(20S) Ginsenoside Rh2-Activated, Distinct Apoptosis Pathways in Highly and Poorly Differentiated Human Esophageal Cancer Cells

Ginsenoside Rh2 (G-Rh2), a rare ginsenoside isolated from red ginseng, has considerable anti-cancer activity and induces apoptosis in a variety of cancer cells, but its activity in esophageal cancer cells is unclear. In this study, we examined the cytotoxic activity of (20S) G-Rh2 in highly differentiated esophageal squamous ECA109 cells and poorly differentiated esophageal squamous TE-13 cells. (20S) G-Rh2 exerted intense cytotoxicity in ECA109 and TE-13 cells with an IC50 of 2.9 and 3.7 μg/mL, respectively. After treatment with G-Rh2, Bcl-2, and Bcl-xL, the two main anti-apoptosis Bcl-2 family proteins upregulated, and Bax and Bak, the two key pro-apoptosis proteins translocated to mitochondria in both cell lines. At the same time, cytochrome c and Smac released from mitochondria, followed by caspase-9 activation, indicating that a mitochondria-mediated intrinsic apoptosis pathway was activated in both cell lines upon treatment with (20S) G-Rh2. It is noteworthy that (20S) G-Rh2 upregulated the transcription and protein expression of two death receptors, Fas and DR5, and subsequently activated Caspase-8 in the TE-13 cells but not in the ECA109 cells. Taken together, we demonstrated the potent anti-esophageal cancer cell activity of (20S) G-Rh2 and showed its working mechanism in two differentiated esophageal cancer cells, which can provide important evidence for developing an effective strategy for anti-esophageal cancer treatment.


Introduction
Esophageal cancer is one of the most malignant tumors in the world, which can be divided into squamous cell carcinoma and adenocarcinoma [1]. Esophageal squamous cell carcinoma (ESCC) is a common type of esophageal cancer in Asia [2] while adenocarcinoma is the main type in western countries [2]. Treatment for esophageal cancer includes chemotherapy, radiation therapy, and surgery; postoperative chemotherapy is considered to be an effective method for the prevention of postoperative recurrence [3]. Due to the lack of early symptoms, esophageal cancer is mostly advanced by the time it is diagnosed, which leads to a poor survival rate after esophagectomy [4]. A study has shown that the 5-year survival rate after three esophagectomies is only about 50% [5]. Although advances in diagnosis, staging, and therapy methods have slightly improved the survival rate of patients in recent years, esophageal cancer is still one of the most fatal cancers in the world.
(20S) G-Rh2 induces apoptosis in a variety of tumor cells, including human leukemia cells, human cervical cancer cells, human lung cancer cells, and human ovarian cancer cells [17], but its effect on esophageal cancer cells is still unclear. In this research, we studied the effect of (20S) G-Rh2 in the highly differentiated esophageal squamous cell ECA109 and poorly differentiated esophageal squamous cell TE-13 and demonstrated that (20S) G-Rh2 can inhibit cell growth in both ECA109 and TE-13 cells by inducing apoptosis with different mechanisms.

(20S) G-Rh2 Inhibits the Cell Growth of ECA109 and TE-13 Cells
We first tested the effect of (20S) G-Rh2 on the cell viability of esophageal cancer cells with a cell viability assay. The results showed that (20S) G-Rh2 has a strong cell growth inhibitory effect in both ECA109 and TE-13 cells, with an IC50 of 2.9 and 3.7 μg/mL, respectively ( Figure 1). Cisplatin was used as a positive drug. and TE-13 cells with an IC50 of 2.9 and 3.7 μg/mL, respectively; (C) cell viability assay of ECA109 and TE-13 cells treated with Cisplatin. Cisplatin showed cell growth inhibition in ECA109 and TE-13 cells with an IC50 of 5.7 and 6.3 μg/mL, respectively.

(20S) G-Rh2 Inhibits the Proliferation of Esophageal Cancer Cells by Inducing Apoptosis
In order to prove that (20S) G-Rh2 inhibits esophageal cancer cell proliferation by inducing apoptosis, ECA109 and TE-13 cells were treated with (20S) G-Rh2, and both of them showed significant morphological characteristics of apoptosis (Figure 2A,B). Then, we studied the apoptotic ratio after (20S) G-Rh2 treatment by flow cytometry. The results showed that after treatment with 7.5 μg/mL (20S) G-Rh2 for 1 h, the apoptotic ratio of ECA109 and TE-13 cells were 34.59% and 18.29%, respectively, which would be 41.64% and 21.97% when the duration of (20S) G-Rh2 treatment was extended to 2 h ( Figure 2C-F). and TE-13 cells with an IC50 of 2.9 and 3.7 µg/mL, respectively; (C) cell viability assay of ECA109 and TE-13 cells treated with Cisplatin. Cisplatin showed cell growth inhibition in ECA109 and TE-13 cells with an IC50 of 5.7 and 6.3 µg/mL, respectively.

(20S) G-Rh2 Inhibits the Proliferation of Esophageal Cancer Cells by Inducing Apoptosis
In order to prove that (20S) G-Rh2 inhibits esophageal cancer cell proliferation by inducing apoptosis, ECA109 and TE-13 cells were treated with (20S) G-Rh2, and both of them showed significant morphological characteristics of apoptosis (Figure 2A,B). Then, we studied the apoptotic ratio after (20S) G-Rh2 treatment by flow cytometry. The results showed that after treatment with 7.5 µg/mL (20S) G-Rh2 for 1 h, the apoptotic ratio of ECA109 and TE-13 cells were 34.59% and 18.29%, respectively, which would be 41.64% and 21.97% when the duration of (20S) G-Rh2 treatment was extended to 2 h ( Figure 2C We also examined the activity of Caspase-3 in ECA109 and TE-13 cells, which were treated with 7.5 μg/mL (20S) G-Rh2 for 1, 2, and 4 h. Caspase-3 activity in ECA109 and TE-13 cells both time-dependently increased, which increased 11-fold to the control after 4 h in ECA109 and increased 12-fold to the control after only 1 h in TE-13 ( Figure 3A,B). As the substrate of Caspase-3, cleaved PARP in both ECA109 and TE-13 cells was also examined by Western blotting (Figure 2G,H). These results demonstrated that (20S) G-Rh2 inhibits the growth of ECA109 and TE-13 cells by inducing apoptosis. We also examined the activity of Caspase-3 in ECA109 and TE-13 cells, which were treated with 7.5 µg/mL (20S) G-Rh2 for 1, 2, and 4 h. Caspase-3 activity in ECA109 and TE-13 cells both time-dependently increased, which increased 11-fold to the control after 4 h in ECA109 and increased 12-fold to the control after only 1 h in TE-13 ( Figure 3A,B). As the substrate of Caspase-3, cleaved PARP in both ECA109 and TE-13 cells was also examined by Western blotting (Figure 2G

(20S) G-Rh2 Activates the Intrinsic Apoptosis Pathway in Esophageal Cancer Cells
We first tested the activation of the intrinsic apoptosis pathway in ECA109 and TE-13 cells. Cells were treated with 7.5 μg/mL (20S) G-Rh2 for 1, 2, and 4 h; then, Caspase-9 activity in the whole lysis was tested by using an Ac-LEHD-AFC kit. We found that the activity of Caspase-9 was time-dependent and increased in both ECA109 and TE-13 cells, and these results were consistent with the results of Western blotting assay ( Figure 3).
The decrease of the mitochondrial membrane potential is an early event of intrinsic apoptosis and is closely related to the release of Smac and cytochrome c. In our study, the mitochondrial membrane potential was detected by using a mitochondrial membrane potential assay kit with JC-1. Briefly, both ECA109 and TE-13 cells were treated with 7.5 μg/mL (20S) G-Rh2 and a JC-1 buffer for 4 h; then, the fluorescence of the JC-1 monomer (green) and aggregates (red in the matrix) were observed by a fluorescence microscope. The red dot-shaped fluorescence on mitochondria gradually diffused, and the green fluorescence increased in both ECA109 and TE-13, indicating the decrease of the mitochondrial membrane potential ( Figure 4A,B). The RT-PCR and Western blotting assay showed upregulated expression levels and mitochondrial localization of Bax and Bak while Smac and cytochrome c translocated from the mitochondria to the cytoplasm ( Figure 4C-E). The expression level of anti-apoptotic proteins was also examined and showed that cIAP-1 and Bcl-2 were downregulated after being treated with (20S) G-Rh2 ( Figure 5). Taken together, it can be concluded that (20S) G-Rh2 induced apoptosis in esophageal cancer cells by activating the mitochondria-mediated intrinsic apoptosis pathway. Figure S1 shows Cells were treated with 7.5 µg/mL G-Rh2 for the indicated times. Cell-free Caspase-8, -9, and -3 activities were detected by using specific substrates: Ac-IETD-AFC (for Caspase-8), Ac-LEHD-AFC (for Caspase-9), and Ac-DEVD-AFC (for Caspase-3); (C,D) Western blot results of cleaved Caspase-8 and cleaved Caspase-9 in ECA109 and TE-13 cells after being treated with (20S) G-Rh2. ** p < 0.01, *** p < 0.001.

(20S) G-Rh2 Activates the Intrinsic Apoptosis Pathway in Esophageal Cancer Cells
We first tested the activation of the intrinsic apoptosis pathway in ECA109 and TE-13 cells. Cells were treated with 7.5 µg/mL (20S) G-Rh2 for 1, 2, and 4 h; then, Caspase-9 activity in the whole lysis was tested by using an Ac-LEHD-AFC kit. We found that the activity of Caspase-9 was time-dependent and increased in both ECA109 and TE-13 cells, and these results were consistent with the results of Western blotting assay ( Figure 3).
The decrease of the mitochondrial membrane potential is an early event of intrinsic apoptosis and is closely related to the release of Smac and cytochrome c. In our study, the mitochondrial membrane potential was detected by using a mitochondrial membrane potential assay kit with JC-1. Briefly, both ECA109 and TE-13 cells were treated with 7.5 µg/mL (20S) G-Rh2 and a JC-1 buffer for 4 h; then, the fluorescence of the JC-1 monomer (green) and aggregates (red in the matrix) were observed by a fluorescence microscope. The red dot-shaped fluorescence on mitochondria gradually diffused, and the green fluorescence increased in both ECA109 and TE-13, indicating the decrease of the mitochondrial membrane potential ( Figure 4A,B). The RT-PCR and Western blotting assay showed upregulated expression levels and mitochondrial localization of Bax and Bak while Smac and cytochrome c translocated from the mitochondria to the cytoplasm ( Figure 4C-E). The expression level of anti-apoptotic proteins was also examined and showed that cIAP-1 and Bcl-2 were downregulated after being treated with (20S) G-Rh2 ( Figure 5). Taken together, it can be concluded that (20S) G-Rh2 induced apoptosis in esophageal cancer cells by activating the mitochondria-mediated intrinsic apoptosis pathway. Figure S1 shows this in detail: (20S) G-Rh2-induced caspase-dependent apoptosis in esophageal cancer cells identified by cell viability assay. this in detail: (20S) G-Rh2-induced caspase-dependent apoptosis in esophageal cancer cells identified by cell viability assay

(20S) G-Rh2 Activates the Extrinsic Apoptosis Pathway of TE-13 Cells
The death receptor signal pathway is another important pathway to induce apoptosis. In our research, we also tested the changes of Caspase-8 activity. We found that after treating with (20S) G-Rh2 for 1 h, Caspase-8 activity in TE-13 increased 7-fold to the control group but did not change in ECA109 ( Figure 3A,B). The result of Western blotting also showed a cleaved Caspase-8 band in the TE-13 cells, which deepened time-dependently ( Figure 3C,D). These results indicated that (20S) G-Rh2 could activate the death receptor apoptosis pathway in TE-13 cells. Subsequently, we investigated the transcription and expression levels of a series of death receptor family members in TE-13 cells by using RT-PCR and Western blotting. The results showed that death receptors Fas and DR5 were significantly upregulated at both the protein and mRNA levels ( Figure 6A,B). Therefore, we speculated that (20S) G-Rh2 can activate the extrinsic apoptosis pathway in TE-13 cells by upregulating the transcription and expression levels of death receptor family members.

(20S) G-Rh2 Activates the Extrinsic Apoptosis Pathway of TE-13 Cells
The death receptor signal pathway is another important pathway to induce apoptosis. In our research, we also tested the changes of Caspase-8 activity. We found that after treating with (20S) G-Rh2 for 1 h, Caspase-8 activity in TE-13 increased 7-fold to the control group but did not change in ECA109 ( Figure 3A,B). The result of Western blotting also showed a cleaved Caspase-8 band in the TE-13 cells, which deepened time-dependently ( Figure 3C,D). These results indicated that (20S) G-Rh2 could activate the death receptor apoptosis pathway in TE-13 cells. Subsequently, we investigated the transcription and expression levels of a series of death receptor family members in TE-13 cells by using RT-PCR and Western blotting. The results showed that death receptors Fas and DR5 were significantly upregulated at both the protein and mRNA levels ( Figure 6A,B). Therefore, we speculated that (20S) G-Rh2 can activate the extrinsic apoptosis pathway in TE-13 cells by upregulating the transcription and expression levels of death receptor family members.

Discussion
Apoptosis plays an important role in the growth and development of multicellular organisms. Excessive apoptosis may lead to degenerative disease while insufficient apoptosis may lead to tumor disease. In cancer cells, dysregulation of apoptosis caused by blocked apoptotic signaling pathways often occurs, and cancer cells gain an excessive survival advantage by disabling apoptotic mechanisms [18]. An apoptosis pathway can be divided into intrinsic apoptosis and extrinsic apoptosis pathways; the former is mitochondria-centered and eventually activates Caspase-3 and Caspase-7 by activating Caspase-9 while the latter starts from the binding of death receptors (members of the TNF receptor

Discussion
Apoptosis plays an important role in the growth and development of multicellular organisms. Excessive apoptosis may lead to degenerative disease while insufficient apoptosis may lead to tumor disease. In cancer cells, dysregulation of apoptosis caused by blocked apoptotic signaling pathways often occurs, and cancer cells gain an excessive survival advantage by disabling apoptotic mechanisms [18]. An apoptosis pathway can be divided into intrinsic apoptosis and extrinsic apoptosis pathways; the former is mitochondria-centered and eventually activates Caspase-3 and Caspase-7 by activating Caspase-9 while the latter starts from the binding of death receptors (members of the TNF receptor superfamily such as TNF-R1, Fas, DR3, DR4, and DR5) to their ligands (mainly members of the TNF family) and then activates Caspase-8 via DISC (death-inducing signaling complex) and eventually activates Caspase-3. Activated Caspase-8 can also transduce apoptotic signals to mitochondria through cascade amplification and then trigger intrinsic apoptotic pathways [19].
In the result of Western blotting, we found that the band of cleaved Caspase-8 in TE-13 cells appeared earlier than the band of cleaved Caspase-9, suggesting that the extrinsic apoptosis pathway was activated first in TE-13 cells and promoted the activation of the intrinsic apoptosis pathway. Both Caspase-8 and Caspase-9 were activated in TE-13 cells while only intrinsic apoptotic pathways were activated in ECA109 cells, which may be the reason for the slower increase of Caspase-3 activity in ECA109 cells.
The response of apoptosis pathways to multiple physiological signals and cytotoxic factors is controlled by Bcl-2 family proteins [20,21]. Oligomeric Bax and Bak destroys the integrity of the mitochondrial outer membrane and releases Smac and cytochrome c to the cytoplasm [22] while Bcl-2 inhibits the mitochondrial localization of Bax and Bak and prevents the release of these pro-apoptotic proteins [23]. In this study, both ECA109 and TE-13 cells showed time-dependent downregulation of Bcl-2 protein levels and activation of intrinsic apoptosis pathways. At the same time, it was also observed that the expression level of Bcl-xl protein was downregulated in TE-13 cells, in which the activity of Caspase-9 was increased more rapidly than in ECA109 cells.
(20S) G-Rh2 has been shown to induce apoptosis in a variety of cancer cell lines by activating mitochondrial or death receptor-mediated apoptosis pathways [14]. Both in vitro and in vivo experiments have shown that (20S) G-Rh2 can inhibit growth and metastasis of tumor cells, so it is considered a promising natural compound for the treatment of cancer. In a previous study, it was demonstrated that (20S) G-Rh2 can activate the extrinsic apoptosis pathway in a p53-dependent manner [24]. However, in this research, both the transcription and expression level of Fas and DR5 in TE-13 were upregulated after being treated with (20S) G-Rh2 although TE-13 is a cell line with dysfunctional p53, suggesting that (20S) G-Rh2 can also trigger the extrinsic apoptosis pathway in a non-p53-dependent way. This result may be related to the fact that (20S) G-Rh2 has multiple intracellular targets and regulates the activity of several transcription factors. These specific mechanism remains need to be studied in future.

Materials
ECA109 and TE-13 cell lines were from the Affiliated Cancer Hospital and the Institute of Guangzhou Medical University. The (20S) G-Rh2 (≥97%) was purchased from Sigma Aldrich and dissolved in 75% ethanol at a concentration of 7.5 mg/mL and stored at −80 • C. The Cisplantin (≥98%) was purchased from Sigma Aldrich and dissolved in a phosphate buffer saline (PBS) at a concentration of 10 mg/mL and stored at −80 • C. Caspase substrates, Ac-DEVD-AFC, Ac-IETD-AFC, and Ac-LEHD-AFC were purchased from Calbiochem (La Jolla, CA, USA). The Mitochondria Isolation Kit was purchased from Pierce (Rockford, IL, USA). The mitochondrial membrane potential assay kit with JC-1 was purchased from Beyotime (Shanghai, China). Antibodies to Smac, Bax, Bak, PARP, Bcl-xL, cIAP-1, cIAP-2, and β-Actin were purchased from SantaCruz Biotechnology (Santa Cruz, CA, USA). Antibodies to Caspase-8, Caspase-9, cytochrome c, XIAP, and COXII were purchased from Cell Signaling Technology (Beverly, MA, USA).

Cell Culture
ECA109 and TE-13 cells were cultivated in Dulbecco's Modified Eagle's Medium (DMEM) with 10% newborn calf serum at 37 • C in a CO 2 incubator with 5% CO 2 . The culture medium contained 100 µg/mL of penicillin and 100 µg/mL of streptomycin .

Apoptosis Analysis
Cells were treated with 7.5 µg/mL (20S) G-Rh2 in fresh serum-free DMEM for 1, 2, and 4 h. Then cells were fixed in 70% ethanol at −20 • C for 15 min and stained with 1 µg/mL DAPI. Fluorescent cells were photographed under a fluorescence microscope (Olympus, Tokyo, Japan)

Western Blotting Assay
Esophageal cells were treated with (20S) G-Rh2. The treated cells were washed with ice-cold PBS twice and solubilized in a lysis buffer containing 20 mM Tris pH 7.5, 2 mM MgCl 2 , 1 mM DTT, 0.5% Triton X-100, 1 mM EGTA, 25 mM NaF, 1 mM Na 3 VO 4 , 50 mM glycerol phosphate, 2 mg/mL leupeptin, 2 mg/mL pepstatin A, 2 mg/mL antipain, and 1 mM PMSF. After incubating on ice for 1 h, the insoluble materials were removed by centrifugation at 12,000× g for 15 min. The equal amount of cell lysate was separated by SDS-PAGE and electrotransferred onto a fixed PVDF membrane. The membrane was blocked with 5% skim milk in PBST (PBS with 0.1% v/v Tween 20) for 1 h at RT and then incubated with antibodies at 4 • C overnight. Then, membranes were washed with PBST and incubated with a horseradish peroxidase-coupled, anti-mouse immunoglobulin G (IgG) or an anti-rabbit IgG secondary antibody (Pierce) for 1 h at RT and then detected with an electrogenerated chemiluminescence (ECL) revelation system (Tanon, Shanghai, China). The relative intensity of the Western blotting assay was calculated by using Image J and displayed under each band. The absolute gray value of the target strip was calculated and then divided by the absolute gray value of its corresponding internal reference (β-actin for proteins in the cytoplasm and whole cell lysate; COXII to obtain the relative intensity for proteins in the mitochondria).

Flow Cytometry and Annexin V Assay
1 × 10 6 exponentially growing esophageal cells were harvested and resuspended with a 400 µL binding buffer in an Annexin V Assay Kit. A total of 200 µL of the cells were incubated with a positive control buffer for 10 min at RT, washed with PBS, and resuspended with the 200 µL binding buffer again. Then, cells were mixed again and separated into 3 parts (130 µL per tube) and added to the 5 µL Annexin V-FITC, PI solution, or PBS and incubated in the dark for 10 min at RT as the control.
Esophageal cells were treated with 7.5 µg/mL (20S) G-Rh2 for 1 or 2 h and then harvested. Cells were washed with PBS and resuspended with a 1 mL binding buffer. A total of 100 µL of the cells were transferred into a new tube and incubated for 10 min in the dark with the 5 µL Annexin V-FITC and then incubated for 5 min with the 5 µL PI solution and added PBS up to 500 µL. The percentage of Annexin V(+) cells was determined by flow cytometry, which indicates the frequency of the total apoptotic cells (Becton Dickinson FACS Calibur Cytometer).

RT-PCR
The sequences of primers used in the RT-PCR assay are in Table S1. Cells were suspended by Trizol (Life Technologies Corporation, California, CA, USA) to exact RNA after harvest. Reverse transcription (RT) was performed using the StarScript II First-strand cDNA Synthesis Kit-II (GenStar, Beijing, China). The RT mixture contained 1µg template RNA, 1 µL oligo (dT) 18 , 10 µL reaction mix, 1µL StarScript II RT mix, and 8 µL H 2 O. The reaction mixture was incubated in a thermocycler programmed at 50 • C for 40 min and then heated at 85 • C for 5 min to denature the StarScript II RT mix.
4.9. RNA-Seq Analysis 5 × 10 6 TE-13 cells in a 6-well plate were harvested in 1 mL Trizol and were used for global transcriptome analysis by Annoroad Gene Technology (Beijing, China). Significant differentially expressed genes between the (20S) G-Rh2-treated and control groups were identified with the cutoff of p < 0.05 and |Log2FoldChange| > 2.

Statistical Analysis
Data were presented as a mean ± standard deviation with Microsoft Office 2013. The statistical significance was calculated with the Student's t-test. Differences were considered statistically significant as follows: * p < 0.05; ** p < 0.01; *** p < 0.001.

Supplementary Materials:
The following supporting information can be downloaded at https:// www.mdpi.com/article/10.3390/molecules27175602/s1. Figure S1: (20S) G-Rh2-induced caspasedependent apoptosis in esophageal cancer cells identified by cell viability assay; Table S1: primers used in this study; Table S2: list of upregulated TFs with the regulating Fas gene; Table S3: list of upregulated TFs with the regulating DR5 gene.