High Expressions of Notch and Survivin in Elderly Patients with Glioma Contribute to an Unfavorable Prognosis

Gliomas account for 24% of all primary brain and central nervous system tumors. To date, elderly patients constitute 10-25% of patients with a diagnosis of glioblastoma multiforme, but limited attention has been put on their optimal treatment, largely due to a very poor expected survival (only 4-6 months). Unraveling the molecular mechanism of gliomas provides an opportunity to develop novel biomarkers and therapeutic targets. In this study, we collected fasting blood samples from elderly patients diagnosed with glioma and who received treatment in our hospital between May 2016 and May 2019 and determined the expression levels of Notch and Survivin proteins in di ﬀ erent clinical stages and their relationship with patient survival. A total of 68 healthy volunteers in this hospital during the same period served as healthy controls. Compared with the healthy controls, the expressions of Notch 1, Notch 2, Notch 3, and Survivin protein in the serum of elderly glioma patients were remarkably increased ( P < 0 : 05 ), but the expression of caspase-3 protein declined ( P < 0 : 05 ). As the clinical stage of the patient advanced, the expressions of Notch 1, Notch 2, Notch 3, and Survivin increased, and this increase was statistically signi ﬁ cant ( P < 0 : 05 ). It was observed that high expressions of serum Notch 1, Notch 2, Notch 3, and Survivin were associated with poor overall survival of elderly patients with glioma. We used γ -secretase inhibitor MRK-003 and speci ﬁ c ligand Jagged1 to alter the Notch pathway in U251 cells. It was revealed that MRK-003 incubation e ﬀ ectively suppressed the mRNA expression of Survivin in U251 cells, but Jagged1 stimulation signi ﬁ cantly promoted the mRNA expression of Survivin in U251 cells. Results of MTT and transwell migration assays revealed reduced U251 cell viability and migration following MRK-003 treatment and enhanced cell viability and migration following Jagged1 stimulation. In conclusion, the ﬁ nding obtained from these results supports that Notch and Survivin proteins contribute to the development of glioma in elderly patients and could serve as prognostic factors.


Introduction
Gliomas represent the most common primary brain and central nervous system tumors worldwide and occur almost exclusively in the following four lobes of the brain: frontal (23.6%), temporal (17.4%), parietal (10.6%), and occipital (2.8%), leading to significant morbidity and mortality in adults, especially elderly people [1,2]. To date, elderly patients already account for 10-25% of all patients with a diagnosis of glioblastoma multiforme, but limited attention has been put on their optimal treatment, largely due to a very poor expected survival ranging only from 4 to 6 months [3]. In this context, the increasing aging of the population will contribute to at least twofold increase in the number of glioma diagnoses in elderly patients in the following two decades [4,5]. Because of the inability to tolerate the treatment, elderly patients with gliomas have far less chance to undergo resection and receive adjuvant therapies than their younger counterparts and typically only receive palliative care [6]. Therefore, the management of gliomas in elderly patients has received much attention and becomes an important and challenging topic in neurooncology.
Unraveling the molecular mechanism of gliomas provides an opportunity to develop novel biomarkers and therapeutic targets. Notch is a crucial evolutionary conserved pathway that engages in the modulation of central cellular processes during embryonic and postnatal development, and it encodes for four paralogs, Notch 1-4 [7]. The Notch pathway inhibits neuronal differentiation in the central nervous system through maintenance of neural stem cells and commitment of neural progenitor cells into the glial lineage. Notch is therefore often implicated in the development of brain tumors, as tumor cells share various characteristics with neural stem and progenitor cells [8]. Survivin is known as a member of the inhibitor of apoptosis family and is nearly undetectable in most normal tissues in adults. It is highly expressed in almost all human malignancies [9]. Recent studies focus on nanoparticles based on Survivin-targeting treatments as brain cancer therapies [8]. Caspase-3 is a member of cysteinyl aspartate-specific proteases that are highly conserved in multicellular organisms and function as a key regulatory molecule in neurogenesis and synaptic activity [10]. In this study, we collected fasting blood samples from elderly patients diagnosed with glioma and who received treatment in our hospital between May 2016 and May 2019 and determined the expression levels of Notch and Survivin proteins in different clinical stages and their relationship with patient survival.

Patient Information.
The study recruited elderly patients with glioma according to predefined inclusion and exclusion criteria. Inclusion criteria are age ≥ 60 years, initial diagnosis of glioma according to the 2007 WHO classification of tumor of the central nervous system [11], and patients' or their family members' signed informed content. Exclusion criteria are patients with other malignant tumors; patients with severe metabolic system diseases such as diabetes; patients with immune system diseases; patients with severe heart, kidney, and lung dysfunction; and patients with severe cognitive dysfunction. Finally, a total of 100 elderly patients with glioma diagnosed and treated in our hospital from May 2016 to May 2019 were recruited into the study. Among these 100 patients, 65 were males and 35 were females, aged 62-77 years old, with an average of 66:6 ± 8:6 years; there were 29 patients in clinical phase I, 21 patients in clinical phase II, 30 patients in clinical phase III, and 20 patients in clinical phase IV. The 68 healthy persons undergoing physical examination in this hospital during the same period were selected as the control group. There was no statistical difference in the average age and gender ratio between the two groups (P > 0:05). A 6-year follow-up was performed on each patient. The study protocol was approved by the Ethics Committee of our hospital.

Blood Sample Collection and Enzyme-Linked
Immunosorbent Assay (ELISA). Fasting venous blood from each elderly patient was sampled and centrifuged at low speed to separate the upper serum for testing. The serum levels of Notch 1, Notch 2, Notch 3, Survivin, and caspase-3 were measured by ELISA methods using commercial available kits (Santa Cruz Company, Elabscience Company, and Omega Company) following the instructions provided by the manufacturers.

Cell
Culture. Human glioma cell line U251 (Shanghai North Connaught Biotechnology Co., Ltd., China) was maintained in DMEM with 10% fetal bovine serum (FBS, Gibco, USA), 100 μg/mL streptomycin, and 100 IU/mL penicillin in a humidified environment in the presence of 5% CO 2 . U251 cells were seeded into the 6-well plate and allowed to settle overnight. Afterwards, the cultured cells were treated with 10 μM γ-secretase inhibitor (GSI) MRK-003 for 48 h to block the Notch signaling pathway [12].

2
Journal of Nanomaterials 2.8. MTT Assay. Following a 48 h period of cell treatment, U251 cell viability was determined using the MTT assay. In brief, U251 cells (5 × 10 3 cells/well) were plated into the 96well plate, and the plate was added with 10 μL of MTT solution (R&D Systems, USA), followed by 4 h of further incubation. The absorbance at 450 nm wavelength (OD450) was measured at indicated time points (24 th and 48 th ) using a plate reader (Thermo Scientific, Watertown, USA).
2.9. Transwell Migration Assay. U251 cell migration was assessed using a transwell (8 μm pore size), and 5 × 10 4 cells were plated into each well of the upper chamber uncoated with the membrane. The lower chamber was added with the medium containing 10% FBS. After cells were incubated for 24 hours, those that migrated from the upper chamber into the lower chamber were fixed with methanol and stained with 0.1% crystal violet dye. A microscopic view was captured under an inverted microscope.
2.10. Statistical Analysis. Data statistics were completed using SPSS 21.0 statistical software (IBM Corp., Armonk, NY, USA). The measurement data were expressed by the mean ± standard deviation. The t-test was used to compare the two groups. One-way analysis of variance was used for multiple-group comparisons. If the overall comparison was different, the F test was used for pairwise comparison. The possibility of a difference less than 0.05 was held to be statistically significant.

Expression Levels of Notch Proteins in Elderly Patients
with Glioma according to Different Clinical Stages. First, we collected blood samples from 100 elderly patients with glioma and performed ELISA methods to examine expression levels of Notch 1, Notch 2, and Notch 3. Results showed that compared with healthy controls, the expression levels of Notch 1, Notch 2, and Notch 3 in elderly patients with glioma were evidently increased (P < 0:05, Table 1). Next, we classified 100 elderly patients with glioma based on their clinical stage. It was revealed that the expression levels of Notch 1, Notch 2, and Notch 3 were associated with the clinical stage of glioma (P < 0:05, Table 1), suggesting that high expression of Notch proteins may contribute to the progression of glioma.

Expression Levels of Survivin and Caspase-3 Proteins in Elderly Patients with Glioma according to Different Clinical
Stages. Subsequently, we examined expression levels of Survivin and caspase-3 in blood samples from 100 elderly patients with glioma by ELISA methods. Results of ELISA revealed that elderly patients with glioma presented an elevated expression level of Survivin concomitant with a declined expression level of caspase-3 compared with healthy controls (P < 0:05, Table 2). In addition, higher Survivin expression levels and lower caspase-3 expression levels reflected the advanced clinical stage of glioma (P < 0:05, Table 2), indicating that Survivin and caspase-3 proteins may be associated with the progression of glioma.

High Expressions of Notch and Survivin Proteins Were
Associated with Poor Overall Survival of Elderly Glioma. The overall survivals of elderly patients with high expression levels of Notch 1, Notch 2, Notch 3, and Survivin were shorter than those with medium expressions (P < 0:05).
Elderly patients with medium expression levels of Notch 1, Notch 2, Notch 3, and Survivin were longer than those with low expressions (P < 0:05). The Kaplan-Meier curve was plotted to depict the overall survival of elderly patients with glioma. As shown in Figure 1, expression levels of Notch 1, Notch 2, Notch 3, and Survivin were correlated with the prognosis of elderly patients with glioma. As expression levels of Notch and Survivin proteins increased, the patient's prognosis became worse.  3 Journal of Nanomaterials displayed that the incubation of cells with MRK-003 effectively suppressed the mRNA expression of Survivin in U251 cells (Figure 2(a)). Next, we are also wondering the effect of Notch activation on Survivin expression in U251 cells. Since the Notch pathway is activated upon specific ligands like Jagged1 binding to the related transmembrane receptors, U251 cells were cultured for 48 h on immobilized Jagged1 ligand and then analyzed for Survivin expression. As expected, an increased mRNA expression of Survivin was detected in U251 cells treated with Jagged1 compared with those treated with IgG-Fc (Figure 2(b)). Notably, the delivery of pN3-N1ICD in combination with the Survivin   Journal of Nanomaterials promoter into U251 cells yielded a 2.4-fold increase in Survivin promoter activity (Figure 2(c)). These findings indicated that Notch activation was associated with Survivin overexpression in glioma.

Alternation of Notch Pathway Affected Glioma Cell
Viability and Migration. Next, we set out to ascertain the effects of the Notch pathway on U251 cell viability and migration. Results of MTT assays revealed reduced U251 cell viability following MRK-003 treatment and enhanced cell viability following Jagged1 stimulation (Figure 3). Results of transwell migration assays demonstrated fewer U251 cells migrating from the upper chamber into the lower one upon MRK-003 treatment and more U251 cells migrating from the upper chamber into the lower one upon Jagged1 stimula-tion ( Figure 4). These findings indicated that activation of the Notch pathway may contribute to the development of glioma. 5 Journal of Nanomaterials clinical value of high expression of Notch and Survivin proteins in elderly patients with glioma and its relationship with the prognosis of patients, compared with healthy controls, the expression of Notch 1, Notch 2, Notch 3, and Survivin proteins in the elderly glioma group significantly increased while the expression of caspase-3 significantly decreased. Besides, with the increase in the clinical stages of patients, the abovementioned protein changes became more apparent, with significant differences between the clinical stages. Therefore, the expression of Notch and Survivin in elderly patients with glioma is closely related to the clinical stage and prognosis of patients.

Discussion
The abnormal expression of Notch protein is closely related to the pathogenesis of various malignant tumors [13,14] and is a potential target of drug therapy [15]. In the investigation of the inhibitory effect of Notch 1 blocker on tumor growth of the osteosarcoma mouse model and related molecular mechanisms, it was discovered that Notch 1 blocker can inhibit the tumor growth of the osteosarcoma mouse model. The molecular mechanisms involved in this process include reducing the production of proproliferation molecules and proinvasion molecules and increasing the production of proapoptosis and invasion-inhibiting molecules [16]. The analysis of the effect of RNAi interference with the expression of Notch 1 gene in melanoma cells on the immune mechanism of mouse tumors also confirmed that siNotch 1 can inhibit the Notch pathway of melanoma cells and then reduce its secretion of TGF-β; as a result, its inhibitory effect on lymphocytes is reduced, the infiltration of gp100 antigen-specific CD8 + T cells in tumor tissues is increased, the secretion of IFN-γ is promoted, the proportion of Treg cells is lowered, and the body's antitumor immune function is enhanced [17]. Previous studies have also proposed that in renal clear cell carcinoma cell lines, overactivated Notch 1 can promote cancer by regulating the PTEN/PI3K/AKT signaling pathway; this provides a new treatment strategy for the treatment of renal clear cell carcinoma [18]. In our study, compared with the control group, the expression of Notch 1, Notch 2, and Notch 3 proteins in the elderly glioma group significantly increased; with the increase in the clinical stages of patients, the abovementioned protein changes became more apparent, with significant differences between the clinical stages. This indicates that the expression of Notch in elderly patients with glioma is closely related to the clinical stage and prognosis of the patients.
Survivin is a member of the inhibitor of apoptosis protein family. It is expressed in a cell cycle-dependent manner during mitosis. It is located in distinct parts of the mitotic apparatus and plays an essential role in cell division and inhibition of apoptosis. Survivin is expressed in most human cancers, but not in normal adult tissues [19,20]. Inhibition of Survivin has been well-studied as a cancer therapeutic. For example, Carrasco and his team found specific inhibition of Survivin expression by LY2181308-sensitized tumor cells to chemotherapeutic-induced apoptosis. Most importantly, they found that LY2181308 yielded antitumor activity in an in vivo human xenograft tumor model, and this model was sensitized to gemcitabine, paclitaxel, and docetaxel [21]. With the increase of age, the mechanism of neurogenesis decrease is still unclear, but it has been reported that it involves the changes of the microenvironment of neural progenitor cells. Astrocytes regulate the cycle of neural progenitor cells by acting on the expression level of Survivin, a known mitotic regulator. Among the cell cycle genes found in the elderly neural progenitor cells, Survivin is the only one that can restore the proliferation of the elderly brain neural progenitor cells. Miranda et al. provide a mechanism for the gradual loss of neurogenesis in the brain in relation to aging and reveal that targeted silencing of Survivin expression directly or through Wnt signaling could stimulate adult neurogenesis [22]. Survivin exhibits its antiapoptotic function in part by inhibiting caspase-3 activity [23]. The results in our study also demonstrate that compared with the healthy control, the expression of Survivin protein in the elderly glioma group was significantly increased while the expression of caspase-3 significantly was reduced; with the increase in the clinical stages of patients, the abovementioned protein changes became more apparent, with significant differences between the clinical stages. It suggests that Survivin and Survivin-dependent caspase-3 proteins are also closely  Journal of Nanomaterials related to the clinical stage and prognosis of elderly patients with glioma.
In summary, the present study showed that Notch and Survivin proteins were overexpressed in elderly glioma and their upregulation may contribute to the development of glioma in elderly patients and could serve as prognostic factors. We also demonstrate that activation of the Notch pathway mediates upregulation of Survivin expression and leads to glioma cell viability and migration. Finally, these data might be helpful in identifying the molecular mechanism involved in the elevated level of Survivin in glioma cells, and targeting these factors might be an important implication in the development of new therapeutic strategies aimed at blocking Notch activation in glioma cells.

Data Availability
The data used to support the findings of this study are included within the article.

Conflicts of Interest
The authors declare that they have no conflicts of interest.