α-Tomatine Inhibits Proliferation, Metastasis, Drug Resistance and Immune Inltration Through the PI3K-AKT-MAPK Axis in Gastric Cancer

Background: α-Tomatine, a naturally existing steroidal glycoalkaloid in immature green tomatoes, had anticarcinogenic performances in various types of cancer cells. Within this study, we aimed to investigate the ecacy and potential molecular mechanism of α-tomatine in gastric cancer (GC) and the association with immune inltration and prognosis. Methods: We used human GC cells MGC803, BGC823, SGC7901 and SGC7901/DDP to evaluate the cell functions of α-tomatine, and the molecular mechanisms of α-tomatine were investigated by qRT-PCR and western blotting analysis. Timer database was used to analyzed the correlation of targets repressed by α-tomatine, immune inltration and prognosis. Results: Results showed that α-tomatine strongly inhibited PI3K-AKT and MAPK signaling pathways, thereby repressing the proliferation and metastasis of GC cells with different differentiations and cisplatin resistance. The inhibitory effect of α-tomatine on TWIST, Slug and PARP in SGC7901/DDP cells also suggested α-tomatine provided a feasible approach for confronting metastasis of clinical cisplatin resistant cases. Immunologically, α-tomatine could partially modulate the process and consequences of immune inltration based on the correlation between 65 genes corresponding to interacting proteins and immune cell inltration. α-Tomatine could improve prognosis of GC patients by inhibiting AKT3, VIM, FN1 and SNAI2, and at least partially counteract immune dysfunction triggered by macrophage inltration. Conclusion: Strategies for treating GC should not be restricted to single, repetitive drug use, they should be predisposed to emerging, multi-target drugs. GC was highly susceptible to α-tomatine suggesting that α-tomatine could be used as an emerging and promising approach to confront GC.

Members of the mitogen-activated protein kinase (MAPK) superfamily are related to elevated morphogenesis, ampli cation, apoptosis, movement and invasion capacity of cells [5][6][7]. The MAPK signaling cascades are made up of three main MAP kinases including extracellular signal-regulated kinases 1 and 2 (ERK1/2), c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK), and p38 MAPK [8][9][10][11]. Multiple phospho-MAP kinase members, modulated by different extramural signals, activate downstream targets so as to take effects in cellular signal transduction. Furthermore, the molecular mechanism of MDR is complex, which is thought to be involved in drug transport, drug pumping, intracellular drug metabolism, DNA damage, DNA repair and apoptosis [12]. Simultaneously, tumorigenesis is not effectively suppressed due to immune dysfunction. "Non-self" tumor cells are speci cally eliminated by the immune system characteristic for a monitor through cellular immune mechanism to maintain the stability of internal environment. However, tumor cells rapidly divide and proliferate in the body thereby accelerating tumor progression when they escape the immune system by various factors. Chemokines and antigens expressed by tumor cells induce mass in ltration of immune cells, whereas tumor cells induce immune escape by down-regulating or blocking tumor antigens and their own low immunogenic [13]. In ltration of immune cells may stimulate activation of tumor-related pathways via secrete cytokines, further exacerbating tumor progression.
α-Tomatine is a sort of glycoalkaloids, naturally extracted in unripe green tomatoes (500 mg/kg of fresh fruit) [14]. As the tomato matures, the ingredient is partially degraded until the ripening level of the red tomato is about 5 mg/kg of fresh fruit [11,15]. α-Tomatine is composed of an aglycone moiety (tomophylline) and a tetrasaccharide moiety (β-glycolipid), which contains two D-glucose molecules, one D-galactose and one D-xylose (Fig. 1A). Previous studies showed that α-tomatine resulted in the negative regulation of proliferation and apoptosis, thereby repressing the ampli cation of human colorectum and liver-derived cancer cells [16,17]. Moreover, α-tomatine modulated T cell-mediated inhibition of mouse lymphoid tumor EG7-Ova and took effects as an anti-in ammatory drug by confronting nuclear factor kappa B (NF-κB) and JNK signaling in macrophages of mouse [18][19][20]. To evaluate the molecular mechanism of α-tomatine against GC, we used human GC cells MGC803, BGC823, SGC7901 and SGC7901/DDP to elucidate the speci c role of α-tomatine in cell function and molecular mechanism, so as to reveal its inhibitory effect on proliferation, metastasis, drug resistance and immune in ltration of GC, and to provide the possibility that α-tomatine could be a potential drug for the treatment of GC.

Apoptosis analysis by ow cytometry
Cell apoptosis were performed by Annexin V-APC/PI staining method (Becton-Dickinson, USA) following the manufacturer's protocol. Cells were suspended in 6-well plates at 1×105 cells/well and incubated with Annexin V-APC and propidium iodide (PI) at 4°C for 30 min. The cell suspensions were then analyzed by Flow cytometry, and repeated all experiment three times.

Wound-healing assay
For cell motility assay, gastric cells (2×105 cells/ml) were seeded in 6-well plates and grown to a density of about 80% to 90%. Before changing the medium, the cells in the well plate were scraped with a yellow sterile micropipette tip to form a constant-width, neat-edge wound area. The scraped cells were then washed three times with PBS, and gastric cells were cultured to various concentrations of α-tomatine (0, 1, 1.5, and 2 μM).
The movements of cells were observed and photographed at 0, 24 and 48 h in serum-free medium. We compared the wound area of α-tomatine-treated pores with those of the control group to quantify migrated cells. 3 positions were randomly selected for each concentration, and the remaining area of the wound was marked with ImageJ at different times.
Subsequently, membranes were washed with TBST three times for 10 min and incubated with appropriate secondary antibody (horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit IgG, cycles of 95℃ for 2 seconds; 60℃ for 20 seconds and 70℃ for 10 seconds. All mRNA expressions were calculated by using the 2−ΔΔCt method, and repeated all experiment three times. The values were presented as mean ± SD.

PPI networks construction
The signi cant associations of protein-protein interactions (PPIs) are provided in STRING database (http://string-db.org/) [21]. The visual explorations of interaction networks were conducted by Cytoscape [22]. In our works, we used multiple protein model in STRING database to build the PPI networks of PI3K-AKT and Ras-Raf-MAPKs signal pathways across 3 functional states in GC. Followed of which, we used Cytoscape to visualize the PPI networks [23]. The medium con dence was set at 0.400.

TIMER database analysis
The analysis of the immune in ltrates across different types of tumor are conducted by The TIMER database (https://cistrome.shinyapps.io/timer/) [24]. We used Diff Exp module to explore differential expressions of 8 genes (PIK3R1, AKT1, AKT2, AKT3, MEK1, MEK2, ERK1 and ERK2) between tumor and its adjacent normal tissue. Gene module to explore the relationship between 8 genes mentioned above and their associated genes and immune in ltration levels of 6 immune cells (B cell, CD4+T cell, CD8+T cell, macrophage, neutrophil, dendritic cell). Survival module to explore the clinical relevance of immune cells (Cox regression analysis and Kaplan-Meier analysis). In addition, we analyzed the association between the expression levels of speci c genes and the in ltration levels of tumor purity and 6 immune cells.
Statistical Analysis SPSS version 13.0 (SPSS, Chicago, USA) was used for all statistical analyses. Data were expressed as the mean ± standard error of mean (S.E.M), and statistical signi cance was carried out using t-tests and one-way ANOVA. Signi cant differences were established at P < 0.05. All analyses were conducted with GraphPad Prism 5 software (GraphPad Software, Inc., San Diego, CA).

The proliferation ability of GC cells was inhibited by α-tomatine
In contrast to normal cells, tumor cells manifest a variety of pathological states, especially a signi cant increase in ampli cation capacity. Cancer is an inevitable consequence when the gene mutation of cells in vivo cannot be effectively inhibited, leading to uncontrolled growth [25]. To investigate the effect of αtomatine on the proliferation of GC cells, the proliferation activity of MGC803, BGC823, SGC7901 and SGC7901/DDP were analyzed by CCK8 kit. The results showed that the proliferation of the GC cells at the different levels of differentiation was signi cantly inhibited by α-tomatine, compared with the control group. The proliferation of these 4 GC cells experienced a signi cant decrease by the intervention concentration of α-tomatine at 1.5 μΜ (Fig. 1B), suggesting that α-tomatine exerted a strong inhibitory effect on GC cells at this concentration. Moreover, the IC50 values of 4 GC cells that we calculated were 1.412 ± 0.108, 1.055 ± 0.047, 1.147 ± 0.142, 1.250 ± 0.156, respectively (Fig. 1C). Intriguingly, the proliferation of 7901/DDP cell was also inhibited by α-tomatine. These results clearly demonstrated that α-tomatine could effectively reduce the ampli cation ability of GC cells with varying degrees of differentiation or resistance to cisplatin.

The apoptosis of GC cells was facilitated by α-tomatine
Apoptosis is a gene-oriented programmed death, which is to effectively eliminate dysfunctional cells [26]. Inhibition of tumor cells from auto-apoptosis often makes them predisposed to neoplastic evolution occurrence so as to induce tumorigenesis and drug resistance. As for cell proliferation, α-tomatine also could affect GC cell apoptosis. Apoptosis of GC cells were measured by ow cytometry to analyze the effect of α-tomatine on apoptosis. We tested the apoptosis of MGC803, BGC823, SGC7901 and SGC7901/DDP treated with 1 μΜ of α-tomatine 24 hours. The results showed that the percentage of early and late withering of the 4 GC cells increased signi cantly after treatment with α-tomatine compared with corresponding control group, suggesting that α-tomatine could signi cantly increase the apoptosis rate of GC cells. The apoptotic rates of MGC803 and SGC7901 cells treated without or with α-tomatine were 6.07% ± 1.63% vs. 22.93% ± 2.75% and 7.53% ± 1.94% vs. 29.70% ± 3.32% ( Fig. 2A and 2B). Similarly, the apoptosis of 7901/DDP cell was also increased by α-tomatine. The above results demonstrated that αtomatine could mediate apoptosis of different degrees of differentiation and cisplatin-resistant GC cells.
α-Tomatine inhibited the migration activity of GC cells Migration, with respect to metastasis, is another important feature of tumor cells [26]. Metastasis is often associated with the progression of gastric cancer. Whether metastasis has become one of the main factors affecting prognosis and survival [27]. We tested the healing area of the scratches of 4 GC cells treated with different concentrations of α-tomatine (0, 0.5, 1 and 2 μΜ) in a serum-free medium and photographed and recorded at 0h, 24h and 48h. The result showed that α-tomatine could effectively increase the healing area of the wound, and the area increases with up-regulation of the concentration of α-tomatine, compared with those of the non-α-tomatine-treated group. (Fig. 3A-D). The effect of αtomatine intervention concentration of 2 μΜ on the migration ability of the 4 GC cells was more than 40%. On the premise that the own proliferation of cells was ruled out as much as possible, these results exhibited that α-tomatine could effectively inhibit the athletic ability of cells thereby inhibiting metastasis of different degrees of differentiation and cisplatin-resistant GC cells.
Landscape of PIK3R1, AKT1, AKT2, AKT3, MEK1, MEK2, ERK1 and ERK2 expression levels in distinct cancers Available evidences manifested that over activation of PI3K-AKT and MAPKs pathways is the crucial mediator of tumor proliferation and metastasis. To investigate features of above pathways in distinct cancers, cognition of the difference of expression levels of core genes is necessary. We analyzed the expression levels of 8 genes including PIK3R1, AKT1, AKT2, AKT3, MEK1 (MAP2K1), MEK2 ((MAP2K2), ERK1 (MAPK3) and ERK2 (MAPK1) in distinct human cancers. Data from the TIMER database suggested that the expression levels of AKT1, MEK1 and ERK2 were signi cantly up-regulated in GC comparing with normal tissue (Fig. 4), as well as in BRCA (bladder urothelial carcinoma), CHOL (cholangiocarcinoma), HNSC (head and neck cancer) comparing with normal tissue. However, the difference of the other genes was not statistically signi cant in GC (data was not shown).
α-Tomatine suppresses the PI3K-AKT signaling pathway and MAPK signal cascade pathway in GC cells The above experiments proved that α-tomatine could inhibit the proliferation and metastasis of GC cells, and the inhibition was positively correlated with higher concentrations. Furthermore, cancer progression is often accompanied by abnormal expression of oncogenes and corresponding proteins. To further clarify the underlying mechanism of α-tomatine, we investigated the effect of α-tomatine on the constitutive activation status of AKT and a major mammalian MAPKs (ERK1/2). Western blot was used to detect the AKT, phospho-AKT, MEK1/2, phospho-MEK1/2, ERK1/2 and phospho-ERK1/2 protein levels in BGC823, SGC7901 and SGC7901/DDP cells treated with α-tomatine (0, 0.5, 1 and 2 μΜ) for 24h. The result demonstrated that intervention of α-tomatine suppressed the AKT, phospho-AKT, MEK1/2, phospho-MEK1/2, ERK1/2 and phospho-ERK1/2 expression levels in a dose-dependent manner (Fig. 5). We further investigated the expression of AKT, ERK1 and ERK2 at mRNA levels. Similarly, the results of RT-PCR indicated that α-tomatine repressed the mRNA expression of AKT, ERK1 and ERK2 in a dose-dependent manner ( Fig. 6A and 6B). These results indicated that α-tomatine could down-regulate the PI3K-AKT signaling pathway and MAPK signal cascade pathway at transcription and protein levels, partially explaining the mechanism by which α-tomatine inhibited the proliferation and metastasis of gastric cancer.
α-Tomatine inhibits TWIST, Slug and PARP mRNA expression in 7901/DDP cells, which are crucial during epithelial-mesenchymal transition Aberrant epithelial-mesenchymal transition (EMT) activation is considered a key step in the promotion of cancer metastasis, as it increases cancer cell motility and invasiveness [28]. EMT is unmasked a crucial approach in the progression and metastasis of tumor [28]. RT-PCR assay was used to investigate the repressed effect of α-tomatine on the transcription levels of those regulators associated with EMT, including TWIST, Slug, PARP, Vimentin, Caspase-3 and N-cadherin [29][30][31][32][33][34]. The results of RT-PCR showed that α-tomatine down-regulated the mRNA expression of TWIST, Slug and PARP in SGC7901/DDP, whereas no signi cant difference was uncovered in the expression of Vimentin, Caspase-3 and Ncadherin, which was not statistically signi cant (data was not shown). Under the effect of the highest concentration (2 μΜ) of tomatine, the expression levels of these three genes decreased by 73%, 62%, and 37%, respectively ( Fig. 7A and 7B). These results illustrated that α-tomatine down-regulated the mRNA expressions of TWIST, Slug and PARP at the transcription level and acted as a crucial role during EMT so as to remedy cisplatin-resistant gastric cancer.
Interaction between PI3K-AKT and MAPKs pathways and proteins related to tumorigenesis, metastasis, metabolism To further clarify and predict the mechanisms associated with PI3K-AKT and MAPK pathways, we selected proteins with different subtypes on these pathways and proteins evidently related to three molecular biological functions involving tumorigenesis, metastasis and metabolism, to construct proteinprotein interaction (PPI) networks. Results showed that signi cantly interrelations were cognized to exist between the majority of the above 65 proteins (Supplementary table 1), typically illustrated by AKT1-FOXO3, AKT3-GSK3B and MAPK3-JUN (Fig. 8A). Considering the interaction between pathway-associated proteins and function-associated proteins, it is reasonable to presume that α-tomatine inhibits the activation of PI3K-AKT and MAPK pathways and proteins interacting with the pathways. The effects of αtomatine are not only restricted to anti-proliferation and anti-metastasis by repressing PI3K-AKT and MAPK pathways, but also modulates proteins involved in tumorigenesis, metastasis, metabolism. Certainly, nor can we exclude that this anti-proliferative and anti-metastatic effect is essentially the effect of these proteins when they act as downstream targets of PI3K-AKT and MAPK pathways.

Correlation between 65 interacting proteins and tumor in ltrating immune cells
Tumor progression is frequently accompanied by immune dysfunction. To investigate whether PI3K-AKT and MAPK pathways and their interacting proteins are associated with immune dysfunction in GC, we analyzed the correlation between genes corresponding to 65 interacting proteins and the in ltration levels of 6 immune cells including B cell, CD4+T cell, CD8+T cell, macrophage, neutrophil and dendritic cell based on TIMER database ( Fig. 8B and supplementary table 2). For each type of immune cell, we selected 6 genes characteristic for the strongest correlation (positive or negative correlation) and statistical difference, and provided the relationship between their expression levels and in ltration level of immune cell in GC (Fig. 8C). Fig. 8B and 8C showed that the expression levels of genes were positively associated with in ltration levels of immune cells, excluding CDK4, CCNA2 and TWIST1 in B cell, pointing to a synergy between these genes and immune cells in GC. Inhibition of those interacting proteins by αtomatine could thus affect the process and consequences of immune in ltration.
In order to investigate the underlying prognosis of immune cells, the Cox regression model was used to analyze 6 immune cells. Result showed that higher in ltration level of macrophage was a risk factor for GC, whereas no statistical differences were found in the other immune cells. We performed the Kaplan-Meier analysis of macrophage, suggesting that higher in ltration level of macrophage was correlated with poorer survival outcomes in GC (Fig. 8D and supplementary table 3). As a result of 8B and 8C, 6 genes strongly associated with macrophages were AKT3, VIM, FN1, MAPK10, SNAI2 and PIK3CG.
Reference indices for further Kaplan-Meier analysis were determined by expression levels of these genes and in ltration level of macrophage simultaneously. Due to their positive correlations, two curves with the same predisposition of expression level and in ltration level (high gene expression + high macrophage and low gene expression + low macrophage) were more comparable. Curves demonstrated that the group of high levels often predicted poor prognosis (Fig. 8D). To evaluate the outcome signi cance of these genes to analysis whether high expression levels of genes predicted poor prognosis, similar to high in ltration level of macrophage. The Cox regression model is used to prove that higher expression levels of genes were risk factors for GC, including AKT3, VIM, FN1, MAPK10 and SNAI2, whereas no statistical differences were found in PIK3CG. Kaplan-Meier analysis indicated that higher expression levels of genes were correlated with poor survival outcomes in GC, including AKT3, VIM, FN1 and SNAI2, whereas no statistical differences were found in PIK3CG and MAPK10 (Fig. 8D and supplementary table 3). Considering that macrophages mediated gene expression in tumor cells by secreting interleukin-6 and other cytokines, α-tomatine could improve prognosis of GC patients by inhibiting AKT3, VIM, FN1 and SNAI2, and at least partially counteract immune dysfunction triggered by macrophage in ltration.

Discussion
GC is one of the most common malignancies. Its tendency to recurrence and metastasis after surgery predicts a generally poor prognosis. To provide a potential treatment for GC, we investigated the mechanism of α-tomatine on human MGC803, BGC823, SGC7901 and SGC7901/DDP cells in vitro proliferation, apoptosis and migration. Result showed that α-tomatine decreased the levels of phosphorylation of AKT, phosphorylated MEK1/2 and phosphorylated ERK1/2 in a dose-dependent manner, suggesting that PI3K-AKT and MAPKs signaling pathways might be the crucial for α-tomatine to exert effects of anti-proliferation and anti-metastasis. During the development and metastasis of cancer, the vast majority of tumor cells move and are predisposed to attack organs and tissues where they occur, resulting in metastasis [35,36]. Combining with our conclusions, the migration capacity of GC cells is controlled at least in part by the PI3K-AKT and MAPKs signaling pathways. In addition, α-tomatine dosedependently inhibited the expression of TWIST, Slug and PARP in SGC7901/DDP. It was shown that αtomatine partially inhibit the EMT pathway of GC resistant cells, leading to resist metastasis.
The inhibitory effects of PI3K-AKT and MAPKs pathways widely investigated should not only be summarized as proliferation and metastasis. PPI networks indicated that they might induce tumorigenesis, metastasis, and metabolism through other interacting proteins with high combined score. These proteins may be directly activated or inhibited by pathways or activate or inhibit pathways, or indirectly regulated by other signal transduction pathways. Systematic and comprehensive construction of a clear mechanism relationship is conducive to improve and enrich the treatment of GC. Immune dysfunction acts as a "promoter" in the development of tumors. When tumor cells secrete a variety of abnormal chemokines to recruit immune cells, they reduce or even eliminate antigens so that the immune response fails to respond effectively. An accumulation of immune cells results in in ltration. Previous studies have shown that IL-17 induces AKT-dependent activation of IL-6-JAK2-STAT3 in hepatocellular carcinoma [37], indicating that immune cells regulate the expression levels of genes in tumor cells by secreting such IL-6 or other cytokines. Our results showing the correlation between the expression levels of genes and the in ltration levels of immune cells also provide an explanation for this possibility. Further prognostic analysis demonstrated that the relationship between the in ltration level of macrophage and prognosis and the relationship between the expression levels of AKT3, VIM, FN1, SNAI2 and prognosis was frequently consistent. Therefore, a linear regulatory relationship may be existed between macrophages and these genes. α-Tomatine could at least partially antagonize the biological changes of tumor cells induced by the in ltration of macrophages, resulting in improving prognosis. Furthermore, the inhibitory effects of α-tomatine on gene might be regulatory factors on highly correlated in ltration levels of immune cells.

Conclusions
In summary, we proposed a schematic explanation of potential mechanisms for the repressive effect of α-tomatine on GC cells (Fig. 9). Above results support that the anti-proliferative and anti-metastatic effects of α-tomatine on GC cells might be accomplished via inactivating PI3K-AKT and MAPKs pathways. In addition, the anti-proliferative and anti-metastatic effects of α-tomatine on cisplatin resistant GC cells provide an underlying manner to clinically reverse drug resistance. The effects of proteins related to PI3K-AKT and MAPKs pathways and the correlation between these proteins, immune in ltration and prognosis also suggest that the inhibitory effects of α-tomatine are not restricted to proliferation and metastasis. The regulatory mechanism of α-tomatine with metabolic process and immune function in GC needs to be further explored. α-Tomatine have started shedding light on promising therapeutic strategies for GC with high metastasis and drug resistance, and could be further experimented through in vivo model to de nitize if it is effective for confronting the proliferation, metastasis, drug resistance and immune in ltration of GC.

Consent for publication
Not applicable.

Availability of data and materials
The data used to support the ndings of this study are included within the article and the supplementary information les.

Competing interests
No con ict of interest exits in the submission of this manuscript, and manuscript is approved by all authors for publication.

Funding
This work was supported by China Postdoctoral Science Foundation (No. 2019M653812XB). The National Science Foundation (81973533). Guangxi University High-level Innovation Team and the Project of Outstanding Scholars Program (2019AC03004) and Guangxi Science and Technology Project (AD19245197). The study funders contributed to the purchase of experimental items and had no role in the design, data acquisition, analyses, or data interpretation of this project.
Authors' contributions ZD: conception, design and performance of the experiment, and wrote the manuscript. MSS and YH: analysis of data and contributed to the conception of the study. YLH, TWZ and MXW: contributed signi cantly to analysis and manuscript preparation. All authors have read and approved the manuscript.    The differential expression between the cancerous tissues and adjacent normal tissues for AKT1, MAP2K1 and MAPK1 (*P <0.05; **P <0.01; ***P <0.001).
GAPDH was used as an internal control.    We used ChemDraw software to draw the mechanisms by which α-tomatine suppresses proliferation, metastasis and drug resistance of GC cells. α-Tomatine represses proliferation and metastasis of GC cells via inhibiting the PI3K-AKT and MAPKs pathways, and represses TWIST, Slug and PARP expressions so as to reverse drug resistance.