Triptolide Inhibited Liver Cancer Growth Based Through SPTLC2-S1P Signal Pathway

14 Background: Hepatocellular carcinoma is a cancer that has a high incidence in men, and its 15 incidence is increasing year by year. Studies show that angiogenesis plays an important role in the 16 formation of tumors, not only providing nutrients to tumor cells, but also closely related to tumor 17 growth and metastasis. So, how to find new anti-vascular and anti-tumor targets for the 18 pathogenesis of liver cancer is a key issue that needs to be resolved. 19 Methods: After treating HUVEC and HepG2 cells with different concentrations of TP, the 20 relationship between TP's anti-vascular and anti-tumor activities and sphingolipids was 21 investigated respectively. Then, the three-dimensional co-culture model was used to explore the 22 correlation between HUVEC and HepG2 cells, and to find the relationship between it and 23 sphingolipids. 24 Results: TP can inhibit the tube formation process of HUVEC cells. Through PCR Array, PCR 25 and Western Blot experiments, it is found that it may achieve this effect by down-regulating 26 SPTLC2. TP can also inhibit the proliferation, migration and invasion of HepG2 cells through the 27 same mechanism. In the three-dimensional co-culture model of HUVEC and HepG2 cells, it was 28 found for the first time that HUVEC can promote the biological process of HepG2 cells. It was 29 found through ELISA and Western Blot experiments that it may be achieved through the 30 S1P/S1PRS pathway, and TP was found in the dosing experiment. It can significantly inhibit the 31 induction of HUVEC on HepG2 cells. 32 Conclusions: These data confirm that the level of SPTLC2 may be related to the anti-vascular and 33 anti-tumor effects of TP. The data also showed that there is a correlation between the viability of 34 HepG2 cells and HUVEC cells, which may be related to the expression of S1P/S1PR S . Ultimately, 35 these data may help discover new anti-tumor targets.


39
Hepatocellular carcinoma is a cancer that has a high incidence in men, and its incidence is increasing year by year [1]. The pathogenesis of liver cancer is a very complicated process 1 composed of a series of related links mediated by various risk factors such as chronic viral 2 hepatitis, alcohol abuse, non-alcoholic steatohepatitis, and type 2 diabetes [2]. Studies show that 3 angiogenesis plays an important role in the formation of tumors, not only providing nutrients to 4 tumor cells, but also closely related to tumor growth and metastasis [3]. In addition, these new 5 vascular tissues can provide blood and nutrients for the continued growth of tumors, and tumor 6 tissues can further promote the regeneration of blood vessels through a variety of ways, thereby 7 forming a vicious circle, so modern medicine believes that cutting off the vascular tissues of 8 tumors can "Starved to death" tumor. So, how to find new anti-vascular and anti-tumor targets for 9 the pathogenesis of liver cancer is a key issue that needs to be resolved. Only by cutting off the 10 nutritional supply of tumor tissue and inhibiting the further growth of tumors can liver cancer be 11 fundamentally overcome. 12 In recent years, with the further study, it is found that in addition to being the basic component 13 of cell membrane, sphingolipids also participate in a variety of signal transduction pathways and 14 play an important role in the occurrence and development of various diseases. The metabolism of 15 sphingolipids is a key pathway in cancer biology, and its metabolites ceramide, sphingosine and 16 sphingosine-1-phosphate (S1P) together regulate tumor cell death, proliferation and drug 17 resistance, as well as host angiogenesis and inflammation [4]. Ceramide is produced by the 18 hydrolysis of sphingolipids, which can also de novo synthesis of ceramide by the precursor 19 dihydroceramide, which is converted to ceramide by dihydroceramide desaturase to induce tumor 20 cell apoptosis [5]. Ceramide is hydrolyzed by a ceramidase to produce sphingosine, which is 21 phosphorylated by sphingosine kinases (SK1 and SK2) to produce S1P. S1P is dephosphorylated 22 by S1P phosphatase 1 and 2 and is degraded by S1P lyase, which cleaves S1P to produce 23 phosphoethanolamine and hexadecenal. In addition to intracellular targets, S1P also binds to and 24 activates the G protein-coupled receptor family, S1P receptor 1-5 (S1PR1-5), which regulates the 25 biological activity of cells. Studies in many cancer cell lines indicate that S1P induces 26 proliferation and inhibits ceramide-induced apoptosis. Ceramide is a key factor in sphingolipid 27 metabolism, and Serine palmitoyltransferase (SPT) is a key enzyme for de novo synthesis of 28 ceramide. SPT in mammals is a heterodimer composed of two subunits, namely SPTLC1 and 29 SPTLC2, and SPTLC3 is the third subunit found in 2009 [6]. SPT is a class of substances with 30 pre-inflammatory and pre-apoptotic properties that can be involved in subsequent responses by 31 activating multiple protein kinases and phosphatases downstream of the inflammatory response, or 32 by generating S1P as a second messenger [7]. 33 Triptolide(TP) is one of the main active ingredients extracted from the roots, stems and leaves 34 of Tripterygium wilfordii. It is a small molecule compound with anti-tumor, anti-angiogenesis, 35 anti-inflammatory and pro-apoptotic effects [8]. TP has anti-liver cancer [9,10], ovarian cancer 36 [11][12][13][14], lung cancer [15][16][17], gastric cancer [18,19], breast cancer [20,21] effects. 37 Based on the above facts, this experiment explores the effects of TP on liver cancer from the 38 three perspectives of anti-vascularity, tumor suppression and tumor microenvironment, and finds 39 the connection between TP's anti-liver cancer effect and sphingolipid. In this study, we first 40 studied the effect of TP on human umbilical vein endothelial cells (HUVEC) and its possible 41 mechanism, explored the effect of TP on angiogenesis, and looked for the effect of TP on 42 angiogenesis New target. Secondly, we studied the effect of TP on liver cancer cell HepG2, and 43 further explored the new target of TP against liver cancer. As we all know, as one of the members 44 of the tumor microenvironment, vascular endothelial cells not only form vascular nutrition tumor 1 tissue, but also penetrate the entire tumor tissue. So does endothelial cell itself have a certain 2 effect on tumor cells? Then, we further explored the interaction between HUVEC and HepG2 cells 3 in the three-dimensional co-cultivation mode and its possible mechanism, and studied the 4 influence of TP on the co-cultivation system. 5 6

7
Cell grouping and transfection 8 Human umbilical vein endothelial cells (HUVEC) were purchased from the North Branch of the 9 Institute of Biotechnology, and the liver cancer HepG2 cells were purchased from the Chinese 10 Academy of Sciences and cultured in 10% (v/v) fetal bovine serum and 1% (v/v) 11 Penicillin-streptomycin in DMEM. Cultures were placed at 37°C in a humidity incubator with 5% 12 CO 2 . When the cells are in good condition and are as long as about 80%, they are used in 13 subsequent experiments. Cultured cells were divided into siR-SPTLC2 group, siR-NC group, 14 SPTLC2 group, SPTLC2-NC group and blank group and transfected with siR-SPTLC2, siR-NC, 15 SPTLC2, SPTLC2-NC, and no treatment was done, respectively. In addition, a medium 16 concentration of celestrol was added to each group.

18
Cell proliferation assay 19 Cell viability was determined by the CCK-8 assay. Briefly, HUVEC and HepG2 cells were 20 adjusted to a density of 4 x 10 4 cells/ml, plated into 96-well plates overnight, 100 μl per well, and 21 treated with different concentrations of celestrol. The HUVEC group was treated with 12.5 nM, 25 22 nM, 50 nM TP, DMSO as the negative control, and endostatin as the positive control (8 mg/L), 23 and the time was set at 24, 48, and 72 hours. The HepG2 group was treated with 1 μM, 2 μM, and 24 4 μM of TP, and DMSO was used as a negative control, and the time was set at 24 and 48 hours.

25
The HUVEC and HepG2 co-culture groups were set to 1 to 4 days, 2 x 10 3 HepG2 cells (200 26 μl/well) were added to the upper chamber of the 24-well co-culture chamber, and 5 x 10 3 HUVEC 27 cells were added to the lower chamber (500 μl/well). After the cells were treated for a specific 28 time, the medium in the 96-well plate was aspirated, and 100 μl of CCK-8-containing medium 29 (CCK-8 reagent: medium = 1:10) was added. Transferred the co-culture group upper chamber to a 30 new 24-well plate containing 500 μl of the above CCK-8 medium. After 1 to 4 hours, measure the 31 absorbance at 450 nm with a microplate reader 32 33 Cell migration and invasion assay 34 The migration ability of the treated HUVEC cells and HepG2 cells was determined using a 35 24-well two-compartment transwell assay. The TP treatment of each group of HUVEC cells and 36 HepG2 were resuspended in serum-free DMEM. 200 μl of the cell suspension was added to the 37 upper chamber of the transwell, and 500 μl of the complete medium was added to the lower 38 chamber. In the co-culture group, the density of HepG2 cells was adjusted to 5×10 4 /200 μl, and 39 the density of the lower chamber HUVEC was adjusted to 2×10 4 /500 μl. After 24 hours, the upper 40 chamber was taken out and washed with PBS three times, 4% tissue cell fixative was fixed for 1 41 hour, washed again with PBS for 3 times, 0.1% crystal violet stained for 30 minutes, PBS was 42 washed 3 times, gently wipe the cells inside the chamber with a cotton swab, and finally 43 photographed with a microscope. After the end of the photographing, each group of chambers was decolorized for 5 minutes in 500 μl of a 10% (v/v) acetic acid solution, and the absorbance was 1 measured at 550 nm. 2 The cell invasion assay was similar to the cell migration assay except that the transwell 3 membrane was pretreated with Matrigel and the HepG2 cell density was adjusted to 3 x 10 5 4 cells/200 μl.

6
Cell adhesion assay 7 The pre-cooled Matrigel was placed in a pre-cooled 96-well plate at 50 ul per well and allowed to 8 air dry. 2% BSA 100 ul was added to each well and blocked for 1 h. 3×10 4 HUVEC cells were 9 seeded in a 96-well plate with 100 μL of cell suspension per well. After 1 hour, the 96-well plate 10 was taken out, washed twice with PBS, 100 ul of the above CCK-8 mixed solution was added, and 11 incubation was continued for 1 to 4 hours in a cell culture incubator, and finally the absorbance at 12 450 nm was analyzed by a microplate reader.

14
Cell tube formation assay 15 Matrigel membrane blocked in 5% nonfat milk in TBST for 1h at room temperature and then incubated 41 with primary antibodies at 4 ℃ overnight. The immunoblots were then incubated with a secondary 42 antibody at room temperature. Finally, the antigen-antibody complex on the membrane was 43 visualized using ECL plus and X-ray film.

Enzyme linked immunoSorbent assay (ELISA) 1
The HUVEC and HepG2 cells co-culture for 1 to 4 days, 2 x 10 5 HepG2 cells (2 ml/well) were 2 added to the upper chamber of the 6-well co-culture chamber, and 1 x 10 5 HUVEC cells (1 3 ml/well) were added to the lower chamber. After the cells were treated for a specific time, the 4 supernatants were collected. It was detected by enzyme-linked immunosorbant assay according to 5 the manufacturer's protocol. 6 7 Statistical analysis 8 All data were represented by mean±SD. GraphPad Prism 5.0 software was applied to statistical 9 analysis, and significance between groups was ascertained by one-way ANOVA compared with 10 least significant difference. When the P-value was less 0.05, the analysis was accepted as 11 statistical difference. 12 13

TP inhibits HUVEC cell proliferation, migration, adhesion and tube formation 15
Firstly, we assessed the effects of TP on HUVEC cells proliferation. Figure 1A presented that TP 16 treatment remarkably inhibited the proliferation of HUVEC cells in a dose and time dependent 17 manner. The proliferation of HUVEC cells were both dramatically decreased after treatment with 18 8 mg/L endostatin and 8 mg/L endostatin + TP 12.5 nM. Considering the results, we decided to 19 use 24h for subsequent experiments. Figure 1B presented that TP treatment significantly inhibited 20 the migration of HUVEC cells in a dose-dependent manner. The migration of HUVEC cells were 21 both dramatically decreased after treatment with 8 mg/L endostatin and 8 mg/L endostatin + TP 22 12.5 nM. The adhenion and tube formation of HUVEC cells after TP treatment were also analyzed 23 in our research. The results of Figure 1C and 1D showed that TP treatment remarkably inhibited 24 HUVEC cells adhenion and tube formation in a dose-dependent manner. The adhenion and tube 25 formation of HUVEC cells were both dramatically decreased after treatment with 8 mg/L 26 endostatin and 8 mg/L endostatin + TP 12.5 nM.

28 TP down-regulates the expression of SPTLC2 in HUVEC cells 29
Firstly, we treated HUVEC cells with 25 nM TP and extracted RNA for PCR-array assay for 30 screening for altered genes. As presented in Figure 2A, SPTLC2 changes the most, which implied 31 that SPTLC2 might be involved in the effects of TP on HUVEC cells proliferation, migration, 32 adhesion and tube formation. As presented in Figure 2B and 2C, we further verify the expression 33 of SPTLC2 by RT-PCR and Western blotting. To verify the roles of SPTLC2 in HUVEC cells proliferation, migration, adhesion and tube 38 formation, SPTLC2 and siR-SPTLC2 were transfected into HUVEC cells, respectively. Figure 3A  39 showed that siR-SPTLC2 transfection down-regulated the expression of SPTLC2, while SPTLC2 40 transfection up-regulated the expression of SPTLC2 in HUVEC cells. Figure 3B presented that 41 HUVEC cells proliferation was notably inhibited by SPTLC2 suppression and markedly enhanced 42 by SPTLC2 overexpression. Figure 3C presented that HUVEC cells migration was notably 43 inhibited by SPTLC2 suppression and markedly enhanced by SPTLC2 overexpression. The results 44 of Figure 3D and 3E showed that SPTLC2 suppression remarkably inhibited HUVEC cells 1 adhenion and tube formation, SPTLC2 overexpression enhanced HUVEC cells adhenion and tube 2 formation. Finally, we explored the mechanism. As shown in Figure 3F, SPTLC2 can affect the 3 expression of S1P. The down-regulation of SPTLC2 can inhibit the expression of S1P, and the 4 up-regulation of SPTLC2 can promote the expression of S1P. Based on this, we believe that 5 SPTLC2 is likely to regulate various biological processes of cells by regulating the expression of 6 S1P. 7 8 TP inhibits HepG2 cell proliferation, migration and invasion 9 Firstly, we assessed the effects of TP on HepG2 cells proliferation. Figure 4A presented that TP 10 treatment remarkably inhibited the proliferation of HepG2 cells in a dose and time dependent 11 manner. Considering the results, we decided to use 24h for subsequent experiments. Figure 4B and 12 4C presented that TP treatment significantly inhibited the migration and invasion of HepG2 cells 13 in a dose-dependent manner. 14 15 TP down-regulates the expression of SPTLC2 in HepG2 cells 16 As presented in Figure 4D and 4E, TP treatment dramatically down-regulated the expression of 17 SPTLC2 in HepG2 cells in a concentration-dependent manner.

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SPTLC2 participates in the effects of TP on HepG2 cell proliferation, migration and 20 invasion 21 To verify the roles of SPTLC2 in HepG2 cells proliferation, migration and invasion, SPTLC2 and 22 siR-SPTLC2 were transfected into HepG2 cells, respectively. Figure 5A showed that siR-SPTLC2 23 transfection down-regulated the expression of SPTLC2, while SPTLC2 transfection up-regulated 24 the expression of SPTLC2 in HepG2 cells. Figure 5B presented that HepG2 cells proliferation was 25 notably inhibited by SPTLC2 suppression and markedly enhanced by SPTLC2 overexpression.

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The results of Figure 5C and 5D showed that SPTLC2 suppression remarkably inhibited HepG2 27 cells migration and invasion, SPTLC2 overexpression enhanced HepG2 cells migration and 28 invasion. Finally, we explored the mechanism. As shown in Figure 5E, SPTLC2 can affect the 29 expression of S1P. The down-regulation of SPTLC2 can inhibit the expression of S1P, and the 30 up-regulation of SPTLC2 can promote the expression of S1P. Based on this, we believe that 31 SPTLC2 is likely to regulate various biological processes of cells by regulating the expression of 32 S1P.

34 HUVEC cells may induce proliferation, migration and invasion of HepG2 cells through the 35
S1P-S1PR S pathway. 36 In order to verify the effect of HUVEC cells on proliferation, migration and invasion of HepG2 37 cells, HUVEC cells and HepG2 cells were co-cultured to further detect the proliferation, migration 38 and invasion of HepG2 cells. As shown in Figure 6A, HUVEC cells promoted the proliferation of 39 HepG2 cells in a time-dependent manner. Figure 6B and 6C presented that HUVEC cells 40 significantly promote migration and invasion of HepG2 cells. To further understand its mechanism 41 of action, we further examined changes in S1P in medium and changes in S1PR S expression in 42 HepG2 cells. As shown in Figure 6D, the level of S1P increased as the co-culture time increased.

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As shown in Figure 6E, S1PR1 and S1PR2 of HepG2 cells also increased with increasing 44 co-culture time. However, S1PR3 has the opposite trend. 1 2

TP inhibits the proliferation, migration and invasion of HepG2 cells induced by HUVEC 3 cells. 4
HUVEC cells were treated with different concentrations of TP and then co-cultured with HepG2 5 cells. As shown in figure 7A, it was found that the proliferation of HepG2 cells decreased with the 6 increase of treatment concentration. As shown in Figure 7B and 7C, the induction of migration 7 and invasion of HepG2 cells by HUVEC cells treated with TP was also attenuated. content of S1P in the culture system was determined by an ELISA assay. (E) S1PR S protein 14 expression in HepG 2 cells was checked by Western blot assay. * P<0.05, ** P<0.01 versus 15 Non-co-culture group.

19
As is known to all, angiogenesis plays an important role in tumor growth and metastasis. In 20 addition to transferring tumor cells, the newly formed vascular tissue can also provide a 1 continuous supply of nutrients to the tumor tissue [22]. Therefore, modern medicine believes that 2 cutting off the tumor vascular tissue can "starve" the tumor. However, as a member of the tumor 3 microenvironment, vascular endothelial cells not only form vascular vegetative tumor tissues, but 4 also run through the entire tumor tissue, so do endothelial cells themselves have some influence on 5 tumor cells? Angiogenesis is associated with many tumors, especially solid tumors such as liver 6 cancer and breast cancer. TP is one of the anti-tumor drugs that has attracted much attention in 7 recent years, which can not only inhibit tumor angiogenesis, but also inhibit the biological 8 processes of various tumors. In recent years, with further studies, it has been found that nerve 9 sphingolipid is not only a basic component of cell membrane, but also involved in a variety of 10 signal transduction pathways and plays an important role in the occurrence and development of 11 various diseases, especially tumors [4]. With this in mind, we hypothesized whether there was 12 some connection between TP's anti-tumor effects and sphingolipid. Based on the above facts, we 13 designed there experiments to explore: Firstly, we explored the mechanism between TP and 14 HUVEC, and explored the connection between TP's inhibition of angiogenesis and nerve 15 sphingolipin; Then, we further explore the connection between the antitumor effects of TP and 16 nerve sphingolipids. Finally, we continued to study the effect of HUVEC on tumor cells and its 17 possible mechanism. 18 Firstly, we verify TP anti-angiogenesis effect in vitro, in order to guarantee the reliability of the 19 data we choose the endostatin as a positive drug, within our experiment data showed endostatin 20 can significantly inhibit the whole process of HUVEC cells into tube (proliferation, migration, 21 adhesion, and angiogenesis), TP the inhibitory effect of HUVEC cells into tube ability compared 22 with the positive control is a bit weak, but still have statistical significance (P < 0.01). So, is there 23 an inevitable connection between TP's antivascular effect and sphingolipid? We screened the 24 changes of 48 nerve spongolipin-related genes by PCR Array experiment. According to the 25 experimental results, we found that compared with the control group, the change of SPTLC2 in 26 the TP group was larger (P<0.01), which is likely to be a new target of TP's anti-angiogenesis. And 27 SPTLC2 is nerve sheath one of the key enzyme of lipid metabolic pathway, the new synthesis of 28 ceramide in SPTLC2 generated under the action of ceramide, and ceramide is closely related to 29 cell apoptosis, it can promote cell apoptosis, ceramide further under the action of ceramide 30 enzyme hydrolysis to produce sphingosine, sphingosine kinase S1P further its phosphorylation, 31 whereas S1P can affect the tumor microenvironment, thus promotes the transfer of tumor and 32 growth [23]. Based on this, we further detected the expression of SPTLC2 by Western Blot and 33 RT-PCR, and the results showed that the expression of SPTLC2 decreased significantly with the 34 increase of TP concentration (P<0.01), which greatly attracted our interest. The transfection model 35 of SPTLC2 was further constructed and the results were detected by Western Blot. After the 36 successful establishment of the model, we further tested the tube-forming ability of the transfected 37 SPTLC2 cells. The results showed that the tubulogenesis of HUVEC cells was significantly 38 improved after SPTLC2 was elevated. However, after knocking down SPTLC2, its pipe forming 39 ability decreased significantly (P<0.01). Based on the above results, we believe that SPTLC2 is 40 likely to be a new target of TP's antivascular effect, and TP is likely to play the antivascular effect 41 by down-regulating SPTLC2. We then further explored the association between SPTLC2 and TP 42 anti-tumor, and our experimental data showed that TP significantly inhibited the proliferation, 43 migration, and invasion of HepG2 cells (P<0.01). Since angiogenesis is closely related to the 44 occurrence and development of tumor, and TP is known to have both anti-vascular and anti-tumor 1 effects, is it possible for TP to play both anti-vascular and anti-tumor effects through the same 2 mechanism? According to this conjecture, we by Western Blot and RT-PCR experiments further 3 testing SPTLC2 expression in HepG2 cells, the results showed with the increase of concentration 4 of TP its expression decreased significantly (P<0.01), it could also be the result hints SPTLC2 TP 5 antitumor effect of new targets, so as in HUVEC cells we also build the model of transfection 6 HepG2 cells. The data showed that when SPTLC2 was elevated, the proliferation, migration and 7 invasion of HepG2 cells were significantly increased. However, after knocking down SPTLC2, it 8 decreased significantly (P<0.01). Based on the above experimental results, we believe that 9 SPTLC2 is likely to be a new target of TP's anti-tumor effect, and TP is likely to play an 10 anti-tumor effect by down-regulating SPTLC2. Finally, we constructed the transfection model of 11 HUVEC and HepG2 cells, and after the model was successfully constructed, we further detected 12 the change of S1P expression. The data showed that the expression of S1P in the two cells 13 decreased after SPTLC2 was knocked down. After up-regulating SPTLC2, the expression of S1P 14 in both cells increased. Based on the above results, we believe that TP may have an anti-vascular 15 and anti-tumor effect by down-regulating SPTLC2 and eventually affecting the expression of S1P. 16 Subsequently, we further explored the effects of endothelial cells as a member of the tumor 17 microenvironment on tumor cells. We found that HUVEC cells promote the biological processes 18 (proliferation, migration, and invasion) of liver cancer cell line HepG2 cells by using a 19 three-dimensional co-culture model, and we found for the first time that endothelial cells promote 20 the biological behavior of breast cancer cells. So, what mechanism is it implemented by? Based on 21 the three-dimensional co-culture model, we speculate that HUVEC cells may secrete a substance, 22 which is likely to be water-soluble and can diffuse freely in the medium, thus acting on HepG2 23 cells and binding to its corresponding receptors on the cell membrane, thus promoting its 24 development. So what is this substance and its receptor? As described in our introduction, S1P, the 25 metabolite of spongolipid, is a water-soluble substance, which can play a variety of roles in the 26 development of various tumors by combining with S1PR S . So, is it through this pathway that 27 HUVEC cells promote HepG2 cells? With these questions in mind, we further detected the content 28 of S1P in the co-culture system, and the data showed that the content of S1P increased 29 significantly with the extension of the co-culture time (P<0.01). Does the corresponding S1PR S on 30 HepG2 cells also change? The changes of S1PR1-3 were further detected by Western Blot. The 31 results showed that S1PR1 and S1PR2 were significantly up-regulated with the extension of 32 co-culture time, while S1PR3 was significantly down-regulated with the extension of co-culture 33 time (P<0.01). Based on the above experimental results, we believe that the promotion effect of 34 HUVEC cells on liver cancer cells is probably realized through the S1P-S1PR S pathway. So can 35 HUVEC cells promote liver cancer cells that can be inhibited by TP? With this problem in mind, 36 we further pretreated HUVEC cells with different concentrations of TP, co-cultured them with 37 HepG2 cells after a certain period of time, and then detected the proliferation, migration and 38 invasion ability of HepG2 cells after a certain period of time. Experimental data showed that with 39 the increase of TP treatment concentration, the ability of HUVEC cells to induce HepG2 cells 40 decreased significantly. Based on this result, we concluded that TP could inhibit the induction of 41 HUVEC cells to tumor cells in addition to its significant anti-vascular and anti-tumor effects. 42 43

Study strengths and limitations 1
This study explored the effects of TP on liver cancer from the perspectives of anti-vascular, 2 anti-cancer and tumor microenvironment, and discovered the connection between the anti-liver 3 cancer effect of TP and sphingolipids. New targets have been discovered for anti-vascular and 4 anti-tumor research, which will further promote the study of tumorigenesis mechanisms. This 5 study has several limitations. The regulatory effect of SPTLC2 in other tumors needs further 6 experimental verification, and the relationship between TP's anti-tumor effect and SPTLC2 in vivo 7 also needs further experimental research. 8 9

Conclusions
10 These data confirm that the level of SPTLC2 may be related to the anti-vascular and anti-tumor 11 effects of TP. The data also showed that there is a correlation between the viability of HepG2 cells 12 and HUVEC cells, which may be related to the expression of S1P/S1PR S . Ultimately, these data 13 may help discover new anti-tumor targets.