced ototoxicity. Through the construction of a sorcin knockdown model, we proved that sorcin downregulation can increase the ototoxicity of cisplatin by increasing apoptosis, ROS accumulation, and MMP damage; this effect occurs through the ERK1/2 signaling pathway. In conclusion, our results suggest that the knockdown of sorcin can increase the ototoxicity induced by cisplatin and help in the development of new protective strategies against HC injury.