Inhibition of 6-P on proliferation of pancreatic cancer cells
The chemical structure of 6-P and its derivatives 6-G and 6-S was displayed in Fig. 1. To investigate the effect of 6-P on proliferation of pancreatic cancer, pancreatic cancer cell lines MIA PaCa-2 and SW1990 were treated with different concentrations of 6-P for 48 h (0, 20, 40, 80 μM) or the same concentration of 6-P for different time frames (0, 24, 48, 72 h). First, CCK-8 assay was performed to evaluate the effect of 6-P on pancreatic cancer cell viability. The results suggested that cell viability significantly decreased with increasing 6-P concentration in MIA PaCa-2 and SW1990 (Fig. 2A, B). In addition, cell colony formation assay indicated the same results that the number of cell colonies was obviously inhibited by culturing with different concentrations of 6-P and the 80 μM concentration showed a highest inhibited effect on cell colonies (Fig. 2C, D). To further evaluate the cytotoxicity and anti-proliferation of 6-P, we used a phase contrast microscope to observe and capture the morphological changes of pancreatic cancer MIA PaCa-2 and SW1990 treated with 6-P. The results uncovered that 6-P caused adherent pancreatic cells to become round, shrink, wiredrawing and separate from the bottom of the culture plates, indicating a significant apoptosis state, especially in concentration of 80 μM or treating with 72 h (Fig. 2E, F).
Inhibition of 6-P on migration and invasion of pancreatic cancer cells
To further validate whether 6-P had the inhibitory effect on migration and invasion of MIA PaCa-2 and SW1990, transwell assay and wound healing assay were performed to evaluate to migrate and invasive ability. The migration and invasion significantly decreased in the concentration of 40 and 80 μM compared with 0 μM, revealing that 6-P could also partly suppress the metastasis of pancreatic cancer cells (Fig. 3A–G). In addition, we tested the epithelial-mesenchymal transition (EMT) using western blot assay to detect the protein levels of E-cadherin, N-cadherin and Vimentin. The results demonstrated that the expression of E-cadherin gradually rose with the increasing concentration of 6-P. Conversely, the expression of N-cadherin and Vimentin gradually reduced with the increasing concentration of 6-P (Fig. 3H, I, Additional File 1). The results suggested an inhibited function of 6-P on EMT of pancreatic cancer cells.
6-P interacts with EGFR to exert suppression functions on proliferation and metastasis of pancreatic cancer cells
In order to explore the potential binding target of 6-P, bioinformatics methods were performed to predict the underlying protein site. First, we downloaded the 3-dimensional structure file of the compound 6-P from PubChem Compound Search database (https://pubchem.ncbi.nlm.nih.gov/). Then, we transeferred the data to SwissTargerPrediction software for predictive analysis and obtained the target protein of the compound 6-P. Subsequently, KEGG pathway enrichment analysis was performed to figure out the involved signaling pathway of these underlying target protein by DAVID database (https://david.ncifcrf.gov/). Finally, we set the standard for judging significant enrichment of pathways with a P value less than 0.05, and the top 12 signal pathways with enrichment number were visualized using R language with clusterProfilerKEGG package. The results of KEGG pathway enrichment analysis indicated that 6-P was significantly correlated with PI3K-AKT signaling pathway and epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor resistance (Fig. 4A). Interestingly, molecular docking analysis with 6-P on the 3-dimensional structure of EGFR suggested that there was an interaction between 6-P and EGFR (Fig. 4B). Combined with KEGG results, we hypothesized that 6-P might occupy key sites of EGFR molecular structure to exert biological regulation functions. Subsequently, western blot assay was performed to detect EGFR expression of pancreatic cancer cells treated with 6-P. The results confirmed that 6-P could decrease expression of EGFR and the inhibition of 6-P on EGFR expression could be partly rescued with supplementary EGFR (Fig. 4C, D, Additional File 1). In addition, we further evaluated the proliferation and metastasis of pancreatic cancer cells treated with 6-P after adding EGFR plasmid to upregulate EGFR expression. The results suggested that the cell proliferation, migration and invasion could also be partly rescued with supplementary EGFR (Fig. 4E–G). And western blot results revealed upregulation of EGFR could reverse 6-P mediated-inhibition of EMT process (Fig. 4H, I, Additional File 1).
6-P-mediated ubiquitination degradation of EGFR leads to inactivate PI3K/AKT signaling pathway
To further investigate the underlying molecular mechanism of 6-P on EGFR, we firstly evaluated the mRNA expression levels of EGFR in pancreatic cancer cells treated with 6-P. The results indicated that there was no significant difference in MIA PaCa-2 or SW1990 cells (Fig. 5A). However, our data suggested 6-P downregulated the protein expression of EGFR. To figure out the reason why 6-P changed the protein expression, not the mRNA expression, 293 T cells were treated with CHX and 6-P for 1 h, 2 h to evaluate the protein stability. The results demonstrated that the de novo synthesis of EGFR in 6-P treatment group reduced more rapidly compared to 6-P non-treatment group, suggesting 6-P decreased the protein stability of EGFR (Fig. 5B, C, Additional File 1). Subsequently, we suspected that downregulated EGFR protein expression was the result of 6-P-involved proteasome-dependent degradation mechanism. To validate the suspicion, a proteasome inhibitor, MG-132 (5 μM), was used to evaluate whether 6-P was involved in EGFR degradation by proteasome-dependent route. The results confirmed that MG132 inhibited EGFR degradation (Fig. 5D, Additional File 1). Then HA-labeled ubiquitin and Flag-labeled EGFR plasmids were co-transferred into the 293 T cells and 6-P was added to treat the cells. Co-immunoprecipitation and SDS-gel electrophoresis were performed to evaluate the levels of EGFR ubiquitination. Interestingly, the treatment of 6-P significantly enhanced EGFR ubiquitination, indicating 6-P promoted proteasome-dependent degradation of EGFR via ubiquitin modification pathway (Fig. 5E, Additional File 1). Subsequently, we detected the PI3K/AKT signaling pathway which was one of the downstream pathways of EGFR to further validate the results of KEGG pathway enrichment analysis. The results suggested that EGFR could activate PI3K/AKT while the activity of PI3K/AKT signaling could be reversed by treating with 6-P, indicating 6-P negatively activate PI3K/AKT signaling pathway (Fig. 5F, G, Additional File 1). Immunofluorescence staining was used to analysis the expression and localization of p-AKT. MIA PaCa-2 and SW1990 cells treated with 6-Paradol showed obvious decrease of p-AKT in comparison with the NC groups (Fig. 5H).
EGFR inhibitor enhanced 6-P mediated-inhibition effect on PI3K/AKT signaling activity
To further confirm that 6-P mediated-EGFR degradation was involved in inhibitory effect on PI3K/AKT signaling, we respectively used EGFR overexpression plasmid and (or) EGFR inhibitor Erlotinib (2 nM) to regulate EGFR expression. The results verified that Erlotinib promoted 6-P mediated degradation of EGFR and inactivity of PI3K/AKT signaling, however, upregulation of EGFR expression could rescue the activity of PI3K/AKT signaling and the expression of EGFR (Fig. 6A, B, Additional File 1). Subsequently, gain- or lose-functional experiments were performed to evaluate the interaction between 6-P and EGFR on proliferation and metastasis of pancreatic cancer cells. The results revealed Erlotinib and 6-P had synergistic effects to exert inhibition on proliferation and metastasis of pancreatic cancer cells, which could be rescued by upregulation of EGFR expression (Fig. 6C–F). Meanwhile, Erlotinib combined with 6-P significantly inhibited EMT process and overexpressed EGFR removed the inhibitory effect on EMT process (Fig. 6G, H, Additional File 1).
6-P significantly suppressed tumor growth in vivo
To explore whether 6-P suppressed tumor growth in vivo, we constructed a subcutaneous tumorigenesis model in nude mice which were orally administered with 6-P (10 mg/kg/d). The results suggested the size of tumor was obviously smaller in 6-P treatment group compared with control groups, indicating a inhibitory function of 6-P on tumor growth (Fig. 7A). Interestingly, at 2 weeks of implantation, the tumor volume could be measured, thus we conducted a experiment for 6-P treatment on tumor-bearing mice. From 1 to 3 weeks after 6-P treatment, the tumor volumes was smaller in 6-P treatment groups and the stripped tumor weight was also lighter in 6-P treatment groups (Fig. 7B, C). Subsequently, IHC analysis was performed to detect relative expression of Ki67, PCNA, N-cadherin, E-cadherin, Vimentin, EGFR, phosphorylated-AKT and phosphorylated-PI3K. The results demonstrated E-cadherin expression was upregulated in 6-P treatment group and the rest of indexes were all downregulated, which were consistent with our previous results in vitro (Fig. 7D, E).
Overall, 6-P might functioned as an anti-tumor drug to inhibit pancreatic cancer cell proliferation and migration in vivo and in vitro by targeting EGFR, inducing EGFR degradation through decreasing the protein stability of EGFR and enhancing the ubiquitin-mediated proteasome-dependent degradation. Eventually, 6-P decreased EGFR expression and inhibited PI3K/AKT signaling to suppress tumor progress (Fig. 8).