# Aprepitant Promotes Caspase-Dependent Apoptotic Cell Death and G2/M Arrest through PI3K/Akt/NF-κB Axis in Cancer Stem-Like Esophageal Squamous Cell Carcinoma Spheres

Authors: Hossein Javid; Amir R. Afshari; Farnaz Zahedi Avval; Jahanbakhsh Asadi; Seyed Isaac Hashemy
Journal: BioMed Research International
 (2021)

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## Abstract

The antagonists of the neurokinin-1 receptor (NK1R) are known for their anti-inflammatory, anxiolytic, antiemetic, and anticancer activities. Aprepitant, a nonpeptide NK1R antagonist, is used in nausea and vomiting, the most common side effects of cancer chemotherapy in patients. It has been established that NK1R activation by substance P (SP), which links cancer promotion and progression to a neurokinin-mediated environment, became one mechanism that corresponds to the mitogenesis of tumor cells. Therefore, this study is aimed at explaining and evaluating the anticancer impacts of aprepitant on esophageal squamous cancer cell (ESCC) spheres by using in vitro experiments, such as resazurin, ROS, annexin-V binding, RT-PCR, and Western blot analysis. As a result, we showed that aprepitant had strong antiproliferative and cytotoxic effects on ESCC cell spheres. Also, aprepitant caused significant G2-M cell cycle arrest depending on concentration increase. Further, exposure of cells to this agent resulted in caspase -8/-9-dependent apoptotic pathway activation by modifying the expression of genes involved in apoptosis. Besides, treatment of the cells by aprepitant abrogates of the PI3K/Akt pathway, as shown by reducing the level of Akt, induces apoptotic cell death. In summary, pharmacological inhibition of NK1R with aprepitant seems to have a significant chance of treating ESCC as a single agent or in conjunction with other chemotherapeutic drugs.


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## Body

## 1. Introduction

Esophageal squamous cell carcinoma (ESCC) is the dominant histological subtype of esophagus cancer, which constitutes more than 90% of this malignancy [1]. Given the ongoing progress of therapies, like surgery and chemoradiation, the overall survival rate of affected patients remains dismal because of treatment failure and high risk of recurrence. It is, therefore, critically necessary to clarify its pathogenesis and to identify efficient agents as emerging chemotherapeutic potential therapies for its prevention, diagnosis, and treatment [2, 3].In most cancers, there is a side population of cells characterized by self-renewal ability, differentiation potential, high tumorigenicity, and therapy resistance, known as cancer stem cells (CSCs) [4, 5]. Unfortunately, the presence of CSCs in ESCC can consequently lead to therapeutic failures [6]. There are still no drugs in clinics available to target CSCs specifically; however, chemotherapy for advanced or recurrent ESCC is the primary approach of palliative treatment [7, 8]. Recently, the expression and secretion of peptides by tumors have been shown to influence the growth and development of cancer. As an undecapeptide of the tachykinin family, substance P (SP) is widely distributed in the central and peripheral nervous systems [9]. Neurokinin-1 receptor (NK1R) shows a preferential affinity for SP, regulating many biological functions, including neurogenic inflammation, pain, and depression [10]. Notably, numerous studies have indicated that the SP/NK1R system may play a significant role in the development of cancer (such as pancreatic cancer, endometrial cancer, colon carcinoma, hepatoblastoma, glioblastoma, and breast cancer), regulating cell proliferation and migration for invasion and metastasis, and controlling cell proliferation for angiogenesis [11–14]. It has been reported that the SP/NK1R system is involved in pancreatic cancer cells via inducing angiogenesis, migration, and tumor proliferation [15]. In contrast, it has been shown that NK1R antagonists could suppress the spread of pancreatic cell cancer, angiogenesis, and pancreatic cancer cell migration [16].Aprepitant has been shown to suppress cancer cell growth as a selective high-affinity antagonist of the human SP/NK1R system [15]. Moreover, the antitumor action of aprepitant has been previously reported by Javid et al. [17]. In a recent report, Berger et al. observed that NK1R is represented in human hepatoblastoma cells, and its inhibition with aprepitant led to significant suppression of the tumor, both in vitro and in vivo [15]. In other research, the inhibition of NK1R by aprepitant has been shown to suppress cell proliferation and induce apoptosis in multiple cancer cells by modifying different signal transduction pathways like phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) and nuclear factor-kappa B (NF-κB) [18, 19]. While several types of research highlighted the role of aprepitant in malignant diseases, its antitumor mechanism of action is little understood, and more studies are ongoing to more closely describe the molecular pathways involved in the apoptosis-inducing and cytotoxic effects of aprepitant. Herein, this study is aimed at showing the cytotoxic effects of NK1R antagonist aprepitant and defining its antitumoral action against ESCC.


## 2. Methods and Materials

### 2.1. Cell Culture, Chemicals, and Reagents

The KYSE-30 human esophageal squamous cell carcinoma cell line was prepared from the National Cell Bank of Iran (NCBI), Pasteur Institute (Tehran, Iran). It was cultured in medium containing a 1 : 1 mixture of RPMI 1640, and Ham’s F-12 (Betacell, Iran) supplemented with 5% fetal bovine serum and 1% penicillin-streptomycin (Grand Island, NY, USA) for 5-6 days until the cells reached the exponential phase of growth (0.6–1×106 cells/mL). SP and aprepitant were bought from Sigma-Aldrich (St. Louis, MO, USA). A stock solution of SP and aprepitant at the concentration of 70 μM and 74 mM, respectively, was prepared through dissolving the compound in sterile dimethyl sulfoxide (DMSO, Gibco, Scotland). It is then diluted to make a working solution and, cells were treated with relevant amounts of the SP and aprepitant solution to attain a concentration of different and specific doses. So, the final concentrations of DMSO in the culture medium did not exceed 0.1% in all the treatments.


### 2.2. ESCC Cell Sphere Formation

Single-cell suspensions derived from adherent cells were transferred to the polymer of 2-hydroxyethyl methacrylate- (poly HEMA-) coated petri dishes at the concentrations of 100000 cells/mL for ESCC cells. The cells were maintained at 37°C with 5% CO2 in serum-free RPMI/F12 medium supplemented with 2% B-27 supplement (Grand Island, NY, USA), 20 ng/mL basic fibroblast growth factor (Grand Island, NY, USA), and 20 ng/mL epidermal growth factor (Sigma-Aldrich Company, USA) to form spheres. To replenish nutrients, the medium was refreshed every two days. After six days, the spheres were separated into the single cells and were cultured subsequently into the new nonadherent petri dishes under the same conditions as before. After three passages, the stem-like properties of sphere cells (tertiary ESCC cell spheres) were analyzed by the following experimental tests.


### 2.3. Cell Viability Assay

As previously described, a colorimetric resazurin assay is based on the intracellular conversion of resazurin (nonfluorescent) to resorufin and dihydro-resorufin (highly fluorescent) in the presence of mitochondrial enzymes of metabolically active cells [20]. Briefly, 2.5×104 ESCC cell spheres were cultured in 96-well plates and administered with various doses of aprepitant 0 (untreated group), 15, 30, 50, 80, and 120 μM for 24 h and 48 hours. Next, the resazurin solution (phosphate buffer saline, 0.01 mg/mL) was added to each well. In the subsequent 3 hours, the absorbance at 600 nm excitation and 570 nm emission was measured on a microplate fluorimeter, and the IC50 value was evaluated using the GraphPad Prism® 6 (GraphPad Software, San Diego, CA, USA) software.


### 2.4. Trypan Blue Assay

The ESCC cell spheres were administered with various doses (0, 15, 30, 50, 80, and 120μM) of aprepitant for either 24 or 48 hours. Then, the viable cell density was carried out with 0.4% trypan blue staining as described [17].


### 2.5. RNA Analysis and Quantitative Reverse Transcription- (qRT-) PCR

RNA was extracted from the administrated cells by SP/aprepitant following manufacturer’s instructions (Qiagen, Valencia, CA, USA), and qRT-PCR (with specific primers for GAPDH, Bax, Bcl-2, p53, p21, and survivin) was carried out as described [17].


### 2.6. ELISA

Following exposure of ESCC cell spheres to aprepitant/SP, the protein concentrations of p53 and p21 were determined in cell supernatants according to manufacturer’s instructions (Biospeis, China), and ELISA assay was done as described [17].


### 2.7. Western Blotting

ESCC cell spheres were seeded out, administered with SP (1000 nM)/aprepitant (30μM), rinsed, and resuspended in ice-cold RIPA lysis buffer. The 30 μg protein samples were measured by a BCA protein assay kit and separated with 10% SDS-PAGE and electrotransferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad, HC, USA). The membranes were blocked with 5% nonfat skim milk for two hours at room temperature and then incubated with specific antibodies against PI3K, Akt, NF-κB P65, and β-Actin (1 : 1000; Cell Signaling Technology, MA, USA) at 4°C overnight followed by incubation with HRP-conjugated secondary antibodies (1 : 3000; Cell signaling Technology, MA, USA). After one-hour incubation, immune complexes were visualized using the Chemiluminescence Detection Kit (Thermo Fisher Scientific, Inc., Waltham, MA, USA), as shown by manufacturer’s directions. Finally, the intensity of protein bands was performed utilizing the ImageJ 1.52a software (NIH, Bethesda, Rockville, MD, USA) and then compared to the beta-actin protein.


### 2.8. Caspase Activity

The1×106 ESCC cell spheres were administered with SP (500 and 1000 nM) in the absence or presence of aprepitant (30 μM). Next, the activities of caspase-8 and caspase-9 were measured in cell supernatants according to manufacturer’s instructions (R&D System, USA), as described previously [17].


### 2.9. ROS Assay

The level of reactive oxygen species (ROS) production was examined by the cellular ROS detection kit following manufacturer’s instructions. Briefly,75×104 ESCC cell spheres were seeded and incubated overnight. After washing, the cells were exposed to the DCFDA solution (20 μM) for 30 min at 37°C. Next, the cells were rewashed and treated with SP (500 and 1000 nM)/aprepitant (30 μM) for 24 hours. The relative fluorescence intensity was measured (Excitation/Emission: 485/535 nm) with the fluorescence plate reader Perkin-Elmer. Tertbutyl hydrogen peroxide (TBHP, 150 μM) was utilized as a positive control.


### 2.10. Annexin V-FITC Assay

Apoptosis of ESCC cell spheres administrated 24 hours with SP (500 and 1000 nM)/aprepitant (30μM) was assessed with the annexin V-FITC kit following manufacturer’s directions (Roche Applied Science, Germany). Finally, flow cytometric analysis was performed on a flow cytometer (BD Bioscience, San Diego, CA, USA), and data analysis was assessed by FlowJo software (Treestar, OR, USA). All the treatments were conducted in triplicates.


### 2.11. Cell Cycle Analysis

The6×105 ESCC cell spheres were treated with SP (500 and 1000 nM)/aprepitant (30 μM) for 24 hours. Next, the cellular DNA content was evaluated with PI staining, as previously described [17].


### 2.12. Statistical Analysis

The experimental data are presented as themean±standarderror of the mean. The values were analyzed using GraphPad Prism® 6.0 software (San Diego, CA, USA) for Windows. The relative ratios of various groups were compared using the ANOVA followed by Bonferroni’s t-test. A statistically significant difference for all data was considered to be a p value < 0.05. All results were assessed in triplicate as compared to the untreated control group.


## 2.1. Cell Culture, Chemicals, and Reagents

The KYSE-30 human esophageal squamous cell carcinoma cell line was prepared from the National Cell Bank of Iran (NCBI), Pasteur Institute (Tehran, Iran). It was cultured in medium containing a 1 : 1 mixture of RPMI 1640, and Ham’s F-12 (Betacell, Iran) supplemented with 5% fetal bovine serum and 1% penicillin-streptomycin (Grand Island, NY, USA) for 5-6 days until the cells reached the exponential phase of growth (0.6–1×106 cells/mL). SP and aprepitant were bought from Sigma-Aldrich (St. Louis, MO, USA). A stock solution of SP and aprepitant at the concentration of 70 μM and 74 mM, respectively, was prepared through dissolving the compound in sterile dimethyl sulfoxide (DMSO, Gibco, Scotland). It is then diluted to make a working solution and, cells were treated with relevant amounts of the SP and aprepitant solution to attain a concentration of different and specific doses. So, the final concentrations of DMSO in the culture medium did not exceed 0.1% in all the treatments.


## 2.2. ESCC Cell Sphere Formation

Single-cell suspensions derived from adherent cells were transferred to the polymer of 2-hydroxyethyl methacrylate- (poly HEMA-) coated petri dishes at the concentrations of 100000 cells/mL for ESCC cells. The cells were maintained at 37°C with 5% CO2 in serum-free RPMI/F12 medium supplemented with 2% B-27 supplement (Grand Island, NY, USA), 20 ng/mL basic fibroblast growth factor (Grand Island, NY, USA), and 20 ng/mL epidermal growth factor (Sigma-Aldrich Company, USA) to form spheres. To replenish nutrients, the medium was refreshed every two days. After six days, the spheres were separated into the single cells and were cultured subsequently into the new nonadherent petri dishes under the same conditions as before. After three passages, the stem-like properties of sphere cells (tertiary ESCC cell spheres) were analyzed by the following experimental tests.


## 2.3. Cell Viability Assay

As previously described, a colorimetric resazurin assay is based on the intracellular conversion of resazurin (nonfluorescent) to resorufin and dihydro-resorufin (highly fluorescent) in the presence of mitochondrial enzymes of metabolically active cells [20]. Briefly, 2.5×104 ESCC cell spheres were cultured in 96-well plates and administered with various doses of aprepitant 0 (untreated group), 15, 30, 50, 80, and 120 μM for 24 h and 48 hours. Next, the resazurin solution (phosphate buffer saline, 0.01 mg/mL) was added to each well. In the subsequent 3 hours, the absorbance at 600 nm excitation and 570 nm emission was measured on a microplate fluorimeter, and the IC50 value was evaluated using the GraphPad Prism® 6 (GraphPad Software, San Diego, CA, USA) software.


## 2.4. Trypan Blue Assay

The ESCC cell spheres were administered with various doses (0, 15, 30, 50, 80, and 120μM) of aprepitant for either 24 or 48 hours. Then, the viable cell density was carried out with 0.4% trypan blue staining as described [17].


## 2.5. RNA Analysis and Quantitative Reverse Transcription- (qRT-) PCR

RNA was extracted from the administrated cells by SP/aprepitant following manufacturer’s instructions (Qiagen, Valencia, CA, USA), and qRT-PCR (with specific primers for GAPDH, Bax, Bcl-2, p53, p21, and survivin) was carried out as described [17].


## 2.6. ELISA

Following exposure of ESCC cell spheres to aprepitant/SP, the protein concentrations of p53 and p21 were determined in cell supernatants according to manufacturer’s instructions (Biospeis, China), and ELISA assay was done as described [17].


## 2.7. Western Blotting

ESCC cell spheres were seeded out, administered with SP (1000 nM)/aprepitant (30μM), rinsed, and resuspended in ice-cold RIPA lysis buffer. The 30 μg protein samples were measured by a BCA protein assay kit and separated with 10% SDS-PAGE and electrotransferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad, HC, USA). The membranes were blocked with 5% nonfat skim milk for two hours at room temperature and then incubated with specific antibodies against PI3K, Akt, NF-κB P65, and β-Actin (1 : 1000; Cell Signaling Technology, MA, USA) at 4°C overnight followed by incubation with HRP-conjugated secondary antibodies (1 : 3000; Cell signaling Technology, MA, USA). After one-hour incubation, immune complexes were visualized using the Chemiluminescence Detection Kit (Thermo Fisher Scientific, Inc., Waltham, MA, USA), as shown by manufacturer’s directions. Finally, the intensity of protein bands was performed utilizing the ImageJ 1.52a software (NIH, Bethesda, Rockville, MD, USA) and then compared to the beta-actin protein.


## 2.8. Caspase Activity

The1×106 ESCC cell spheres were administered with SP (500 and 1000 nM) in the absence or presence of aprepitant (30 μM). Next, the activities of caspase-8 and caspase-9 were measured in cell supernatants according to manufacturer’s instructions (R&D System, USA), as described previously [17].


## 2.9. ROS Assay

The level of reactive oxygen species (ROS) production was examined by the cellular ROS detection kit following manufacturer’s instructions. Briefly,75×104 ESCC cell spheres were seeded and incubated overnight. After washing, the cells were exposed to the DCFDA solution (20 μM) for 30 min at 37°C. Next, the cells were rewashed and treated with SP (500 and 1000 nM)/aprepitant (30 μM) for 24 hours. The relative fluorescence intensity was measured (Excitation/Emission: 485/535 nm) with the fluorescence plate reader Perkin-Elmer. Tertbutyl hydrogen peroxide (TBHP, 150 μM) was utilized as a positive control.


## 2.10. Annexin V-FITC Assay

Apoptosis of ESCC cell spheres administrated 24 hours with SP (500 and 1000 nM)/aprepitant (30μM) was assessed with the annexin V-FITC kit following manufacturer’s directions (Roche Applied Science, Germany). Finally, flow cytometric analysis was performed on a flow cytometer (BD Bioscience, San Diego, CA, USA), and data analysis was assessed by FlowJo software (Treestar, OR, USA). All the treatments were conducted in triplicates.


## 2.11. Cell Cycle Analysis

The6×105 ESCC cell spheres were treated with SP (500 and 1000 nM)/aprepitant (30 μM) for 24 hours. Next, the cellular DNA content was evaluated with PI staining, as previously described [17].


## 2.12. Statistical Analysis

The experimental data are presented as themean±standarderror of the mean. The values were analyzed using GraphPad Prism® 6.0 software (San Diego, CA, USA) for Windows. The relative ratios of various groups were compared using the ANOVA followed by Bonferroni’s t-test. A statistically significant difference for all data was considered to be a p value < 0.05. All results were assessed in triplicate as compared to the untreated control group.


## 3. Results

### 3.1. The KYSE-30-Derived Sphere Represents CSC-Like Characteristics

To confirm the ESCC cell sphere enrichment, the KYSE-30-derived sphere in passage three was evaluated to their original adherent cells. As depicted in Figure1(a), the cell line showed the most well-shaped sphere appearance in passage 3. Further gene expression analysis indicated that the master regulators of the pluripotency genes (SOX2 and OCT4) are markedly upregulated in ESCC cell spheres as compared to KYSE-30 attached cells (Figure 1(b)). The sphere cells characterized by pluripotency gene overexpression and anchorage-independent proliferation were chosen as cancer stem cell-like cells (CSC-LCs) in the next experiments.Figure 1
CSC characterization of the KYSE-30 cell spheres. The sphere enrichment process of CSCs is shown (a), the sphere was developed from passage 1 to 3. (b) Upregulation of the pluripotency regulators (SOX2 and OCT4) in spheres of KYSE-30 cells compared to the attached cells.∗∗p<0.01. Gene expression was normalized to the GAPDH gene expression as a reference gene, followed by the 2−Δct formula.
(a)(b)


### 3.2. Aprepitant Effectively Mitigates Cell Viability and Metabolic Activity of ESCC Cell Spheres

Growth inhibition of the ESCC cell spheres by aprepitant was analyzed with the resazurin proliferation assay. ESCC cell spheres were incubated with increasing concentrations of NK1R antagonist aprepitant for 24 and 48 h. As shown in Figure2, we observed a remarkable dose- and time-dependent growth inhibition and metabolic activity inhibition after treatment by aprepitant. Aprepitant showed robust growth inhibition, with 64.21% inhibition at 30 μM and 47.25% at 60 μM. Thus, the concentration required for a 50% reduction in optical density (IC50) observed in the controls treated with aprepitant was 48.21 μM for 24 h. The 30 μM of aprepitant was selected as the experimental concentration based on the dose-dependent viability change.Figure 2
Investigating the effect of aprepitant on cell growth and metabolic activity of ESCC cell spheres. Incubation of the cells with increasing concentrations of aprepitant (0–120μM) for 24 and 48 h reduced the cell viability and metabolic activity in a dose- and time-dependent manner.
(a)(b)


### 3.3. Aprepitant Induces Apoptosis and G2-M Phase Arrest in ESCC Cell Spheres

The cell line has been cultivated with the aprepitant and stained with Annexin-V/FITC, following confirmation of significant growth inhibition of ESCC cell spheres with the NK1R antagonist. After the administration of aprepitant (30μM), a considerable number of apoptotic cells were found in the ESCC cell spheres after 24 hours (Figure 3). After treatment with agonist SP (as an antiapoptotic agent) at 500 and 1000 nM doses, we observed no significant increases in apoptotic rates compared to the control group. Further, the antiapoptotic effects of SP were reversed after treatment with SP plus aprepitant, significantly.Figure 3
NK1R blockage by aprepitant had a suppressed growth on ESCC cell spheres through a caspase-dependent apoptotic pathway. (a, b) The percentages of annexin-V and annexin-V/PI double-positive inhibitor-treated cells were increased in response to drug treatment after 24 hours, as compared with the untreated group. (b) Also, ESCC cell spheres were subjected to the caspase assay to examine the contribution of caspases-8/-9 to aprepitant-induced apoptosis. Aprepitant exerted a substantial increase in caspase-8/-9 activity, which suggested that the apoptotic effect of aprepitant on the ESCC cell spheres was caspase-dependent. (∗) indicates a statistically significant difference compared to controls, while the (#) sign indicates a statistically significant change compared to other treatment groups.
(a)(b)(c)We also examined the caspase-8/-9 activities as the main executors for apoptotic processes to validate findings obtained from the flow cytometry study. Of particular interest is that after 24 hours of aprepitant treatment (30μM) compared with the control group, we also observed a dramatically improved enzymatic activity of caspase-8/-9, thus providing additional evidence that NK1R antagonist may contribute to the induction of ESCC cell spheres through caspase-mediated apoptosis.Recent findings have demonstrated that the treatment of cancer cells with NK1R antagonists, may arrest the progression of the cell cycle, most importantly by down/upregulating the essential proteins that regulate the growth of cells [21]. Notably, DNA content analysis showed a significant increase in the proportion of the G2-M phase of the cell cycle (Figure 4), suggestive of the proapoptotic capability of aprepitant in ESCC cell spheres, which is in accordance with the findings obtained from the annexin-V staining experiment. Moreover, we observed a substantial decrease in the cell proportion in the S phase of the cell cycle with the NK1R antagonist.Figure 4
(a) Flow cytometry analysis of cell cycle development of ESCC cell spheres treated with aprepitant. The frequency of cells in G1 (green peak), S (yellow peak), and G2/M (blue peak) was calculated with FlowJo software. (b) Aprepitant raises the percentage of the cell population in G2-M cells (c) and decreases the percentage of cell accumulation in S-phase. (∗) indicates a statistically significant difference compared to controls, while the (#) sign indicates a statistically significant change compared to other treatment groups including SP 500 nM and SP 1000 nM.
(a)(b)(c)


### 3.4. Aprepitant Did Not Alter the Transcriptional Activity of p53 Apoptosis-Related Genes

Regarding cell growth regulation, the p53 gene is often used to facilitate programmed cell death by transcriptionally triggering proapoptotic genes such as Bax, Bad, Bid, and p21 [22]. In this light, we examined whether NK1R antagonist treatment influenced the mRNA level of genes involved in apoptosis, based on the protein concentration and gene expression of p53 and proapoptotic p53 target genes p21 and Bax in ESCC cell spheres utilizing ELISA and qRT-PCR, respectively. As shown in Figure 5, our results demonstrated that aprepitant (30 μM) treatment plus SP (500 and 1000 nM) did not significantly alter the protein expression level of p53 and p21 as well as the transcriptional activity p53 proapoptotic target genes, such as p53, p21, and Bax.Figure 5
(a) Following exposure to aprepitant (30μM) for 24 hours, total lysates from ESCC cell spheres were collected and ELISA assay was performed using human p53 and P21 ELISA kit. (b) The mRNA expression levels of three genes (p53, p21, and Bax) were analysed in each group after normalizing the cycle threshold (Ct) values to GAPDH as the internal housekeeping gene. The apoptosis-related genes of p53 were not substantially enhanced in ESCC cell spheres by aprepitant. ELISA and qRT-PCR analyses showed that aprepitant plus SP treatment has no meaningful enhancement impact on proapoptotic target genes of p53 transcription activity. (∗) indicates a statistically significant difference compared to controls, while the (#) sign indicates a statistically significant change compared to SP 500 nM treatment group.
(a)(b)


### 3.5. Effects of Aprepitant on ROS Levels in ESCC Cell Spheres

To determine whether the cytotoxic effects of aprepitant on the killing of ESCC cell spheres were accompanied by decreased ROS levels, we decided to determine the levels of ROS (Epoch, BioTek® instruments, Inc., USA) in response to aprepitant and SP in treated versus untreated cells. As Figure6 reveals, the treatment with aprepitant (30 μM, 24 hours) in comparison with the SP alone (500 and 1000 nM) led to a significant reduction in the levels of ROS, demonstrating antioxidative effects of aprepitant in ESCC cell spheres. In comparison to the control group, it was also noteworthy that ROS was elevated by SP at a concentration of 1000 nM. Also, compared to the control group, the combination of SP and aprepitant dramatically reduced the production of ROS caused by SP. Therefore, ROS is not one of the main aprepitant-mediated cytotoxicity pathways in ESCC cell spheres.Figure 6
Aprepitant effects on the ROS levels in the ESCC cell spheres. Our results indicate that the aprepitant decreases the level of reactive oxygen (ROS) in 24 hours. The cells were treated by aprepitant (30μM) for 24 h. ROS levels were measured by a fluorimeter. The intensity of the fluorescence has improved dramatically relative to the control group in the positive control tert-butyl hydroperoxide (TBHP, 150 μM). At 24 hours after treatment, aprepitant significantly reduced the ROS induced by the substance P (SP). (∗) indicates a statistically significant difference compared to controls, while the (#) sign indicates a statistically significant change compared to other treatment groups including SP 500 nM and SP 1000 nM.


### 3.6. Aprepitant Regulates Cytotoxicity through the PI3K/Akt/NF-κB Signaling Pathway

Numerous molecular experiments have shown that stimulation of NK1R is directly correlated with the NF-κB signaling axis and that SP/NK1R controls apoptosis in malignant cells by targeting this axis [23]. We assessed the quantity of NF-κB p65 protein from drug-treated ESCC cell spheres using Western blot analysis, to evaluate whether blockage of NK1R could increase the cytotoxicity of aprepitant through suppression of NF-κB. Furthermore, the RT-PCR study of antiapoptotic NF-κB target genes has examined the impact of aprepitant on the NF-κB axis. We found that while 1000 nM of SP had a stimulatory effect on NF-κB levels and its downstream target genes (survivin and Bcl-2), aprepitant (30 μM) remarkably reduced the protein level of NF-κB and its antiapoptotic target genes. Besides, these effects were significantly ameliorated by treatment with aprepitant, confirming that aprepitant has antioxidative effects, at least partially, through the modulation of NF-κB activity (Figure 7).Figure 7
Aprepitant mitigates the PI3K/Akt/NF-κB signaling pathway, as well as the expression of antiapoptotic NF-κB target genes in ESCC cell spheres. (a) Western blotting found that the protein concentration of PI3K, Akt, and NF-κB 65 in the aprepitant (30 μM) group was substantially reduced in comparison with the control group (p<0.05 and p<0.01). (b) Moreover, the administration of aprepitant (30 μM) showed a markedly reduced relative mRNA expression of NF-κB antiapoptotic target genes, including survivin and Bcl-2. The levels of expression of all the target genes were normalized by GAPDH mRNA levels (p<0.05 and p<0.01). (∗) indicates a statistically significant difference compared to controls, while the (#) sign indicates a statistically significant change compared to other treatment groups including SP 500 nM and SP 1000 nM.
(a)(b)Numerous findings show that Akt activation, as the most significant downstream effector of the PI3K signaling pathway, is one of the critical pathways implicated in pathogenesis in the ESCC [17]. On the other hand, recent studies have revealed that stimulation of NK1R in human cancer cells increases the phosphorylation and the activity of Akt. Notwithstanding this, it was challenging to examine whether inhibition of NK1R using aprepitant participates in the inhibition of Akt in ESCC cell spheres. Interestingly, an apparent reduction of the Akt level was found in the inhibitor-treated cells. More notably, aprepitant (30 μM) could decrease the elevation of the Akt level induced by SP (1000 nM). These effects were also observed in PI3K levels, indicating that aprepitant effectively abolishes the proliferation of ESCC cell spheres through PI3K/Akt protein levels inhibition (Figure 7).


## 3.1. The KYSE-30-Derived Sphere Represents CSC-Like Characteristics

To confirm the ESCC cell sphere enrichment, the KYSE-30-derived sphere in passage three was evaluated to their original adherent cells. As depicted in Figure1(a), the cell line showed the most well-shaped sphere appearance in passage 3. Further gene expression analysis indicated that the master regulators of the pluripotency genes (SOX2 and OCT4) are markedly upregulated in ESCC cell spheres as compared to KYSE-30 attached cells (Figure 1(b)). The sphere cells characterized by pluripotency gene overexpression and anchorage-independent proliferation were chosen as cancer stem cell-like cells (CSC-LCs) in the next experiments.Figure 1
CSC characterization of the KYSE-30 cell spheres. The sphere enrichment process of CSCs is shown (a), the sphere was developed from passage 1 to 3. (b) Upregulation of the pluripotency regulators (SOX2 and OCT4) in spheres of KYSE-30 cells compared to the attached cells.∗∗p<0.01. Gene expression was normalized to the GAPDH gene expression as a reference gene, followed by the 2−Δct formula.
(a)(b)


## 3.2. Aprepitant Effectively Mitigates Cell Viability and Metabolic Activity of ESCC Cell Spheres

Growth inhibition of the ESCC cell spheres by aprepitant was analyzed with the resazurin proliferation assay. ESCC cell spheres were incubated with increasing concentrations of NK1R antagonist aprepitant for 24 and 48 h. As shown in Figure2, we observed a remarkable dose- and time-dependent growth inhibition and metabolic activity inhibition after treatment by aprepitant. Aprepitant showed robust growth inhibition, with 64.21% inhibition at 30 μM and 47.25% at 60 μM. Thus, the concentration required for a 50% reduction in optical density (IC50) observed in the controls treated with aprepitant was 48.21 μM for 24 h. The 30 μM of aprepitant was selected as the experimental concentration based on the dose-dependent viability change.Figure 2
Investigating the effect of aprepitant on cell growth and metabolic activity of ESCC cell spheres. Incubation of the cells with increasing concentrations of aprepitant (0–120μM) for 24 and 48 h reduced the cell viability and metabolic activity in a dose- and time-dependent manner.
(a)(b)


## 3.3. Aprepitant Induces Apoptosis and G2-M Phase Arrest in ESCC Cell Spheres

The cell line has been cultivated with the aprepitant and stained with Annexin-V/FITC, following confirmation of significant growth inhibition of ESCC cell spheres with the NK1R antagonist. After the administration of aprepitant (30μM), a considerable number of apoptotic cells were found in the ESCC cell spheres after 24 hours (Figure 3). After treatment with agonist SP (as an antiapoptotic agent) at 500 and 1000 nM doses, we observed no significant increases in apoptotic rates compared to the control group. Further, the antiapoptotic effects of SP were reversed after treatment with SP plus aprepitant, significantly.Figure 3
NK1R blockage by aprepitant had a suppressed growth on ESCC cell spheres through a caspase-dependent apoptotic pathway. (a, b) The percentages of annexin-V and annexin-V/PI double-positive inhibitor-treated cells were increased in response to drug treatment after 24 hours, as compared with the untreated group. (b) Also, ESCC cell spheres were subjected to the caspase assay to examine the contribution of caspases-8/-9 to aprepitant-induced apoptosis. Aprepitant exerted a substantial increase in caspase-8/-9 activity, which suggested that the apoptotic effect of aprepitant on the ESCC cell spheres was caspase-dependent. (∗) indicates a statistically significant difference compared to controls, while the (#) sign indicates a statistically significant change compared to other treatment groups.
(a)(b)(c)We also examined the caspase-8/-9 activities as the main executors for apoptotic processes to validate findings obtained from the flow cytometry study. Of particular interest is that after 24 hours of aprepitant treatment (30μM) compared with the control group, we also observed a dramatically improved enzymatic activity of caspase-8/-9, thus providing additional evidence that NK1R antagonist may contribute to the induction of ESCC cell spheres through caspase-mediated apoptosis.Recent findings have demonstrated that the treatment of cancer cells with NK1R antagonists, may arrest the progression of the cell cycle, most importantly by down/upregulating the essential proteins that regulate the growth of cells [21]. Notably, DNA content analysis showed a significant increase in the proportion of the G2-M phase of the cell cycle (Figure 4), suggestive of the proapoptotic capability of aprepitant in ESCC cell spheres, which is in accordance with the findings obtained from the annexin-V staining experiment. Moreover, we observed a substantial decrease in the cell proportion in the S phase of the cell cycle with the NK1R antagonist.Figure 4
(a) Flow cytometry analysis of cell cycle development of ESCC cell spheres treated with aprepitant. The frequency of cells in G1 (green peak), S (yellow peak), and G2/M (blue peak) was calculated with FlowJo software. (b) Aprepitant raises the percentage of the cell population in G2-M cells (c) and decreases the percentage of cell accumulation in S-phase. (∗) indicates a statistically significant difference compared to controls, while the (#) sign indicates a statistically significant change compared to other treatment groups including SP 500 nM and SP 1000 nM.
(a)(b)(c)


## 3.4. Aprepitant Did Not Alter the Transcriptional Activity of p53 Apoptosis-Related Genes

Regarding cell growth regulation, the p53 gene is often used to facilitate programmed cell death by transcriptionally triggering proapoptotic genes such as Bax, Bad, Bid, and p21 [22]. In this light, we examined whether NK1R antagonist treatment influenced the mRNA level of genes involved in apoptosis, based on the protein concentration and gene expression of p53 and proapoptotic p53 target genes p21 and Bax in ESCC cell spheres utilizing ELISA and qRT-PCR, respectively. As shown in Figure 5, our results demonstrated that aprepitant (30 μM) treatment plus SP (500 and 1000 nM) did not significantly alter the protein expression level of p53 and p21 as well as the transcriptional activity p53 proapoptotic target genes, such as p53, p21, and Bax.Figure 5
(a) Following exposure to aprepitant (30μM) for 24 hours, total lysates from ESCC cell spheres were collected and ELISA assay was performed using human p53 and P21 ELISA kit. (b) The mRNA expression levels of three genes (p53, p21, and Bax) were analysed in each group after normalizing the cycle threshold (Ct) values to GAPDH as the internal housekeeping gene. The apoptosis-related genes of p53 were not substantially enhanced in ESCC cell spheres by aprepitant. ELISA and qRT-PCR analyses showed that aprepitant plus SP treatment has no meaningful enhancement impact on proapoptotic target genes of p53 transcription activity. (∗) indicates a statistically significant difference compared to controls, while the (#) sign indicates a statistically significant change compared to SP 500 nM treatment group.
(a)(b)


## 3.5. Effects of Aprepitant on ROS Levels in ESCC Cell Spheres

To determine whether the cytotoxic effects of aprepitant on the killing of ESCC cell spheres were accompanied by decreased ROS levels, we decided to determine the levels of ROS (Epoch, BioTek® instruments, Inc., USA) in response to aprepitant and SP in treated versus untreated cells. As Figure6 reveals, the treatment with aprepitant (30 μM, 24 hours) in comparison with the SP alone (500 and 1000 nM) led to a significant reduction in the levels of ROS, demonstrating antioxidative effects of aprepitant in ESCC cell spheres. In comparison to the control group, it was also noteworthy that ROS was elevated by SP at a concentration of 1000 nM. Also, compared to the control group, the combination of SP and aprepitant dramatically reduced the production of ROS caused by SP. Therefore, ROS is not one of the main aprepitant-mediated cytotoxicity pathways in ESCC cell spheres.Figure 6
Aprepitant effects on the ROS levels in the ESCC cell spheres. Our results indicate that the aprepitant decreases the level of reactive oxygen (ROS) in 24 hours. The cells were treated by aprepitant (30μM) for 24 h. ROS levels were measured by a fluorimeter. The intensity of the fluorescence has improved dramatically relative to the control group in the positive control tert-butyl hydroperoxide (TBHP, 150 μM). At 24 hours after treatment, aprepitant significantly reduced the ROS induced by the substance P (SP). (∗) indicates a statistically significant difference compared to controls, while the (#) sign indicates a statistically significant change compared to other treatment groups including SP 500 nM and SP 1000 nM.


## 3.6. Aprepitant Regulates Cytotoxicity through the PI3K/Akt/NF-κB Signaling Pathway

Numerous molecular experiments have shown that stimulation of NK1R is directly correlated with the NF-κB signaling axis and that SP/NK1R controls apoptosis in malignant cells by targeting this axis [23]. We assessed the quantity of NF-κB p65 protein from drug-treated ESCC cell spheres using Western blot analysis, to evaluate whether blockage of NK1R could increase the cytotoxicity of aprepitant through suppression of NF-κB. Furthermore, the RT-PCR study of antiapoptotic NF-κB target genes has examined the impact of aprepitant on the NF-κB axis. We found that while 1000 nM of SP had a stimulatory effect on NF-κB levels and its downstream target genes (survivin and Bcl-2), aprepitant (30 μM) remarkably reduced the protein level of NF-κB and its antiapoptotic target genes. Besides, these effects were significantly ameliorated by treatment with aprepitant, confirming that aprepitant has antioxidative effects, at least partially, through the modulation of NF-κB activity (Figure 7).Figure 7
Aprepitant mitigates the PI3K/Akt/NF-κB signaling pathway, as well as the expression of antiapoptotic NF-κB target genes in ESCC cell spheres. (a) Western blotting found that the protein concentration of PI3K, Akt, and NF-κB 65 in the aprepitant (30 μM) group was substantially reduced in comparison with the control group (p<0.05 and p<0.01). (b) Moreover, the administration of aprepitant (30 μM) showed a markedly reduced relative mRNA expression of NF-κB antiapoptotic target genes, including survivin and Bcl-2. The levels of expression of all the target genes were normalized by GAPDH mRNA levels (p<0.05 and p<0.01). (∗) indicates a statistically significant difference compared to controls, while the (#) sign indicates a statistically significant change compared to other treatment groups including SP 500 nM and SP 1000 nM.
(a)(b)Numerous findings show that Akt activation, as the most significant downstream effector of the PI3K signaling pathway, is one of the critical pathways implicated in pathogenesis in the ESCC [17]. On the other hand, recent studies have revealed that stimulation of NK1R in human cancer cells increases the phosphorylation and the activity of Akt. Notwithstanding this, it was challenging to examine whether inhibition of NK1R using aprepitant participates in the inhibition of Akt in ESCC cell spheres. Interestingly, an apparent reduction of the Akt level was found in the inhibitor-treated cells. More notably, aprepitant (30 μM) could decrease the elevation of the Akt level induced by SP (1000 nM). These effects were also observed in PI3K levels, indicating that aprepitant effectively abolishes the proliferation of ESCC cell spheres through PI3K/Akt protein levels inhibition (Figure 7).


## 4. Discussion

Given that NK1R is considered highly involved in the formation and growth of many carcinomas, intense research is being done to block this receptor with NK1R antagonists for possible anticancer uses [19]. In most of the cancers, there is a side population of cells characterized by self-renewal ability, differentiation potential, high carcinogenicity, and therapy resistance, known as cancer stem cells [4, 5]. ESCC, as world’s sixth leading cause of cancer mortality, is histologically the main form of esophageal cancer. In the treatment of ESCC patients, it is possible that the ESCC cell spheres are of elevated NK1R expression as compared to the healthy cells. Aprepitant (Emend®), an NK1R-specific high-affinity antagonist with no toxicity, is widely used for the treatment of chemotherapy-induced nausea and vomiting (CINV) [24]. The safety of aprepitant against human fibroblasts (the IC50 for fibroblasts are three times higher than the IC50 for tumor cells) has been shown. Further, the IC50 for nontumor cells is 90 μM, but the IC100 for tumor cells is 60 μM, approximately [25, 26].Interestingly, several studies are suggesting this medication has an efficient antitumor impact on a wide range of cancers, including melanoma, lung, hepatoblastoma, pancreas, and breast cancer [27]. Specific molecular pathways are still poorly explained by which aprepitant induces antitumor impacts on various cancer cells. Hence, the effects and molecular pathways of aprepitant in the ESCC cell spheres were analyzed throughout this study.In this context, the concentration-dependent growth inhibition of aprepitant on ESCC cell spheres was discovered. Dysregulation of the cell cycle is a notable feature of cancer, and cell cycle arrest could be an essential guide for anticancer medications [28]. The progression of cell cycles may be regulated by the complexes of cyclin-dependent kinases (CDK). The p21 directly links and prevents kinase activities, which lead to cell cycle arrest, and p53 is known to trigger cell cycle arrest as the tumor suppressor protein [17]. Earlier research has shown that aprepitant prevented cell proliferation in G2/M and significantly downregulated cyclin B1, as well as upregulated p21 in oral squamous cell carcinoma lines (WSU-HN6, UM1, SCC25, and WSU-HN4) [29–31]. Furthermore, the protein behavior can be altered due to posttranslational modifications such as phosphorylation, sulfation, and glycation, which change a local charge of the protein region [32, 33]. Therefore, many changes in gene expression recorded between experiment groups are due to posttranslational modifications, not detected by analyses of RNA. Our studies have shown a similar pattern of no changes in protein expression level in various groups. So, differences in a group of mRNA gene expressions (between SP500 nM and SP500nM+AP30μM (can be ignored. Accordingly, our study showed that aprepitant could cause G2/M cell cycle arrest in ESCC cell spheres, in a CDK-independent manner.Moreover, our study showed that aprepitant could substantially improve the anticancer effects by apoptosis induction. Flow cytometry analysis revealed that apoptosis was markedly induced as compared with the control group by aprepitant treatment in ESCC cell spheres. Two major pathways have currently been established which connect apoptosis: (a) intrinsic or mitochondrial and (b) extrinsic or death-receptor-related [27]. Hence, a caspase activity analysis was conducted to explore caspase activation as a potential mechanism influencing the increased apoptosis level observed in aprepitant-treated ESCC cell spheres. In ESCC cells treated with aprepitant, a substantial increase in caspase-8/-9 activity was reported, which was in line with the results obtained by FACS analysis.The function of caspases and proapoptotic mitochondrial cascades in control of apoptosis is well established, but the involvement of ROS is elusive for the regulation of apoptosis. A wide range of anticancer agents has been shown to kill cancer cells efficiently and to sensitize different cancer cells by modulating ROS production to other chemotherapeutic agents [34, 35]. Most chemotherapies also fatally influence tumor cells by raising the oxidative stress in these cells, thus stressing them beyond their compensatory capacity, and pharmacological agents, which have increased ROS development in cancer cells, are approved agents for cancer therapy [36]. Based on these findings, our data showed that aprepitant could significantly decrease the generation of ROS induced by SP in ESCC cells.In previous decades, a variety of studies have shown that in several forms of malignant tumors, the pathway of PI3K/Akt signaling is aberrant [37–39]. PI3K/Akt pathway activation promotes cell differentiation, development, and angiogenesis and seems to be fundamental for apoptosis, which is considered to be a potential target anticancer therapy [40]. Here, we investigated the role of aprepitant in the PI3K/Akt signaling cascade to understand the mechanisms of aprepitant-inhibited cell growth and induced apoptosis in ESCC. Our findings revealed that apoptotic cell death by inhibition of NK1R was seen, at least partly, through abrogation of the PI3K/Akt pathway, as shown by the significant reduction of the Akt protein level. The PI3K/Akt pathway is of great importance in CSCs, including the maintaining of colony-formation ability and proliferation. Akazawa et al. have consistently demonstrated that NK1R inhibition contributes to decreased Akt activity through the PI3K pathway [18].Many studies have shown that Akt controls the transcriptional behavior of NF-κB via phosphorylation and subsequent degradation of IκBα, permitting NF-κB to be imported at the nuclear level, followed by the binding and triggering of its antiapoptotic target genes [41]. In this regard, it was interesting to determine if the Akt/NF-κB axis may be used to mediate aprepitant-induced apoptosis. Our findings revealed that aprepitant decreases the protein expression level of NF-κB and the mRNA level of antiapoptotic NF-κB target genes like survivin and Bcl-2, suggesting that the apoptotic process in ESCC cell spheres is, at least partly, through the NF-κB-dependent pathway induced by aprepitant.The conclusive proof was documented that a strong dependence on the interaction between p53 and NF-κB signal pathways seems to be because of the susceptibility of cancer cells to apoptosis [42]. Furthermore, two of the most critical regulatory pathways in cells have been identified to be PI3K/Akt and p53, where Akt repression is related to increased p53, subsequent apoptosis, and arrest in growth. In contrast to those as mentioned above, our results reveal that p53 protein expression and mRNA levels of p53 proapoptotic target genes, including p21 and Bax, have not been altered by aprepitant (30 μM). These data proved that aprepitant could exert its apoptotic effects in cell spheres of the ESCC through a p53-independent apoptotic path.In summary, findings from the present study have demonstrated that aprepitant exerts antitumor activities like proliferation inhibition, cell cycle arrest, and induction of apoptosis against ESCC cell spheres. This study also proposes mechanical pathways, which inhibit NK1R through a possible caspase-dependent mechanism that increases apoptotic cell death. Further, aprepitant showed, at least partly, its anticarcinogenicity by inhibiting the PI3K/Akt signaling pathway. Therefore, more preclinical studies will highlight the mechanisms for the molecular involvement of aprepitant cytotoxicity.



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*Source: 8808214-2021-12-09.xml*