17-Allylamino-demethoxygeldanamycin Used Alone or in Combination with Sodium Orthovanadate Promotes Apoptosis and Inhibits Invasion of SH-SY5Y Cells by Modulating PIWIL2

Neuroblastoma (NB) is one of the most common extracranial solid tumors of childhood and accounts for 15% of cancer deaths. Even with the multimodality treatment protocols, the advanced-stage tumor overall 5-year survival rate is less than 50%. Therefore, novel drug therapy targeting cellular signal transduction pathways regulating the apoptotic cascade may be important for the treatment of drug-resistant NB. In our previous studies, we have demonstrated that 5 μ M sodium orthovanadate (SOV) induced the apoptosis of SH-SY5Y cells. 17-Allylamino-demethoxygeldanamycin (17-AAG) is a geldanamycin- (GA-) derived heat shock protein 90 (Hsp90) inhibitor, and it has been shown to have potent antitumor activity in head and neck cancers. However, the e ﬀ ect of 17-AAG on the apoptosis of NB cells has not been reported. Therefore, the purpose of this study was to determine the e ﬀ ects of 17-AAG and SOV on the growth and invasion of SH-SY5Y cells in vitro and explore the related mechanism. In this study, we ﬁ rst investigated the antiviability e ﬀ ect of 17-AAG on SH-SY5Y cells, then studied the cell apoptosis and invasion in ﬂ uenced by 17-AAG and SOV, and assessed the role of PIWI-Like2 (PIWIL2) and piRNA-PIWI signaling in it. The results showed that 5 μ M 17-AAG inhibited cell growth and viability and induced apoptosis in SH-SY5Y cells. Both 17-AAG and SOV reduced the level of PIWIL2 and Bcl-xl proteins and inhibited the invasion of SH-SY5Y cells. In addition, the combined use of the two drugs had greater e ﬀ ect than the single use of any drug.


Introduction
Neuroblastoma (NB) is one of the most common extracranial solid tumors of childhood and accounts for 15% of cancer deaths [1][2][3]. Even with the multimodality treatment protocols, including chemotherapy, surgery, radiation therapy, and immunotherapy, the advanced-stage tumor overall 5year survival rate is less than 50% [4][5][6]. Cases of high-risk NB are associated with frequent relapses and tumors that are resistant to alternative chemotherapy regimens. It is reported that the altered cellular responses to apoptosis are thought to play an important role in drug resistance in high-risk NB [7]. Therefore, novel drug therapy targeting cellular signal trans-duction pathways regulating the apoptotic cascade may be important for the treatment of drug-resistant NB.
The human NB cell line SH-SY5Y is a commonly used cell line in studies related to NB [8,9] and neurodegenerative disease, such as Alzheimer's disease (AD) [10] and Parkinson's disease (PD) [11,12]. For example, SH-SY5Y cells were usually used as a model system in vitro to investigate the effect and mechanism of new drugs and provide a basis for the formulation of drug strategy and the treatment of diseases. Here, we focus on the treatment of NB and study the effect of 17-allylamino-demethoxygeldanamycin (17-AAG) and sodium orthovanadate (SOV, molecular formula: Na 3 VO 4 ) on the apoptosis and invasion of SH-SY5Y cells.
AAG is a geldanamycin-(GA-) derived heat shock protein 90 (Hsp90) inhibitor, which is a less toxic and more stable analog of GA [13]. Hsp90, a ubiquitous chaperone protein, plays significant roles in the organization and maturation of client proteins involved in the progression and survival of tumor cells and has become a therapeutic target for cancer treatment. 17-AAG binds to Hsp90, destroys the Hsp90-client protein cycle, and results in the degradation of Hsp90's client proteins. Several preclinical and clinical studies demonstrated that 17-AAG as a single agent or in combination with other anticancer agents has potent antitumor activity in a range of human cancers [14][15][16][17]. However, it has not been reported whether 17-AAG can induce apoptosis of NB cells or not.
SOV is a proverbial vanadium compound and has many biological activities [7]. Many studies have shown that SOV displays antineoplastic activities against various types of cancers, such as hepatocellular carcinoma [18,19], anaplastic thyroid carcinoma [20], oral squamous cell carcinoma [21], EGFR-mutated lung adenocarcinoma [22], and other tumors [23,24]. In addition, SOV also has neuroprotective and antidiabetic effects [25,26]. In our previous study, we have demonstrated that 5 μM SOV induced the apoptosis of SH-SY5Y cells [27]. The results suggested that SOV could be a promising agent to treat the central nervous system tumors.
Multiple studies have shown that the combined use of 17-AAG and other antitumor reagents enhanced its antitumor efficacy. As an antitumor agent, can SOV combined with 17-AAG inhibit tumor development to a greater extent? Therefore, on the basis of previous studies, we also used SH-SY5Y cells as a cell model for relevant studies. Furthermore, studies have shown that SOV can play an antitumor role by inhibiting the piRNA-PIWI signaling pathway [28,29], so we want to know whether SOV and 17-AAG can inhibit the proliferation and invasion of SH-SY5Y by inhibiting PIWI protein-related signaling pathways.
In this study, we mainly investigated the role of 17-AAG alone or combined with SOV in inhibiting the viability and invasion of SH-SY5Y cells and also explored the potential mechanism of action.

Materials and Reagents.
The human NB SH-SY5Y cell line was purchased from the Cell Bank of Chinese Academy of Sciences (Cat. No.: SCSP-5014, Shanghai, China). The cells were resuspended and plated on a 10 cm petri dish with Dulbecco's modified Eagle's medium (DMEM; Hyclone), supplemented with 10% fetal bovine serum (FBS; Gibco), 100 U/ml penicillin, and 100 μg/ml streptomycin (Sigma Chemical Co., St. Louis, Missouri), and were grown in a 5% CO 2 incubator at 37°C. After culturing for 72 h, the nonadherent cells were removed. The plastic-adherent cells were treated with 0.25% trypsin containing 1 mM EDTA and passaged when they reached about 90% confluent density and subcultured continuously.       Transwell plates were incubated at 37°C in a humidified atmosphere containing 5% CO 2 in air for 24 hours, and then, the noninvaded cells on the upper side of the chamber were wiped with cotton swabs. The cells that had migrated from the Matrigel into the pores of the inserted filter were fixed with 90% ethanol for 30 minutes and stained with 0.1% crystal violet dye for 10 min. The number of cells invading through the Matrigel was counted under an inverted microscope, and nine fields per well were counted. Each assay was repeated three times.

17-AAG Reduces the Expression of PIWIL2 and Bcl-xl
Proteins. Western blot was performed to determine if 5 μM 17-AAG inhibited the expression of PIWIL2 and Bcl-xl proteins in SH-SY5Y cells. The results showed that the expression of PIWIL2 and Bcl-xl proteins in SH-SY5Y cells reduced after treatment of 5 μM 17-AAG, 5 μM SOV, and the mixture of the two drugs (Figure 3(a)). In addition, the protein levels in the 5 μM (17-AAG+SOV) group reduced more than those in 5 μM 17-AAG or 5 μM SOV groups, which indicated that the combined effect is greater than the use of the single drug. Quantification analysis revealed the above results (Figure 3(b), * P < 0:05, * * P < 0:01).

17-AAG or SOV Treatment Inhibits the Invasion of SH-SY5Y Cells. SH-SY5Y cells that migrated into the lower
It can be concluded that both 17-AAG and SOV treatment inhibited the invasion of SH-SY5Y cells, and the combined effect of them was greater than the single use of any drug.

Discussion
Here, we describe the effects of 17-AAG used alone or in combination with SOV on apoptosis and invasion of SH-    BioMed Research International SY5Y cells and explore the level of PIWIL2 expression as a promising mechanism. Our research has achieved certain results and provided a therapeutic strategy for the treatment of tumors. HSP90 is a molecular chaperone required for the stability and function of many proteins. The survival, growth, and invasive potential of cancer cells can be enhanced by HSP90. And HSP90 inhibitors are promising new anticancer agents, among which 17-AAG is in clinical evaluation now [30]. 17-AAG has exhibited lower toxicity and improved stability and induced reduction of key regulators of signal transduction in many human tumors, including colon and breast cancers [31,32].
In our study, we found that 17-AAG inhibited cell growth and viability in SH-SY5Y cells at different concentrations ( Figure 1). When we treated SH-SY5Y cells with 5 μM 17-AAG, the cells' viability reduced sharply at 24, 48, 72, and 96 h ( Figure 1). So, we used this concentration for further tests. The results of apoptosis and invasion assay showed that the single use of 17-AAG or SOV induced significant apoptosis and inhibited the invasion in SH-SY5Y cells at 24 h, 48 h, and 72 h, compared with those in the control groups (Figures 4 and 5). And the combined use of the two drugs had a more significant effect than the single use of any drug, which demonstrated that 17-AAG and SOV have a synergistic inhibitory effect on SH-SY5Y cell development. The antitumor effects of 17-AAG or SOV were demonstrated in multiple studies [14,19,24], and our study confirmed the advantages of their combined usage.
It has been reported that PIWI proteins played a role in germ cell proliferation, differentiation, germline stem cell maintenance, and transposon control in germline from Drosophila to mammals. The PIWIL2 gene is mainly expressed in the testis or embryonic cells among normal tissues but widely expressed in tumors [33,34]. Increasing evidence has proven that PIWI proteins are linked to the hallmarks of cancer, such as cell proliferation, antiapoptosis, and invasion. This provides new possibilities for anticancer therapies through the targeting of PIWI proteins [35,36]. Bcl-xl is one of the leading members of the Bcl-2 protein family and exerts its antiapoptotic role to mediate the interaction with the proapoptotic members of the same Bcl-2 family [37,38]. Lee et al. demonstrated that PIWIL2 inhibited apoptosis and stimulated proliferation through activation of Stat3/Bcl-xl and enhancement of Stat3/cyclin D1 signaling pathways [39,40]. In our previous studies, we have known that SOV inhibited the growth of SH-SY5Y cells via the piRNA-PIWI signal, which is consistent with the report of Izumi et al. in the silkworm ovary-derived BmN4 cells [41]. Then, we want to verify if 17-AAG can enhance this effect. The result of Western blot showed that the expression of PIWIL2 and Bcl-xl proteins reduced when treated with 17-AAG, SOV, and the mixture of the two drugs ( Figure 3). Besides, the result of the apoptosis assay showed that the single use of 17-AAG or SOV induced significant apoptosis in SH-SY5Y cells at 24 h, 48 h, and 72 h, compared with those in the control groups ( Figure 4). And the combined use of the two drugs had more significant effect than the single use of any drug, which demonstrated that 17-AAG could enhance the apoptosis effect induced by SOV in SH-SY5Y cells. In addition, the invasion assay showed that 17-AAG or SOV treatment inhibited the invasion of SH-SY5Y cells, compared with control groups, and the combined use of the two drugs had greater effect than the single use of any drug ( Figure 5). Therefore, we speculated that 17-AAG plays its role by modulating PIWIL2 via the piRNA-PIWI signal pathway.
The limitations of this study may include the mechanism of molecular action which remains to be further studied. In addition, we have only conducted in vitro experiments, and in vivo and clinical trials are essential if clinical transformation is to be achieved. Furthermore, there are some shortcomings in 17-AAG itself. At present, it is mainly improved by combining nanomaterials [42,43], and related researches can be carried out in the later stage. Despite the fact that this study is preliminary, we clearly indicated that 17-AAG used as a single agent or combined with SOV had antineoplastic effect on SH-SY5Y, which may offer a new strategy to treat neuroblastoma or other central nervous system tumors/diseases.

Conclusions
In conclusion, 17-AAG can promote apoptosis and inhibit invasion of SH-SY5Y cells. Furthermore, a synergistic inhibitory effect of 17-AAG combined with SOV has been shown on SH-SY5Y cells. This synergistic effect was mediated through induction of apoptosis by downregulating PIWIL2related pathways.

Data Availability
Readers can access all the data supporting the conclusions of the study online.