Genipin can inhibit the phosphorylation of STAT-3 (Tyr) and decrease the expression of STAT-3 target gene in HCC cells
As a transcription factor, STAT-3 can regulate cell proliferation and angiogenesis through modulating its down-stream target genes such as Bcl-2, VEGF and SOCS-3. To screen novel STAT-3 inhibitory agents, the STAT-3 luciferase reporter system was applied to screen target agents from our internal chemicals library(Fig 1a. up panel). We screened 537 compounds and eventually identified genipin as a novel natural agent for inhibiting STAT-3 signal pathway. Genipin exhibited significant STAT-3 suppressive activity in MHCC97L and HepG2 cells (Fig 1a. down panel). While phosphorylated Y705 has been widely acknowledged to be essential for STAT-3’s transcriptional activity; the function of phosphorylated S727 still controversial, as this modification has been reported to have both up- and downregulatory effects on STAT3’s transcriptional activity. Thus, for validating the STAT-3 suppressive effects, p-STAT-3 (Y705) and p-STAT-3 (S727) expression were examined by western blot after genipin treatment. Our results showed that genipin (20 μM) remarkably inhibited the activation of pSTAT-3 (Y705) but failed to affect the protein expression of STAT-3 and p-STAT-3 (S727) (Fig 1b). Cytoplasmic STAT-3 exported to the nucleus is a critical step for regulating its down-stream genes expression. Both immunofluorescent staining and western blotting results confirmed that genipin inhibited nuclear translocation of STAT-3 (Fig 1c). Furthermore, STAT-3 DNA-binding ability was inhibited by genipin treatment according to the electrophoretic mobility shift assay results (Fig 1d). Protein tyrosine phosphatases (PTPases) are a group of enzymes that are able to eliminate the DNA binding of STAT-3 , thus, we intended to explore whether genipin could inhibit STAT-3 by PTPases in HCC. PTEN, SHP1 and SHP2 are key regulatory PTPases in STAT-3 signal transduction pathways , however, the protein expression of these PTPases has no obvious changes after genipin treatment(supplementary Fig 1a). In addition, to further confirm whether genipin inhibits STAT-3 specifically, we also evaluated the activity of STAT-5, STAT-1, STAT-2, mTOR and MAPK signal pathways by western blotting. Our results indicated that genipin failed to affect the phosphorylation of STAT-5, STAT-1 and STAT-2 as well as the expression of the related proteins in mTOR and MAPK signal pathways (supplementary Fig 1b,c). Up to present, we could conclude that genipin suppressed STAT-3 phosphorylation and nuclear translocation as well as inhibited its DNA-binding ability. STAT-3 dimers exported to the nucleus can activate the promoter of STAT-3 target genes and up-regulates the protein expression of these tumor-related genes such as Survivin, Bcl-2, MMPs, SOCS3 and VEGF . Western blot assay results further confirmed that genipin treatment decreased the expression of STAT-3 target genes in HCC cells (Fig 1e). In addition, chromatin immunoprecipitation assay indicated that genipin inhibited the binding affinity of STAT-3 with Bcl-2, SOCS3 and VEGF(Fig 1f). In summary, above data revealed that genipin could inhibit STAT-3 phosphorylation(Y705) and suppress its target genes expression in liver cancer .
Genipin binds to the SH2 domain in STAT-3
Next, we explored whether genipin could directly interact with STAT-3 by in silico assay. As shown in Fig 3a, genipin was docked nicely into the SH2 domain of STAT-3(PDB Id: 1GB1). PHE716, LYS626, GLN635, SER636, GLU638, ARG609, LYS591, VAL637,PRO639 and TRP623 of STAT-3 formed strong interactions with genipin. To further confirm whether genipin can directly bind to STAT-3-SH2 domain, GST-tagged STAT-3-SH2 domain (42kD) was purified from E.coli (Fig 2b). Then, surface plasmon resonance (SPR) assay was performed to determine the binding affinity between genipin and STAT-3-SH2. SPR analysis results indicated that STAT-3-SH2 bound to genipin with a relative low dissociation constant (KD) value (KD = 2.3 μM) (Fig 2c). The activation of STAT-3 required phosphorylation on tyrosine and forming a dimer via phosphotyrosine/SH2 domain interaction. Our results showed that genipin distinctly suppressed the interaction between purified STAT-3-SH2 and STAT-3 by GST pull-down assay (Fig 2d). Next, Flag-tagged and HA-tagged STAT-3 vectors were constructed and transfected into MHCC97L cells for validating whether genipin inhibits the dimerization of STAT-3. Our results suggested that HA-STAT-3 co-immunoprecipitated with Flag-STAT-3 in MHCC97L cells and genipin blocked the interplay dose-dependently (Fig 2e ). In addition, genipin also inhibited STAT-1: STAT-3 heterodimers formation (Supplementary Fig 2a).These results indicated that genipin might directly bind to the STAT-3-SH2 domain, and inhibit the dimerization of STAT-3 or STAT-1: STAT-3. EGFR can also bind to STAT-3-SH2 domain and activate STAT-3. GST pull-down results indicated that purified STAT-3-SH2 interplayed with EGFR and genipin exposure (20 μM) suppressed the complex formation (Fig 2f). Then, we further explored whether genipin could induce the dissociation of EGFR-STAT-3 complex. We found that treatment with EGF can increase the binding ability of STAT-3 to EGFR in HCC cells while treatment with genipin significantly suppressed these interactions (Fig 2g). These results demonstrated that genipin directly bound with STAT-3-SH2 domain.
Genipin inhibits HCC cells proliferation and angiogenesis in vitro
Above results clearly demonstrated that genipin can inhibit STAT-3 activation. To evaluate the anticancer effect of genipin, we examined its potential suppressive effect on HCC cells proliferation by MTS assay. To our surprise, genipin remarkably inhibited HepG2 and MHCC97L cells viability dose-dependently. But, no significant inhibition effect on normal liver cells LO2 was observed in our study(Fig 3a). Western blotting results showed that the phospho-STAT-3 (Tyr-705) level was decreased after genipin treatment in HCC cells but kept unchanged in normal liver cells (LO2) (Supplementary Fig 3a). Next, we further explored whether STAT-3 inhibition is related with impaired cancer cells proliferation. STAT-3 vectors were transfected into MHCC97L cells, overexpression of STAT-3 obviously reversed genipin-mediated tumor growth inhibition and STAT-3 target genes suppression (Fig 3b). Furthermore, genipin (10 μM) could induce apoptotic cell death in HCC cells as indicated by western blotting and Annexin V/7AAD assay (Fig 3c). Then, we further determined whether genipin inhibited colony formation in MHCC97L and HepG2 cells. As shown in Fig 3d, genipin suppressed colony formation in MHCC97L and HepG2 cells in a dose-dependent manner. Accumulating evidence suggests that STAT-3 plays a critical role in angiogenesis under both pathological and physiological conditions in addition to cell proliferation and survival . It has been widely recognized that angiogenesis plays a pivotal role in cancer development as malignant tumor need sufficient blood provision if the tumor is to grow beyond a few cubic millimeters in volume . One of the most widely applied in vitro experiments to model the reorganization stage of angiogenesis is the tube construction assay. In our study, genipin failed to affect HUVECs viability (Supplementary Fig 4a) or capillary-like structure construction (Supplementary Fig 4b) in culture medium. However, less well-formed capillary-like structures were built for HUVECs in the MHCC97L-conditioned medium after genipin (10, 20 μM) treatment (Fig 3e). In conclusion, above results revealed that genipin might inhibit HCC proliferation and angiogenesis.
Genipin suppresses HCC cells invasion and reverses the EMT process
The spread and metastasis of cancer cells may occur via invading the surrounding tissues and intravasating into blood or lymphatic circulation through the endothelium . Herein, cell invasion ability was analysed by Transwell assay using MHCC97L and HepG2 cells. Our results showed that genipin (10 μM) inhibited HCC cells invasion dose-dependently (Fig 4a). Cancer invasion requires extracellular matrix (ECM) and basement membrane degradation. Thus, fluorescent-gelatin degradation assay was applied to examine whether genipin suppresses ECM degradation by HCC cells. Our results suggested that MHCC97L cells significantly promoted ECM degradation in control group, while genipin (20, 50 μM) treatment reversed ECM degradation by HCC cells (Fig 4b). Three-dimensional (3D) culture is an artificially created environment which provides functional and structural aspects of cancer development. In this study, our 3D culture results showed that genipin (20, 50 μM) remarkably suppressed HCC cells invasion via the surrounding Matrigel (Fig 4c). Epithelial–mesenchymal transition (EMT) is a key process in cancer metastasis by which epithelial cells lose their polarity and cell-cell adhesion, and obtain invasive and migratory properties. During the EMT process, the expression of several epithelial and mesenchymal biomarkers significantly changed. Interestingly, genipin treatment notably decreased the expression of vimentin, fibronectin and N-cadherin while increased the expression of E-cadherin in HCC cells (Fig 4d).
Genipin suppresses cancer progression in HCC xenograft tumor models
For further exploring whether genipin suppresses HCC progression in vivo, we established orthotopic mice xenograft models with MHCC97L cells. Then, DMSO (0.1%) (vehicle) or genipin were administrated daily by intraperitoneal injection. Fig 5a showed that genipin treatment (25, 50 mg/kg) notably decreased tumor weight, which indicated that genipin could inhibit HCC progression in vivo. In addition, genipin treatment also significantly decreased the number of metastasis nodules in lung (Fig 5b, c). Further studies demonstrated that genipin suppressed the protein expression of phospho-STAT-3 (Y705) and inhibited the expressions of STAT-3 target genes in primary liver tumor tissues (Fig 5d, e). Furthermore, decreased vascular density were detected by CD31 stanning in HCC tissues in genipin-treated mice (Fig 5e). The survival rate of mice was analysed for evaluating whether the metastasis inhibition effects of genipin could improve the overall survival rate. Our results showed that genipin significantly improve the survival rate of tumor-bearing mice. No mice was survival in vehicle group (n=8) on day 40, whereas six mice were survival on day 40 and day 50 after genipin (50 mg/kg) treatment (Fig 5f). In conclusion, above results suggested that genipin could inhibit HCC metastasis and improve the overall survival rate in orthotopic transplantation HCC mice models.
Anti-HCC effect of genipin in a patient-derived HCC xenograft mice model
Patient-derived xenograft (PDX) mice model may keep more similarities to the human cancers compared to normal cell-lines xenograft mice model. Previous studies have shown that PDX mice model may be useful for screening novel anti-cancer agents . Herein, seven human surgical HCC tissue samples along with the peripheral normal liver tissues were collected from primary HCC patients (supplementary table 1). Firstly, we detected the protein expressions of STAT-3 and p-STAT-3 (Y705) in these surgical samples. Our results indicated that the expressions of STAT-3 and p-STAT-3 (Y705) were notably reduced in HCC peripheral normal liver tissues compared to tumor tissues (Fig 6a). These results suggested that the activation of STAT-3 is up-regulated in tumor cells derived from HCC patient. After establishing the PDX mice model, we examined the protein expression of STAT-3 and p-STAT-3 (Y705) in tumor-bearing mice. No obvious changes have been found in the expression of p-STAT-3 (Y705) in F0, F1, F2 and F3 passages (Fig 6b). Above results indicated that the activity of STAT-3 has not been changed in patient-derived HCC xenograft mice after serial passages culture. The F3 passages mice were divided into DMSO (0.1%) and genipin (25, 50 mg/kg/day) treatment groups(n=8). After genipin treatment, the HCC growth in mice was significantly suppressed (Fig 6c). The tumor volume in genipin treatment (25, 50 mg/kg/day) group was 597.43 and 401.26 cubic millimeters, respectively. In contrast, the tumor volume in vehicle treatment group was 1452.24 cubic millimeters (Fig 6d). In addition, Tumor weight in liver remarkably decreased after genipin (25, 50 mg/kg/day) treatment (Fig 6e). Interestingly, genipin also decreased the protein levels of p-STAT-3 (Y705) and STAT-3 target genes (Bcl-2, VEGF, Survivin) in mice model (Fig 6f). immunohistochemistry assay further confirmed the decreased expression of p-STAT-3 (Y705) as well as the tumor vascular density (CD 31+) in HCC samples from PDX mice after administration with genipin (25, 50 mg/kg/day) (Fig 6g).
Genipin inhibits the proliferation of other cancer cells
Considering that STAT-3 signaling regulate oncogenic pathway in various tumor cells, we hypothesized that genipin might also inhibit other cancer cells growth. Fig 7a showed that genipin (20, 50 μM) exposure resulted in the growth inhibition of various kinds of cancer cells . In addition, genipin notably suppressed STAT-3 signal pathway in these tumor cells. Fig. 7b showed that the activation of p-STAT-3(Y705) was significantly inhibited by genipin treatment in various non-HCC cancers.
The potential toxicity of genipin on tumor-bearing mice
For evaluating the potential toxicity of genipin in vivo, we further examined the effects of genipin on kidney and liver functions in tumor-bearing mice. No obvious changes on serum creatinine, blood urea nitrogen, aspartate transaminase (AST) and alanine transaminase (ALT) levels between genipin and DMSO group have been detected (p >0.05) (Supplementary Table 2). In addition, body weight changes in mice were detected every 7 days. No significant loss of body weight has been detected after genipin treatment (Supplementary Fig 5a). Furthermore, H&E staining results indicated no obvious histological changes between genipin-treated mice and control mice (Supplementary Fig 5b). In conclusion, these data suggested that genipin exhibit no significant adverse effects on mice at the therapeutic dosage.