Effects of FTY720 activation of PI3K signaling pathway to induce autophagy on proliferation and migration of gastric cancer cells

doi:10.21203/rs.3.rs-3215759/v1

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Research Article

Effects of FTY720 activation of PI3K signaling pathway to induce autophagy on proliferation and migration of gastric cancer cells

https://doi.org/10.21203/rs.3.rs-3215759/v1

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Background

Fingolimod (FTY720), a synthetic analogue of myriocon, has been the subject of numerous studies investigating its antitumor activity. Autophagy, a distinctive cellular process in eukaryotic cells, has been extensively explored in cancer metastasis research.The mechanisms by which FTY720 regulates autophagy in gastric cancer are still not clear.Therefore, exploring the relationship between FTY720 and autophagy regulation will help provide a new perspective for GC treatment.

Methods

We are using gene database analysis to investigate the impact of FTY720 on tumor progression. We aim to elucidate the effects of FTY720 on the biological behavior of gastric cancer cells SGC-7901 and HGC-27 in vitro, particularly focusing on the regulation of autophagy, as well as its influence on tumor growth in vivo. We will integrate histological and molecular methods to assess the biological behavior and explore the molecular mechanisms both in vitro and in vivo.

Results

Based on the database used in this investigation, it was discovered that FTY720 might inhibit the proliferation of cancer cells via inducing autophagy.FTY720 functions as a regulatory factor to promote autophagy expression, prevent GC cell proliferation, migration, and invasion in culture, and slow the formation of tumors in vivo.Experiments using the autophagy inhibitor (HCQ) revealed that FTY720's regulatory inhibition was dramatically reduced.Additionally, we showed that FTY720 significantly boosted PI3K phosphorylation levels although its total protein levels in GC cell lines did not alter appreciably.

Conclusion

In conclusion, our study demonstrated that FTY720 activates the PI3K pathway to cause autophagy, limit GC cell migration and invasion, and induce autophagy. It also suggested that FTY720 signaling molecules may be used as prospective GC therapeutic targets.

Fingolimod

Autophagy

PI3Kpathway

Proliferation

Migration

Invasion

Gastric cancer (GC) is a widespread malignancy and ranks among the top four most common cancers globally [1–2]. Although screening efforts in high-prevalence regions have helped decrease the mortality and morbidity rates associated with GC, the overall 5-year survival rate remains low [3].Despite advancements in laparoscopic surgery, patients with advanced GC continue to have a grim prognosis [4–5]. Hence, it is essential to investigate the molecular mechanisms underlying the occurrence and progression of GC in order to identify prognostic biomarkers and potential drug targets. Such insights would greatly aid in the development of individualized therapy approaches.

The antitumor activity of fingolimod(FTY720) has recently gained significant attention in numerous studies. FTY720 is a synthetic analogue of ISP-1 (myriocon), a fungal metabolite commonly found in traditional herbs [6]. Initially developed as an immunosuppressant, FTY720 inhibits tumor cell migration and homing by interacting with the sphingosin-1-phosphate (S1P) receptor after converting to FTY720-phosphate [7–8]. Moreover, FTY720 has demonstrated its ability to induce apoptosis in various human cancer cell lines, such as breast, prostate, lung, and ovarian cancers [9–10]. These apoptotic effects are mediated by protein kinase C δ, phosphatidylinositol 3-kinase (PI3K), protein phosphatase 2A, and mitogen-activated protein (MAP) kinase [11–14].

Autophagy, a distinctive process observed in eukaryotic cells, has been the subject of research for over 60 years. Recently, it has garnered significant attention, particularly in the realm of cancer metastasis [15–16]. In the tumor microenvironment, autophagy induced by various factors has shown the ability to impede tumor cell proliferation and migration [17–20]. Studies have demonstrated that carnosin can modulate the PI3K/AKT/mTOR pathway to regulate autophagy and hinder the proliferation of cancer cells, thereby creating barriers against tumor growth and metastasis [21]. Previous studies conducted by our research group have revealed a close relationship between the occurrence and development of GC and the phosphatidylinositol 3-kinase (PI3K) pathway. In addition, our research group utilized transcriptome high-throughput sequencing technology and discovered that a high concentration of FTY720 in GC cells leads to an increase in autophagy flux. This study aims to further investigate the regulation of autophagy expression by the PI3K signaling pathway activated by FTY720, as well as its impact on the proliferation, invasion, and migration of GC cells. The findings from this research could provide new insights into the pathogenesis, diagnosis, and treatment of GC.

2.1 Materials

2.1.1 Cell lines

Human gastric cancer cells HGC-27 and SGC-7901 were purchased from China's typical culture Preservation Center and cultured in our laboratory.

2.1.2 Reagent consumables

FTY720, autophagy inhibitor 3-MA and HCQ were purchased from MCE.The pathway inhibitor (Thioridazine HCl) was purchased from GLPBIO.The RPMI1640 culture medium, pancreatic enzyme, fetal bovine serum (FBS) were purchased from Gibco.Penicillin/streptomycin solution, RIPA cell lysate (strong), BCA protein concentration determination kit, CCK8 reagent and apoptosis staining kit were purchased from Biyuntian Biotechnology Company.Mouse monoclonal antibodies to PI3K, p-PI3K, LC3, Beclin-1, P62, GAPDH, Ki-67 were purchased from Proteintech.

Company.SV Total RNA Isolation System was purchased from biosharp.PrimeScriptTM RT reagent Kit with gDNA Eraser (Per-fect Real Time), quantitative PCR kit TB Green® Premix Ex TaqTMⅡ (Tli)RNaseH Plus was purchased from Novoprotein Technology Co., LTD.ECL-plus chemiluminescence kit was purchased from Shanghai Yase Biotechnology Co., LTD. PVDF membrane was purchased from Millipore Company.Matrigel glue was purchased from BD.Other reagents are imported or domestic analysis pure.

2.1.3 Instruments

CFX Connect real-time fluorescence quantitative PCR instrument, Imark enzyme labeling instrument were purchased from the United States Bio-Rad company, CO2 constant temperature cell incubator and Western Blot exposure machine were purchased from the United States Shellab Company, etc.

2.2 Methods

2.2.1 Biological information retrieval

The chemical interaction search tool STITCH(http://stitch.embl.de/) was used to search for the association between FTY720 and autophagy expression.Autophagy expression related pathways were retrieved using KEGG database.

2.2.2 Cell culture

Human gastric cancer cells HGC-27, SGC-7901 were cultured in RPMI1640 culture medium containing 10% fetal bovine serum, penicillin 100 U/ ml, streptomycin 100 µg/ ml, and incubated at 37℃, 5% CO₂ in a constant temperature cell culture incubator, and then digested and subcultured for the subsequent experiments when the cells were grown to the appropriate degree of fusion.

2.2.3 Western Blot

The protein was extracted after the cells were overgrown, and the obtained protein was quantified by BCA method.The same amount of protein samples were taken for SDS-PAGE electrophoresis, and the proteins were transferred to 0.45µm PVDF membrane after electrophoresis.Then the PVDF membrane was immersed in the sealing solution and sealed for 60 minutes.After the closure was completed, the corresponding primary antibody was diluted and the PVDF membrane was incubated at 4℃ overnight.The film was washed the next day and incubated in a room temperature shaker with secondary antibody dilution for 90min.ECL-plus detects protein signals on PVDF membranes, exposes and develops them.

2.2.4 qRT-PCR

Total RNA was extracted, the concentration and purity of RNA were determined, and the RNA was reversely transcribed into cDNA.Using GAPDH as the internal reference, real-time fluorescence quantitative PCR was performed according to the operation method of the kit to detect the expression of the target gene.The PCR amplification conditions were as follows: predenaturation at 95℃ for 2min;Denaturation at 95℃ for 1min, annealing at 95℃ for 20s, extension at 60℃ for 1min, a total of 40 cycles.Dissolution cycle at 65 ~ 95℃.According to the kit instructions, the qRT-PCR reaction was performed using TBGreen®PremixExTaqTM and repeated three times.The mRNA expression level of each gene was calculated using a relatively quantitative method (2^−ΔΔCt).Corresponding primer sequences are shown in Diagram. S1.

2.2.5 Cell Proliferation (CCK8/ Cell cloning)

HGC-27 or SGC-7901 cells were inoculated into 96-well plates at a density of 3.0×10³ cells per well, and FTY720 was added to the required experimental holes 24 h later.After 24 hours, 10µl CCK8 reagent was added to each well, and OD value was detected at 450 nm.HGC-27 or SGC-7901 cells were inoculated into 6-well plates at a density of 2×10⁵ cells per well, and FTY720 was added to the required experimental holes 24 h later.24h later, the pores were treated with 4% paraformaldehyde solution and 0.1% crystal violet for 10 min respectively. After washing and drying with PBS, the photos were taken under the microscope and counted.

2.2.6 Cell scratches

After the cell density was adjusted to 2.0×10⁶ cells/well, the plate was placed in the 6-well plate, and FTY720 was added to the required experimental holes respectively 24 hours later. When the cells were observed to fuse into a single layer state under the microscope, the sterile 200 microgun head was uniformly marked in the holes and continued to be cultured in the incubator.At 0h and 24h, the width area of the scratch was observed by an inverted microscope, and the obtained images were processed by ImageJ software and the scratch mobility was calculated.

2.2.7 Transwell chamber

Matrigel solution was added into serum-reducing medium at a ratio of 1:7 and thoroughly mixed, and then added to the upper chamber of the chamber for incubation for 1h;The cells of each group were paved with a density of 1.0×10⁴ cells/well, and the corresponding Wells were treated with drugs, and 600µL culture medium was added to the lower chamber for 24h.The non-invasive cells in the upper layer of the chamber were removed and treated with 4% paraformaldehyde solution and 0.1% crystal violet for 10min respectively. After washing and drying with PBS, the cells were photographed and counted under the microscope.

2.2.8 Cell apoptosis

After the cell density was adjusted to 2.0×10⁶ cells/well, the plate was placed in the 6-well plate, and drug treatment was added to the required experimental holes 24 hours later.After the time required for treatment, the cells were treated according to the instructions of apoptotic TUNEL kit and Hoechst staining kit, and photographed by fluorescence microscope.

2.3 Animal model experiment

The 4-week-old female nude mice were obtained from Hangzhou Ziyuan Laboratory Animal Technology Co., LTD. (Hangzhou, China) and fed under pathogen free conditions.The nude mice were randomly divided into two groups (5 in each group), and the cells of SGC-7901 (5×10⁶ in each group) were injected subcutaneously into the armpit and back of the nude mice.After 6 hours, FTY720[0.5mg/(kd·d)][22–23] was intraperitoneally injected once every 24 hours until 7 days.Mice in Control group were intraperitoneally injected with the same amount of saline once each time.After 20 days, mice were executed by cervical dislocation under anaesthesia (0.7% pentobarbital sodium).Xenotumor excised, photographed and examined.Double-blind method was used in this experiment.

2.4 Statistical treatment

All statistical data were analyzed by Prism9.0 (GraphpadSoftware) and ImageJ software.Two-sample t test, ANOVA, and least significant difference were used to analyze the data.All experiments were repeated for 3 times, and P < 0.05 was considered to be statistically significant.

1.Effect of FTY720 on proliferation of GC cells

The effects of FTY720 activation of the PI3K signaling pathway on the induction of autophagy and its impact on the proliferation and migration of GC cells have been the focus of recent research. FTY720, a synthetic analogue of ISP-1 (myriocon)(Fig. 1A), has been shown to inhibit tumor cell proliferation [24]. Through analysis of the STITCH database, it was found that FTY720 mediates the Beclin-1 pathway, which in turn affects MKI-67 expression (Fig. 1B). These findings suggest that FTY720 may regulate autophagy flux and influence the proliferation of tumor cells. To investigate this further, GC cells were treated with varying concentrations of FTY720 (umol/L) for 48 hours. CCK-8 results demonstrated a dose-dependent inhibition of proliferative activity in SGC-7901 and HGC-27 cell lines, both derived from gastric cancer (Fig. 1C). Furthermore, cell cloning experiments revealed a significant reduction in proliferation activity for both SGC-7901 (Figs. 1D,E) and HGC-27 (Figs. 1F,G) cells. Based on the above data analysis, we propose that a concentration of 6umol/L FTY720 should be used for subsequent experiments to investigate its effects on autophagy, proliferation, and migration of GC cells.

2. Effect of FTY720 on autophagy of GC cells

In light of the findings from a previous study that demonstrated FTY720's ability to induce autophagy in glioblastoma and ovarian cancer cells [25], we aimed to investigate the potential association between FTY720 and autophagy in GC cells. To test our hypothesis, we treated SGC-7901 and HGC-27 cells with a concentration of 6umol/L of FTY720 for 24 hours. Subsequently, we assessed the expression levels of autophagy-related proteins LC3, Beclin-1, and P62. The results obtained from Western Blot analysis revealed a significant up-regulation of LC3 and Beclin-1 protein expression in SGC-7901 cells following FTY720 treatment, while P62 protein expression levels were notably down-regulated (Figs. 2A,B). These findings strongly suggest the occurrence of autophagy. Similarly, HGC-27 cells exhibited similar patterns of protein expression (Figs. 2C,D).

To investigate the impact of FTY720 on autophagy expression at the transcriptional level, we treated SGC-7901 and HGC-27 cells with 6umol/L FTY720 for 24 hours. Analysis of qRT-PCR results revealed that FTY720 significantly upregulated the mRNA expression of autophagy-related proteins (LC3, Beclin-1), while downregulating the mRNA expression of P62 (Figs. 2E,F). These findings, in conjunction with the aforementioned experiments, suggest that FTY720 can induce autophagy flux in GC cell lines.

3.The PI3K signaling pathway is involved in the autophagy and proliferation of FTY720 regulated GC cells

Phosphatidylinositol 3-kinase (PI3K) is a crucial intracellular signaling pathway [26–27]. Upstream signaling molecules have the ability to regulate the PI3K signaling pathway, thus influencing various downstream effectors [28]. According to the KEGG database, the PI3K signaling pathway is involved in the formation of autophagy (Fig. 3A). Hence, we hypothesize that FTY720 can activate the PI3K signaling pathway in GC to regulate autophagy downstream. To test this hypothesis, we examined the activity of the aforementioned signaling pathways (determined by PI3K and p-PI3K levels) as well as the levels of the autophagy protein LC3 in SGC-7901 and HGC-27 cells treated with FTY720 for 24 hours.The Western Blot results revealed an up-regulation of LC3 and p-PI3K protein levels in SGC-7901 cells, while the total PI3K protein levels remained unchanged (Figs. 3B,D). Similarly, LC3 and p-PI3K protein levels were up-regulated in HGC-27 cells, while total PI3K protein levels showed no significant change (Figs. 3C,E). Based on these findings, we can conclude that phosphorylated PI3K protein activated by FTY720 regulates downstream effectors in GC cells.

To investigate the effect of FTY720 on the transcriptional level of PI3KmRNA, we treated SGC-7901 and HGC-27 cells with 6umol/L FTY720 for 24 hours. Analysis of qRT-PCR results revealed that FTY720 significantly increased the expression of p-PI3K mRNA in SGC-7901 cells, while the expression of total PI3KmRNA was not significantly affected (Fig. 4A). Similar results were observed in HGC-27 cells (Fig. 4B). Based on these experiments, it can be concluded that FTY720 induces the expression of p-PI3K in GC cell lines.In addition,the experimental groups were divided into Control group, FTY720 group, pathway inhibitor group, and FTY720 + pathway inhibitor group. We tested the effects of the PI3K pathway on autophagy and proliferation after a 24-hour treatment. Western Blot results showed that the addition of pathway inhibitor (10umol/L) inhibited Beclin-1 expression in SGC-7901 cells, while KI-67 expression was up-regulated (Figs. 4C,D). Similarly, Beclin-1 expression was inhibited in HGC-27 cells, while KI-67 expression was up-regulated (Figs. 4E,F). Therefore, we demonstrated that FTY720 activates the PI3K signaling pathway in GC, which regulates downstream autophagy and affects GC cell proliferation.

4.Inhibition of autophagy, effect of FTY720 on proliferation of GC cells

Autophagy is a catabolic process that serves as an adaptive survival mechanism activated during cellular stress [29]. It is not only a lysosomal degradation pathway, but also plays a role in the tumorigenesis/progression and treatment resistance of many cancers [30]. Therefore, we conducted further investigation into the impact of autophagy in FTY720 on GC cell proliferation. To minimize the influence of autophagy inhibitors on cells, we treated the cells with autophagy inhibitors 3-MA (2.5mmol/L) [31] and HCQ (20umol/L) [32] for 36 hours.CCK-8 results demonstrated that the inhibitory effect of FTY720 on GC cells was alleviated upon the addition of autophagy inhibitors (Figs. 5A,B). Similarly, Western Blot results revealed a significant up-regulation of KI-67 protein expression in SGC-7901 cells after stimulation with autophagy inhibitors (Figs. 5C,E). KI-67 protein expression was also observed to be upregulated in HGC-27 cells (Figs. 5D,F). In comparison to 3-MA, HCQ exhibited a more pronounced inhibitory effect with minimal impact on the cells themselves. Consequently, HCQ was chosen as the optimal autophagy inhibitor for further investigation into the migration and invasion behavior of GC cells in the subsequent study of FTY720.

5.Effects of FTY720 on migration, invasion and apoptosis of GC cells

According to the results of previous experiments, it was observed that FTY720 promoted the expression of autophagy in GC cells. However, the effect of FTY720 on the biological behavior of GC cells after promoting autophagy remains unclear. To investigate this, we divided the experiment into three groups: Control group, FTY720 group, and FTY720 + HCQ group. The cell scratch test results, as depicted in Figs. 6A and 6C, showed that FTY720 inhibited cell migration in SGC-7901 cells. Similarly, Figs. 6B and 6D demonstrated that FTY720 also inhibited cell migration in HGC-27 cells, indicating that FTY720 mediated autophagy to suppress GC cell mobility. Furthermore, the Transwell cell experiment revealed that the cell invasion ability was significantly enhanced in the Control group and FTY720 + HCQ group compared to the FTY720 group (Figs. 6E,F, SGC-7901; 6E,G, HGC-27). Additionally, we examined the effect of FTY720-induced autophagy expression on GC cell apoptosis. Figures 6H and 6I showed that FTY720 induced autophagy expression, thereby promoting apoptosis.

We concluded that FTY720 induced autophagy to inhibit proliferation, migration and invasion of GC cells in vitro.

6. FTY720 inhibits tumor growth in vivo

To investigate the inhibitory effects of FTY720 on tumorigenicity in vivo, we utilized a mouse model of subcutaneous tumorigenesis with SGC-7901 cells. It is worth noting that SGC-7901 cells exhibit a greater propensity for proliferation, migration, and invasion compared to HGC-27 cells. The results demonstrated that FTY720 significantly inhibited tumor growth in mice, while having minimal impact on the overall growth of the mice themselves(Fig. 7A). Furthermore, we employed Western Blot analysis to assess the levels of autophagy-associated proteins and tumor growth factors in the subcutaneous tumors. The results depicted in Figs. 7B and 7C revealed a significant upregulation of autophagy protein expression in the tumor tissues of the FTY720 experimental group, while the expression of tumor growth factors was downregulated. These outcomes strongly suggest that FTY720 exerts its inhibitory effects on tumor growth in vivo through the induction of autophagy.

FTY720 has shown potential for use in various cancer treatments. In vivo, FTY720 can be converted into FTY720 (S)-phosphate (FTY2-P) by sphingosine kinase 720. This conversion leads to the inhibition of tumor cell activity by suppressing Sphingosine kinase 1, a proto-oncogene associated with cancer in both laboratory studies and clinical settings. Moreover, FTY720 (S)-phosphate stimulates the internalization of S1PR1 on cells [33–34].In this study, we investigated the effects of FTY720, a synthetic analogue of ISP-1, on the proliferation of GC cells. Different concentrations of FTY720 were used to treat the GC cells, and the results demonstrated that FTY720 effectively inhibited their proliferation. The inhibitory activity of FTY720 was found to be positively correlated with the concentration used. Autophagy, a highly conserved eukaryotic cell recycling process, has been shown to play a crucial role in cell survival and maintenance [35–36]. Through retrieval from the STITCH database, it was found that FTY720 can regulate the expression of the autophagy-related gene Beclin-1 and affect the tumor metastasis factor KI-67. Our findings confirm that FTY720 inhibits the proliferation of GC cells and is associated with the modulation of autophagy flux.

Several signaling pathways, including NF-kB[37], HER2/PI3K/AKT[38], and ERK[39], have been reported to play a role in regulating the expression of autophagy. According to the KEGG database, it is suggested that PI3K may be involved in the regulation of autophagy. In this study, we aimed to investigate the involvement of the PI3K signaling pathway in regulating autophagy in GC.The study aimed to verify the inhibitory effect of FTY720 on GC cells (SGC-7901 and HGC-27 cells) by using the autophagy inhibitor HCQ. The researchers investigated the ability of FTY720 to induce autophagy and inhibit the proliferation, migration, and invasion of GC cells through cell scratch, Transwell, and apoptosis tests. Finally, we confirmed the inhibitory effect of FTY720 on tumor growth in vivo.However,the specific mechanism and signaling pathway of in vivo study still require further exploration. In conclusion, our findings indicate that FTY720 activates the PI3K signaling pathway, leading to the expression of autophagy and inhibition of GC cell progression. Therefore, therapeutic strategies focused on generating FTY720 or its analogues hold promise as a treatment for GC.

In summary, our current research provides the first evidence that FTY720 inhibits the growth of GC cells, partially through the activation of the PI3K signaling pathway. Additionally, our results suggest that autophagy plays a significant role in the regulation of FTY720. Based on these findings, we propose that FTY720 or its analogs, as well as modulators of autophagy gene expression, could be utilized as target molecules for the development of novel therapeutic approaches in GC.

Fingolimod (FTY720),Hydroxychloroquine(HCQ) ,Gastric cancer (GC), 3-Methyladenine(3-MA), Mitogen-activated protein (MAP) ,Fetal Bovine Serum (FBS)

*Ethics approval and consent to participate:This study involving animal experiments complied with animal ethics. Bengbu Medical College Animal Ethics Approval Number [2023] No. 446, No. 574. All methods were carried out in accordance with relevant guidelines and regulations.All methods are reported in accordance with ARRIVE guidelines (https://arriveguidelines.org) for the reporting of animal experiments.

*Consent for publication:Not applicable.

*Availability of data and materials: All data generated or analysed during this study are included in this published article and its supplementary information files.

*Competing interests:The authors declare that they have no competing interests

*Funding:Natural Science Research Project of Colleges and Universities in Anhui Province (KJ2021ZD0090). Graduate Scientific Research innovation project of Bengbu Medical College(Byycx22089,Byycx22090).

*Authors' contributions:

Ling: Primarily involved in experiment execution, manuscript writing, and data analysis.Zhu: Mainly involved in experiment design and guidance.Guan: Mainly involved in experimental data analysis.Ji: Involved in cell experiments and animal experiments.Zhao: Involved in animal experiments.All authors read and approved the final manuscript.

*Acknowledgements:Not applicable.

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No competing interests reported.

S1.png
Diagram: S1: primer sequence.The above sequences are provided by Shenggong Bioengineering (Shanghai) Co., LTD.
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Effects of FTY720 activation of PI3K signaling pathway to induce autophagy on proliferation and migration of gastric cancer cells

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Abstract

Figures

1 BACKGROUND

2 METHODS

2.1 Materials

2.1.1 Cell lines

2.1.2 Reagent consumables

2.1.3 Instruments

2.2 Methods

2.2.1 Biological information retrieval

2.2.2 Cell culture

2.2.3 Western Blot

2.2.4 qRT-PCR

2.2.5 Cell Proliferation (CCK8/ Cell cloning)

2.2.6 Cell scratches

2.2.7 Transwell chamber

2.2.8 Cell apoptosis

2.3 Animal model experiment

2.4 Statistical treatment

3 RESULTS

1.Effect of FTY720 on proliferation of GC cells

2. Effect of FTY720 on autophagy of GC cells

4.Inhibition of autophagy, effect of FTY720 on proliferation of GC cells

5.Effects of FTY720 on migration, invasion and apoptosis of GC cells

6. FTY720 inhibits tumor growth in vivo

4 DISCUSS

5 CONCLUSION

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