Wharton Jelly Stem Cells inhibits AGS Gastric Cancer Cells through Induction of Apoptosis and Modication of MAPK and NF ‐ κB Signaling Pathways

Background and aim: Gastric cancer) GC) is one of the most common cancer with high mortality worldwide. The human Wharton's jelly stem cells (hWJSCs) can inhibit several cancer cells through several molecular pathways. Therefore, the present study aimed to investigate anticancer effects of hWJSCs conditioned medium (hWJSC-CM) and cell-free lysate (hWJSC-CL) against of GC cell line AGS and underlying signaling pathways. Methods: In this study, we evaluated the effects of hWJSC-CM and hWJSC-CL on viability, proliferation, migration, invasion, apoptosis, and MAPK and NF ‐ κB signaling pathways in AGS cells. Moreover, mRNA expression of genes involved in apoptosis (BAX, BCL2, SMAC, and SURVIVIN), as well as expression of proteins involved in NF-κB and MAPK signaling pathways were evaluated. Results: The obtained results showed that the hWJSC-CM and hWJSC-CL decreased viability, migration, and invasion of GC cell line AGS in a concentration and time dependent manner. We observed that the hWJSC-CM and hWJSC-CL induced apoptosis pathway through regulation of apoptosis involved genes mRNA expression. In addition, the hWJSC-CM and hWJSC-CL suppressed NF-κB signaling pathways as well as promoted MAPK signaling pathways. Conclusions: In general, our study suggested that the hWJSC-CM and hWJSC-CL inhibits proliferation and viability of GC cell line AGS through induction of apoptosis, as well as modication of NF-κB and MAPK signaling pathways.


Introduction
Gastric cancer (GC) is a prevalent cancer and third cause of cancer death worldwide [1]. The prevalence of GC is increasing in the Asian countries and poor early diagnosis cause to lower survival [2]. Nowadays, chemotherapy followed by surgery are the common treatment methods for GC. However, development of drug-resistant in patients with GC decreased e cacy of chemotherapy agents [3]. Thus, use of novel anticancer agents with natural source are required for treatment and prolonged survival of GC patients [4].
Recently, stem cells therapy is an applicable and favorites approach in treatment of various human cancers. The human Wharton's jelly stem cells (hWJSCs) derived from human umbilical cord are an important source of mesenchymal stem cells (MSCs) with signi cant clinical bene ts, which was usually discarded after delivery [5]. The hWJSCs are multipotent with high proliferative potential, and have mesenchymal and embryonic stem cell characteristics [6]. Unlike to other MSCs, the hWJSCs do not cause tumor formation trough immune systems suppression [7]. Moreover, the hWJSCs can be harvested painless in a large population from umbilical cords. The stemness characteristics of hWJSCs maintain for many passages in culture [6,7]. Previous studies reported that the hWJSCs have an anti-proliferative effects against several human cancer cells [8,9]. Moreover, the evidence suggested that the hWJSCs inhibited proliferation of cancer cells through apoptosis induction [10]. However, underlying mechanisms of the hWJSCs anticancer effects have not been identi ed. Moreover, the effect of hWJSCs has not been evaluated on GC.
Due to the characteristics of MSCs and its anti-cancer effect as well as less side effects of cell therapy than other treatments such as surgery and chemotherapy, the present study investigated the anti-cancer effects of hWJSCs against GC cell line and underlying signaling pathways.

Cell culture
The human umbilical cords were received from healthy women at full-term delivery time, and the hWJSCs were derived according to our previous study [8]. In addition, the GC cell line AGS was purchased from Cell Bank of Pasteur Institute, Tehran, Iran. Both hWJSCs and AGS cells culture was performed using Roswell Park Memorial Institute (RPMI)-1640 medium containing 10% fetal bovine serum (FBS) and 1% penicillin (100 units/mL) and streptomycin (100 µg/mL). The cells incubation was performed at standard condition (37 o C with 95% humidity and 5% CO 2 ).

Preparation of hWJSC-CM and hWJSC-CL
The conditioned medium and cell lysates were prepared from cultured hWJSCs with 70% con uence, according to our previous study [8]. Briley, the culture medium was substituted with antibiotic and FBS free medium, and the hWJSCs conditioned medium (hWJSC-CM) was collected after 72 hours. Next, the cultured hWJSCs were washed once with calcium and magnesium free phosphate buffered saline (PBS (-) ), dissociated using trypsin-EDTA, and centrifuged. The obtained cells pellet mixed with the cell lysis buffer with protease inhibitor. Next, cells suspension was centrifuged and supernatant was collected as hWJSCs cell lysate (hWJSC-CL). The both hWJSC-CM and hWJSC-CL were ltersterilized, and the pH were determined, and stored at -20°C.

Cancer cells viability assay
The GC cell line AGS were seeded in a 96-well plates at density of 1.5×10 4 cells/well in 200 μl culture medium, and then incubated for 24 hours. Next, the cancer cells were treated with various concentrations of hWJSC-CM (38, 40, 42, 44, and 46%) and hWJSC-CL (11,12,13,14, and 15%) for 24-72 hours. The treated cancer cells were washed once with PBS, and then 200 µL MTT solution (5 mg/mL MTT in culture medium) was added to the each well and incubated for 4 hours at standard condition. Next, 50 µL dimethyl sulfoxide (DMSO) was added and incubated further for 30 minutes. The optical densities (OD) at 570 nm wavelength were detected using a plate reader for each well. The relative cancer cell viability was calculated as following: cell viability=(OD 570 of treated samples / OD 570 of untreated samples)×100.

Wound-healing migration assay
The cancer cells were seeded in a 6-well plates at density of 1.5×10 5 cells/well in 2 ml complete culture medium until cells reached 85% con uence. Next, a wound was produced by scraping with a sterile pipette tip in middle of each well, and detached cells were washed with PBS. The cancer cells were treated with hWJSC-CM (44%) and hWJSC-CL (14%), and incubated in standard condition for 48 hours. Finally, the migrated cancer cells were counted by inverted phase-contrast microscope at 0-20 hours.

Transwell invasion assay
The cancer cells were seeded in upper chamber of a transwell chamber at density of 2×10 4 cells/well in FBS serum-free culture medium and treated with hWJSC-CM (44%) and hWJSC-CL (14%). Moreover, complete culture medium supplemented with 10% FBS was added to lower chamber. After incubation for 24 hours at standard condition, the migrated cancer cells to lower chamber were xed in 4% paraformaldehyde for 15 minutes and stained with 0.1% crystal violet for 10 minutes. Finally, cancer cells in lower chamber were counted in 5 random elds by inverted phase-contrast microscope.

Cancer cells apoptosis assay
The cancer cells were seeded in a 6-well plates at density of 1.5×10 5 cells/well in 2 ml complete culture medium for 24 hours. The cancer cells were treated with hWJSC-CM (44%) and hWJSC-CL (14%), and incubated in standard condition for 48 hours. Next, the cancer cells were dissociated using trypsin-EDTA and resuspended in binding buffer (400 μL). Next, the FITC-conjugated Annexin-V (5 μl) and propidium iodide (PI) (10 μl) were added and incubated at room temperature in dark for 5 minutes. Finally, apoptosis of cancer cells was determined by ow cytometry instrument (Becton Dickinson Bioscience).

Gene expression assay by Real-Time PCR
The cancer cells were seeded in a 6-well plates at density of 1.5×10 5 cells/well in 2 ml complete culture medium for 24 hours. The cancer cells were treated with hWJSC-CM (44%) and hWJSC-CL (14%), and incubated in standard condition for 48 hours. Next, the cancer cells were dissociated using trypsin-EDTA and resuspended in TRIzol reagent for extraction of total RNA. The complementary DNA (cDNA) was synthesized from 2 µg total RNA using reverse transcription reagents. The quantitative Real-Time polymerase chain reaction (qRT-PCR) was performed with SYBR green master mix and speci c primers of BAX, BCL2, SMAC, and SURVIVIN genes ( Table 1). The ACTIN (β-actin) gene was used as endogenous control. The calculations were performed by 2 -ΔΔCt (Livak) formula.

Proteins expression assay by western blotting
The cancer cells were seeded in a 6-well plates at density of 1.5×10 5 cells/well in 2 ml complete culture medium for 24 hours. The cancer cells were treated with hWJSC-CM (44%) and hWJSC-CL (14%), and incubated in standard condition for 48 hours. Next, the cancer cells were dissociated using trypsin-EDTA, and lysed in ice-cold RIPA lysis buffer containing protease inhibitor. The cell lysates centrifuged and supernatant collected. The protein levels of p-JNK, JNK, p-p38, p38, p-p65, and p65 were evaluated using western blot analysis. The protein content of cells (~100 μg) were separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by transferring to polyvinylidene di uoride (PVDF) membrane. The membrane was blocked using non-fat dry milk (5%) and then incubated with primary antibodies at 4°C for 24 hours. The horseradish peroxidase (HRP) conjugated antibody was used as secondary antibody for 30 minutes at room temperature. The protein detection was performed using enhanced chemiluminescent (ECL) detection system.

Statistical analysis
The all performed experiments were repeated for three times, and obtained data were presented as mean ± standard deviation (SD). The Student's t-test, Tukey (post-hoc), and one-way analysis of variance (ANOVA) were used to statistical analysis using the Graph Pad Prism software. The p-value<0.05 was considered as signi cant.

Cancer cells migration and invasion
In the wound-healing migration assay, the GC cells showed signi cantly decrease in migration following treatment with the hWJSC-CM (44%) and hWJSC-CL (14%) as compared with untreated cancer cells ( Figure 2). In the transwell invasion assay, the hWJSC-CM (44%) and hWJSC-CL (14%) signi cantly inhibited invasion of GC cells as compared with untreated cancer cells (Figure 3). However, treatment of cancer cells with hWJSC-CL (14%) showed statistically signi cant decreases in cancer cell migration and invasion compared to hWJSC-CM (44%).

Cancer cells apoptosis
The apoptosis quanti cation demonstrated that the early and late apoptosis rate of GC cell line signi cantly increased following treatment with hWJSC-CM (44%) and hWJSC-CL (14%). However, treatment of cancer cells with hWJSC-CL (14%) showed statistically signi cant increase in apoptosis rate of cancer cells compared to hWJSC-CM (44%) (Figure 4). The evaluation of apoptosis-related genes mRNA expression demonstrated that the hWJSC-CM (44%) and hWJSC-CL (14%) signi cantly increased mRNA expression of BAX and SMAC genes as well as signi cantly decreased mRNA expression of BCL2 and SURVIVIN genes. We observed that modi cation of apoptosis-related genes mRNA expression in GC cells by hWJSC-CL (14%) was signi cantly more than hWJSC-CM (44%) ( Figure 5).

MAPK and NF-κB signaling pathways
To investigate whether hWJSC-CM (44%) and hWJSC-CL (14%) change viability, migration, invasion, and apoptosis of GC cell line AGS cells, we investigated the proteins levels involved in MAPK and NF-κB signaling pathways. In the NF-κB signaling pathway, the obtained results demonstrated that the phosphoand total-p65 proteins expression were decreased signi cantly in the hWJSC-CM and hWJSC-CL treated cancer cells (Figure 6). In the MAPK signaling pathway, the hWJSC-CM and hWJSC-CL were increased the active phospho-and total-p38 and phospho-and total-JNK levels in treated cancer cells. These ndings suggested that the NF-κB and MAPK signaling pathways play an important role in anticancer activity of hWJSC-CM and hWJSC-CL in GC cancer cells. However, we observed that modi cation of MAPK and NF-κB signaling pathways in GC cells by hWJSC-CL (14%) was signi cantly more than hWJSC-CM (44%) (Figure 7).

Discussion
The GC is the third cause of cancer mortality worldwide with a poor prognosis and relatively short survival [11]. Despite the great progress in therapeutic methods, prognosis of GC patients remains poor, due to frequent metastasis, drug resistance, and relapse of GC [12]. Therefore, further studies on critical mechanisms in GC progress are imperative. The MSCs including the umbilical cord hWJSCs have been recently used as anticancer agents several human malignancies [8,13]. The hWJSCs harvest painlessly from human umbilical cord which are usually discarded, highly proliferative, widely multipotent, and have long telomeres [14]. The hWJSCs are potentially quite applicable to human patients without a complete genetic match since they are unlikely to induce an acute in ammatory response [15].
The uncontrolled proliferation and high viability of different human cells as well as metastasis to adjacent tissues are the most important cause of cancer progression and mortality [16,17]. In the present study, we evaluated viability, proliferation, migration, and invasion of GC cell line AGS treated with hWJSC-CM and hWJSC-CL, as compared untreated controls. The obtained results demonstrated that the hWJSC signi cantly decreased the GC cells viability, proliferation, migration, and invasion in a time and concentration dependent manner. In this regards, the previous studies reported that the hWJSC inhibits viability, proliferation, migration, and invasion of various cancer cells [7,14]. In a study by Kalamegam et al. reported that the hWJSC-CM and hWJSC-CL suppress cellular growth, proliferation, viability, and migration of ovarian cancer cells through several mechanisms [13]. However, the underlying mechanisms of cancer cells death in the presence of umbilical cord hWJSC remains unknown. Therefore, identi cation of various underlying anticancer mechanisms of hWJSCs against cancer cells is one of the main objective in several previous studies [8,13].
The inhibition of apoptosis is one of the most important pathological processes in progress of various cancers. Thus, induction of apoptosis is a crucial approach for increase cancer cells death following treatment with various anticancer agents [18,19]. In the present study, the effects of hWJSC-CM and hWJSC-CL was evaluated on apoptosis of GC cell line AGS using Annexin V-FITC/PI. The obtained results demonstrated that the apoptosis rates of GC cells signi cantly increased following treatment with hWJSC-CM and hWJSC-CL. In this regards, the previous studies reported that hWJSC-CM and hWJSC-CL cause to induction of apoptosis in several cancer cells [7,14]. In a study by Elias et al. reported that the hWJSCs increased apoptosis rate in the squamous cell carcinoma cell line HEP-2 [20].
The apoptosis process in human is regulated trough interaction of several genes family members [21]. The high expression of BAX and SMAC genes as well as low expression of BCL2 and SURVIVIN genes causes activation of apoptosis onset [22,23]. In this regards, our study demonstrated that the hWJSC-CM and hWJSC-CL signi cantly upregulated mRNA expression of BAX and SMAC genes (as pro-apoptotic genes) as well as downregulated mRNA expression BCL2 and SURVIVIN genes (as anti-apoptotic protein) in GC cells. In a study by Ghorbani and Maleki reported that the hWJSCs induces apoptosis through upregulation and down-regulation of BAX and BCL2 genes expression, respectively [24]. In another study by Elias et al. reported that the hWJSCs up-regulated and down-regulated mRNA expression of P53 and BCL2 genes that leads to induction of apoptosis [20]. These evidences suggested that the hWJSCs induces apoptosis pathway in various cancer cells through modi cation and regulation of apoptosisrelated genes expression.
The MAPK signaling pathway play an important role in viability, proliferation, metastasis, and invasion of cancer cells [25]. The inhibition of MAPK signaling pathway decrease e ciency of various anticancer agents and therapeutic methods [26]. The present study demonstrated that the MAPK signaling pathway was activated by hWJSC-CM and hWJSC-CL in GC cells. On the other hands, the NF-κB signaling pathway play a crucial role in cancer cells proliferation, viability, and carcinogenesis [27]. The inhibition of the NF-κB signaling pathway induces apoptosis and inhibits invasion and migration in various cancer cells [28]. In the present study, we showed that the hWJSC-CM and hWJSC-CL signi cantly inhibits the NF-κB signaling pathway in the GC cells. These results suggested that the MAPK and NF-κB signaling pathway may be involved in inhibitory effects of hWJSC-CM and hWJSC-CL against proliferation, viability, migration, and invasion of GC cells.

Conclusions
In general, our study presents a novel therapeutic approach to treatment of patients with GC. Our study showed that the hWJSC-CM and hWJSC-CL inhibits the viability, proliferation, migration, and invasion of GC cell line AGS through several molecular mechanisms. The hWJSC-CM and hWJSC-CL induces apoptosis in GC cells through modi cation of apoptosis-related genes expression. Moreover, the NF-κB, MAPK signaling pathways may be involved in inhibitory effects of hWJSC-CM and hWJSC-CL in GC cells.
Therefore, the hWJSC can be used in future to control and even treatment of patients with GC. However, further studies are required to identi cation the exact effects of hWJSCs on physiopathology of GC.
Declarations Table   Table 1. The sequences and characteristics of used primers for quanti cation of genes mRNA expression.  The primary cultured hWJSCs with broblasts characteristic in early passages (A) and long broblasts characteristic in later passages (B). The viability of GC cell line AGS treated with hWJSC-CM (C) and hWJSC-CL (D) at 24, 48, and 72 hours. The anticancer activity of hWJSC-CL was signi cantly more than hWJSC-CM.

Figure 2
The GC cell line AGS migration evaluated with wound healing migration assay following treatment with hWJSC-CM (44%) and hWJSC-CL (14%) for 10 to 20 hours. The inhibition of migration in treated cancer cells with hWJSC-CL was signi cantly more than hWJSC-CM.

Figure 3
The GC cell line AGS invasion evaluated with transwell invasion assay following treatment with hWJSC-CM (44%) and hWJSC-CL (14%) for 48 hours. The inhibition of invasion in treated cancer cells with hWJSC-CL was signi cantly more than hWJSC-CM.

Figure 4
The GC cell line AGS apoptosis evaluated with annexin V-FITC/PI assay following treatment with hWJSC-CM (44%) and hWJSC-CL (14%) for 48 hours. The induction of apoptosis in the cancer cells treated with hWJSC-CL was signi cantly more as compared with hWJSC-CM.

Figure 5
The mRNA expression analysis of apoptosis related BAX, SMAC, SURVIVIN, and BCL-2 genes by qRT-PCR following treatment with hWJSC-CM (44%) and hWJSC-CL (14%) for 48 hours. The modi cation of apoptosis related genes mRNA expression in the cancer cells treated with hWJSC-CL was signi cantly more as compared with hWJSC-CM.