Isoliensinine suppressed gastric cancer cell proliferation and migration by targeting TGFBR1 to regulate TGF-β-smad signaling pathways

Abstract

Background: Gastric cancer (GC) ranks as the fifth most prevalent cancer globally, and its pronounced invasiveness and propensity to spread provide significant challenges for therapy. At present, there are no efficacious medications available for the treatment of patients with GC. Isoliensinine (ISO), a bisbenzylisoquinoline alkaloid, was isolated from Nelumbo nucifera Gaertn. It possesses anti-tumor, antioxidant, and other physiological effects. Nevertheless, there is currently no available study on the impact of ISO on GC, and further investigation is needed to understand its molecular mechanism.

Methods: ISO target points and GC-related genes were identified, and the cross-target points of ISO and GC were obtained. We then examined cross-targeting and found genes that were differentially expressed in GCs. Kaplan-Meier survival curves were used to screen target genes, and the STRING database and Cytoscape 3.9.1 were used to construct protein-protein interactions and drug-target networks. In addition, molecular docking studies confirmed the interactions between ISO screen targets. Finally, in vitro tests were used to establish the impact of ISO on GC cells.

Results: Through bioinformatics research, we have identified TGFBR1 as the target of ISO in GC. In addition, we noticed a substantial inhibition in GC cell proliferation, migration, and invasion activities following ISO treatment. Moreover, we noticed that ISO treatment effectively suppressed TGF-β-induced epithelial-mesenchymal transition (EMT) and activation of the TGF-β-Smad pathway. Furthermore, we discovered that siTGFBR1 nullified the impact of ISO on TGF-β-triggered migration, invasion, and activation of the TGF-β-Smad pathway.

Conclusion: Our research suggests that ISO specifically targets TGFBR1 and regulates the TGF-β-Smad signaling pathway to suppress the proliferation and migration of GC cells.

Keywords: epithelial-mesenchymal transition (EMT); gastric cancer; isoliensinine; metastasis; proliferation; transforming growth factor-β (TGF-β).

The study’s flow chart utilizes network pharmacology, molecular docking, and experimental verification approaches to predict the anti-GC activity of ISO.

FIGURE 1

The target of ISO in GC was analyzed using bioinformatics. (A) Diagram illustrating the overlapping areas between the ISO target and GC co-target using a Venn diagram. (B, C) The diagram depicts the relationship between ISO and GC in a PPI network. (D) The Kaplan-Meier survival curves of TGFBR1 in GC were analyzed. Genes with no association with TGFBR1 are represented in blue, genes with a negative correlation are represented in red, and genes with a positive correlation are depicted in green. (E) Differential expression analysis of TGFBR1 in cancer and para-cancerous tissue, red represents cancer tissue, and gray represents para-cancerous tissue. (F) The molecular docking data for TGFBR1 indicate a binding energy of −6.64 kcal/mol and the potential formation of at least two hydrogen bonds with TGFBR1. (G) 2D visualization of molecular docking results using online tools.

FIGURE 2

The impact of ISO on the growth, movement, and infiltration of stomach cancer cells. (A, B) CCK-8 is used to assess the cell survival of HGC27 and AGS GC cells after treatment with ISO at various concentrations and time gradients. (C) After 7 days of treatment with ISO (DMSO, 1, 2, 4, 8 μM), colony development and statistical results were observed. (D, E) Following a 2 h pretreatment with 10 μM ISO, TGF-β stimulation for 24 h, the rate of cell penetration was measured using transwell. The statistics show the number of HGC27 and AGS cells that invade and migrate within 24 h. (F, G) Utilizing a quantity of 10 μM of ISO Following a pretreatment period of 2 h, TGF-β was administered for 10 h and 24 h using a wound healing test to assess the impact of ISO on the migratory capacity of HGC27 and AGS cells. The statistical results are shown as the ratio of cell migration compared to the initial migration area at 0 h. Results are expressed as means ± SEM, n = 3. * P < 0.05, *** P < 0.001 when compared to DMSO group. # P < 0.05, ### P < 0.001 when compared to the Control group (not A, B).

FIGURE 3

Western blotting is used to measure the impact of ISO on the EMT of gastric cancer. (A) Pretreatment with different concentrations of ISO (0, 10, 20, 30, 40 μM) for 2 h, followed by TGF-β induction for 24 h, and Western blotting to detect the levels of E-cadherin, N-cadherin, Vimentin and Snai1 in HGC27 cells and AGS cells. (B) Statistical data on the relative protein expression in HGC27 and AGS cells. Results are expressed as means ± SEM, n = 3. * P < 0.05, ** P < 0.01 and *** P < 0.00, compared to the DMSO group. # P < 0.05, ## P < 0.01, ### P < 0.001 when compared to Control group.

FIGURE 4

Immunofluorescence detection the impact of ISO on the EMT process in gastric cancer. (A) Immunofluorescence detection of E-cadherin, N-cadherin, Vimentin and Snai1 levels in HGC27 cells and AGS cells after two h pretreatment with 10 μM ISO and TGF-β induction for 24 h. (B) Quantitative assessment of the intensity of fluorescence. Results are expressed as means ± SEM, n = 3.** P < 0.01 and *** P < 0.001, when compared to DMSO group.## P < 0.01, ### P < 0.001 when compared to Control group.

FIGURE 5

Western blotting assesses the impact of ISO on the proteins involved in the TGF-β-Smad pathway. (A) Pretreatment with different concentrations of ISO (0, 10, 20, 30, 40 μM) for 2 h, followed by TGF-β induction for 24 h, and Western blotting to detect the levels of p-Smad2, Smad2, and Smad4in HGC27 cells and AGS cells. (B) The HGC27 and statistical data pertain to the relative protein expression in AGS cells. Results are expressed as means ± SEM, n = 3. ** P < 0.01, *** P < 0.001and ns, not significant (P > 0.05) when compared to the DMSO group. # P < 0.05, ## P < 0.01, ### P < 0.001 when compared to Control group.

FIGURE 6

Immunofluorescence was used to evaluate the influence of ISO on the proteins relevant to the TGF-β-Smad pathway. (A) Immunofluorescence detection of p-Smad2, Smad2, and Smad4 levels in HGC27 and AGS cells after two h pretreatment with 10 μM ISO for two h and TGF-β induction for 24 h. (B) Immunofluorescence was used to detect the impact of ISO on the proteins involved in the TGF-β-Smad pathway. Results are expressed as means ± SEM, n = 3. ** P < 0.01 and *** P < 0.001, compared to the DMSO group. # P < 0.05, ## P < 0.01 and ### P < 0.001 when compared to Control group.

FIGURE 7

Following siTGFBR1, the impact of ISO on the TGF-β-Smad pathway and the migration and invasion of gastric cancer cells was assessed. (A) Western blotting to detect the knockout efficiency of TGFBR1 by three siRNAs. Statistical data on the relative protein expression of TGFBR1 after siRNA treatment. (B, C) Western blotting was performed to detect the presence of P-Smad2 and Smad2 proteins following treatment with siTGFBR1 and ISO 10 μM. The symbol “+” indicates treatment, whereas the symbol “-“ indicates no treatment. Statistical data on the expression of P-Smad2 and Smad2 proteins following various treatments. (D, E) The number of transwell migration and invasion cells following TGF-induction under various treatment conditions. The statistics show the number of HGC27 and AGS cells that invade and migrate within 24 h. (F, G) The effect of ISO on the migratory ability of HGC27 and AGS cells 10 h and 24 h after TGF-β stimulation was examined using a wound healing assay under different treatment settings. The statistical data are shown as cells’ migration area coverage rate relative to the initial time point (0 h). Results are expressed as means ± SEM, n = 3. *** P < 0.001and ns, not significant (P > 0.05) when compared to DMSO group. ### P < 0.001 when compared to the Control group.

FIGURE 8

ISO inhibited the growth and movement of triple-negative breast cancer cells by targeting TGFBR1 to regulate the TGF-β-Smad signaling pathways.