Gracillin shows potent efficacy against colorectal cancer through inhibiting the STAT3 pathway

Colorectal cancer (CRC) accounts for about 10% of all annually diagnosed cancers and cancer‐related deaths worldwide. STAT3 plays a vital role in the occurrence and development of tumours. Gracillin has shown a significant antitumour activity in tumours, but its mechanism remains unknown. The human CRC cell lines HCT116, RKO, and SW480 and immunodeficient mice were used as models to study the effects of gracillin on cell proliferation, migration and apoptosis. These were evaluated by cell viability, colony formation, wound‐healing migration and cell apoptosis assays. Luciferase reporter assay, and immunostaining and western blot analyses were used to explore the specific mechanism through which gracillin exerts its effects. Gracillin significantly reduces viability and migration and stimulates apoptosis in human CRC cells. It also significantly inhibits tumour growth with no apparent physiological toxicity in animal model experiments. Moreover, gracillin is found to inhibit STAT3 phosphorylation and STAT3 target gene products. In addition, gracillin inhibits IL6‐induced nuclear translocation of P‐STAT3. Gracillin shows potent efficacy against CRC by inhibiting the STAT3 pathway. It should be further explored as a unique STAT3 inhibitor for the treatment of CRC.


| INTRODUC TI ON
Colorectal cancer (CRC) accounts for about 10% of all annually diagnosed cancers and cancer-related deaths worldwide. 1 It is the second most common cancer diagnosed in women and the third most common in men. 2 In the United States, increased screening and the removal of precancerous adenomas resulted in a reduced incidence of CRC. The incidence rate, however, is still growing in several European and Asian countries because of the prevalence of risk factors, such as unhealthy diet, smoking and obesity. 3 Hence, exploration and application of new biomarkers for diagnosis, prognosis and treatment are still needed to improve tumour management and patients' survival.
Signal transducer and activator of transcription 3 (STAT3) plays important roles in tumours through its effects on cell proliferation, apoptosis, survival, angiogenesis, metastasis and immunoregulation. 4,5 Mounting evidence shows that constitutively activated STAT3 contributes to tumour development and progression in most cancers, including breast, prostate, gastric, pancreas, colorectum, cervix, ovary, lung, melanoma and blood cancers. [6][7][8][9][10] Stimulated by cytokines and growth factors, STAT3 is phosphorylated at tyrosine residue 705 (Tyr705), leading to its dimerization followed by nuclear entry, where it binds to specific elements in the genomic DNA and activates gene transcription. 4,11,12 Therefore, STAT3 is an attractive target for the development of novel antitumour drugs. Various STAT3 inhibitors have been identified in the past 15 years. 11,13 However, few have moved into clinical trials, and none has thus far been approved for clinical use.  15 It has been reported that saponins have antitumour, antimicrobial, antioxidant, pro-apoptotic and anti-inflammatory properties. 16 Recently, gracillin was shown to have significant antitumour activity in many cancers. [17][18][19] It is reported that gracillin induces apoptosis in HL60 human leukaemic cells via oxidative stress and cell-cycle arrest at the G1 stage. 17 Min et al showed that complex II mediates gracillin interference with the mitochondrial function and results in decreased mitochondrial membrane potential, oxidative phosphorylation and ATP synthesis, and increased mitochondrial reactive oxygen species (ROS) production and apoptotic death in cancer cells. 19 However, its antitumour effect and mechanism of action have not been fully characterized. In this study, cell and animal models were used to analyse the antitumour activity of gracillin in CRC and to elucidate its molecular mechanisms of action.
We found that gracillin can inhibit the STAT3 signalling pathway in CRC cells.

| MTT cytotoxicity assay
Viability of the human CRC cells was evaluated by the MTT assay.
Cells (3000-5000 cells/well) were seeded in 96-well plates and cultured overnight. After being treated with appropriate drugs for 48 hours, cells were incubated with 20 μL MTT (5 mg/mL) for 4 hours. The medium was then replaced with 150 μL DMSO in each well. The plate was agitated on a plate shaker for 5 minutes and then scanned for spectrophotometric absorbance at 490 nm. The half-maximal inhibitory concentration (IC 50 ) value was calculated using the GraphPad Prism 7 software.

| Colony formation assay
Human CRC cells were seeded for colony formation in 6-well plates at a density of 1000 cells per well and incubated overnight at 37°C in a 5% CO 2 atmosphere. Different concentrations of gracillin in DMSO (0, 2.5, 5.0 or 10.0 µmol/L) were added. After 7-10 days (medium exchanged every two days), colonies were washed with PBS and then fixed with 4% paraformaldehyde.
After carefully washing three times with PBS, the colonies were stained with crystal violet for 10 minutes and observed under a light microscope.

| Wound-healing assay
Cell migration was evaluated by the wound-healing assay. RKO cells were plated in 6-well plates at an appropriate seeding density of 1 × 10 6 cells/well. When the cells grew to 80%-90% confluence, the cell monolayers were mechanically scarred with a sterile 200 μL pipette tip along the centre of the well to generate a clean, straight wound area. Different concentrations of gracillin (1.0, 2.5 or 5.0 µmol/L) and 2% FBS were added to the culture media, and the plated were incubated for 24 hours. The scars were observed and measured at 0 hour and 24 hours by light microscopy.

| Cell apoptosis assay
Cell apoptosis assay was performed as previously described. 7 Apoptosis was evaluated using an apoptosis detection kit (BD Biosciences). RKO and HCT116 cells were seeded into 6-well plates

| Western blot analysis
Human CRC cells were seeded in 6-well plates (500 000/well) and incubated overnight at 37°C in an atmosphere of 5% CO 2 . Different concentrations of gracillin were added into the wells. Total protein was extracted and separated by SDS-PAGE. Following electrophoresis, the proteins in the gel were transferred to polyvinylidene difluoride membranes. After blocking for 1 hour with 5% non-fat milk, the membranes were incubated overnight at 4°C with specific primary antibodies. The next day, the blots were incubated with relevant secondary antibodies. Visualization of the target proteins was enhanced with Electrochemiluminescence (ECL) substrate reagents.

| STAT3 luciferase report assay
The STAT3 luciferase reporter plasmid (pGLSTAT3-Luc) was used to detect STAT3 activation, as was previously described. 8 Briefly, HCT116 cells were seeded in 24-well plates and incubated for 24 hours before transfection. The cells were co-transfected with pGLSTAT3-Luc and pRL-TK (a plasmid encoding Renilla luciferase) using Lipofectamine 3000 (Invitrogen) for 6 hours. Finally, the cells were treated with different concentrations of gracillin for 12 hours.
Luciferase activity was assessed by SpectraMax ID3 (Molecular Devices). The inhibition of STAT3 activity by gracillin was calculated as the ratio between the value of firefly and Renilla luciferase activity. Each experiment was carried out in triplicate in three independent experiments.

| Immunofluorescent staining
Briefly, HCT116 cells were seeded onto glass coverslips. When they reached 70% confluence, they were washed once in PBS and then fixed with 4% paraformaldehyde for 15 minutes at room temperature. Coverslips were washed three times in excess of PBS, permeabilized with −20°C 100% methanol for 30 minutes and then blocked in 1% bovine serum albumin (BSA) for 30 minutes. The cell preparations were incubated overnight in the dark at 4°C with a specific primary anti-P-STAT3 antibody (1:100 in 3% BSA). On the following day, the cell preparations were incubated at room temperature for 1 hour with PE-labelled goat anti-rabbit IgG antibody (1:200). After that, the nuclei were stained with DAPI. The images were acquired by confocal microscopy (Leica) using a × 63 oil lens.

| Cytoplasmic and nuclear protein extraction
The cytoplasmic and nuclear proteins of HCT116 cells were isolated by NE-PER Nuclear & Cytoplasmic extraction kit (Thermo Fisher Scientific). Different concentrations of gracillin were added to the plates. Cells were stimulated with IL6 for 20 minutes before being harvested according to the manufacturer's protocol. Protein expression in the cytoplasmic and nuclear fractions was detected using immunoblot analysis.

| Animal experiments
All animal experiments were conducted following the institu-

| Haematoxylin and eosin (H&E) staining
Tissue slices from the hearts, lungs, livers and kidneys were fixed in 4% paraformaldehyde and embedded in paraffin. They were then sectioned (5μm), deparaffinized, rehydrated and then stained with eosin and haematoxylin. Images were acquired with a light microscope.

| Statistical analysis
Data are expressed as mean ± SD of three independent experiments. The different groups were compared by Student's t test or one-way analysis of variance using the GraphPad Pro 7.0 software (GraphPad). Differences were considered significant when P < .05.

| Gracillin inhibits proliferation and migration of human CRC cells
We used the MTT and colony formation assays to identify gracillin's inhibitory effect on the human CRC cells ( Figure 1A). As shown in Figure 1B Furthermore, we observed a significant inhibitory effect on human CRC cells' colony formation in a dose-dependent manner ( Figure 1C). These results suggest that gracillin has a considerable inhibitory effect on human CRC cells' proliferation in vitro. We also performed a wound-healing assay to investigate the effect of gracillin on the migration ability of human CRC cells. As shown in Figure 1D, the migration rates in the gracillin-treated groups were significantly lower than the migration rate in the control group.
These results strongly suggest that gracillin inhibits the proliferation and migration of human CRC cells.

| Gracillin induces apoptosis in human CRC cells
To assess the apoptosis-induction ability of gracillin, RKO and

| Gracillin inhibits the STAT3 signalling pathway in human CRC cells
The docking result ( Figure 3A)  we detected STAT3 constitutive activation in HCT116 and RKO cells by using the luciferase reporter assay. We found that gracillin significantly inhibits P-STAT3 activation in a dose-dependent manner ( Figure 3B and Figure S2A). Western blot assay was employed to determine the expression of the STAT3 signalling pathway-associated proteins. In agreement with the data obtained using the luciferase reporter assay, the P-STAT3 level and the levels of STAT3-related proteins (Mcl-1, VEGF and Survivin) were down-regulated by gracillin in human CRC cells in a dose-dependent manner ( Figure 3C).
To investigate the selective inhibitory effect of gracillin, we evaluated the effect of gracillin on the activation of P-STAT3, P-STAT1, P-STAT4 and P-STAT5 in CRC cells. As shown in Figure 3D and Figure S3, gracillin did not reduce the protein levels of phosphorylated STAT1, STAT4 and STAT5. HCT116 cells were transfected with three different sequences of si-STAT3 (si#1, si#2 and si#3). Among them, si#3 showed the highest STAT3 inhibitory activity ( Figure 3E).
It was therefore selected for subsequent transient transfection assays. To confirm the ability of gracillin to inhibit cell proliferation and to test whether this effect was STAT3-dependent, we examined the inhibitory effect of gracillin on the proliferation of HCT116 cells, which were depleted or not of STAT3 ( Figure 3F). Gracillin did not affect the siSTAT3-transfected cells. In sharp contrast, gracillin was able to inhibit cell proliferation in HCT116 cells transfected with a control siRNA (and therefore expressing STAT3). In brief, these results suggest that gracillin exerts its inhibitory effect through the STAT3 signalling pathway.

| Gracillin inhibits nuclear translocation of STAT3 in human CRC cells
We first investigated whether the STAT3 activation stimulating by IL6 could be disturbed through the treatment of gracillin. As shown in Figure 4A, gracillin can inhibit IL6-induced STAT3 activation in three CRC cell lines. We next determined whether gracillin treatment affected STAT3 nuclear translocation in HCT116 cells. As expected, exposure of HCT116 cells to 25 ng/mL IL6 for 30 minutes resulted in increased translocation of P-STAT3 (green fluorescence) from the cytoplasm to the nucleus (blue fluorescence, DAPI stained). Gracillin was found to inhibit this translocation ( Figure 4B,C). These results further confirm the inhibitory effect of gracillin on IL6-induced nuclear translocation of P-STAT3. These results strongly suggest that the antitumour effect of gracillin is mediated by blocking the STAT3 signalling pathway.

| Gracillin inhibits CRC tumour growth in a xenograft model
To have increased and that of Bcl-2 has decreased in tumours of the gracillin-treated mice. Moreover, gracillin treatment caused no observable toxicity to the heart, liver, kidneys or lungs as compared with that in the vehicle group ( Figure 5F), thus demonstrating its excellent safety profile.

| D ISCUSS I ON
The recently discovered cancer-promoting functions of STAT3 further emphasize the importance of targeting STAT3 activities related to the mitochondria, epigenetic regulation, cancer stem cells, premetastatic niches and more. 22,23 The STAT3-Fam3a axis has been reported to promote the progression of the muscle stem cell myogenic lineage by enhancing the mitochondrial respiration. 24 STAT3 can mediate the epigenetic silencing of tumour suppressor genes (TSG). Gambi and his colleagues confirmed that oncogene-driven constitutive STAT3 acetylation was responsible for the silencing of TSG.
Furthermore, they showed that the Sin3a transcriptional repressor protein is an obligatory partner of STAT3 in promoting genes repression, revealing the mechanisms involved in STAT3-mediated transcriptional regulation. 25 Agarwal et al found that G-CSF promotes tumorigenicity and metastasis of neuroblastoma through STAT3dependent cancer stem cell activation. 26 Liu et al described a novel function of STAT3 in regulating the pH by interacting with the vacuolar H+-ATPase on lysosomes. This discovery proposes a new anticancer strategy by limiting this function. 27 It was also shown that the inhibition of the STAT3 pathway by a small-molecule inhibitor or siRNA significantly enhanced the stimulator of interferon genes (STING) signalling, which was induced by the STING agonist c-diAM (PS)2. Important to note that these STAT3 inhibitors did not induce the STING signalling by themselves. 28 All these recent discoveries Regrettably, thus far, no STAT3 inhibitor has been approved for cancer therapy. To develop a more successful treatment for cancer, we have done much work on finding an effective STAT3 inhibitor. 29,30 Agents derived from natural sources have attracted much attention because of their safety, efficacy and immediate availability. 4 They are the best sources for drugs and can lead to novel drug discovery. 31 Some natural products and derivatives have been found to possess STAT3 inhibitory effect, such as rhein, 7,8 alantolactone 32 and others. Here, we identified a new STAT3 inhibitor. In this study, we used CRC cells and animal models to examine the antitumour effects of gracillin and its downstream regulatory mechanism. The results show that gracillin significantly inhibits the proliferation and migration ( Figure 1) and induces apoptosis in CRC cells (Figure 2).

F I G U R E 5
Gracillin inhibits CRC tumour growth in a xenograft model. A, BALB/c nude mice were administered gracillin intraperitoneally (1 or 2 mg/kg) every other day. Tumour volume (V) was calculated based on its length (A) and width (B), according to the following formula: Tumour weight (n = 6). C, Representative images of the solid tumour in the control and gracillin-treated groups. D, Mice total bodyweight. E, Proteins extracted from tumour tissues were evaluated by the Western blot assay. F, Tissue samples derived from the hearts, livers, kidneys and lungs of mice from the three groups and stained with H&E. All images were captured using light microscopy. Data are presented as means of three experiments, and error bars represent SD (*P < .05, ***P < .001, ****P < .0001) In animal experiments, we found that gracillin could significantly inhibit tumour growth without any apparent physiological toxicity ( Figure 5).
Moreover, gracillin was found to inhibit STAT3 phosphorylation in CRC cells, as evaluated by the STAT3-dependent dual-luciferase reporter system. Gracillin inhibits not only STAT3 but also regulates the expression of STAT3 target gene products, including Bcl-2, Mcl-1, Survivin and VEGF ( Figure 3). Moreover, gracillin did not affect siSTAT3 cells, indicating that the ability of gracillin to inhibit cell proliferation depends on STAT3. In addition, we found, using immunofluorescence staining, that gracillin inhibits the IL6-induced nuclear translocation of P-STAT3 ( Figure 4). Our data demonstrate that gracillin shows potent efficacy against CRC by inhibiting the STAT3 pathway. Moreover, we have shown that gracillin has few side effects on the mice at the therapeutic concentration used in this study. Reciprocal cross talk between the STAT3 and EGFR pathways is a key molecular mechanism leading to resistance in cancer cells. 5,32,33 We plan to investigate the possible combination of gracillin with other clinical cancer drugs to improve its antitumour potency and verify its effect on CRC.

| CON CLUS IONS
Our findings provide a convincing proof that gracillin possesses a potent anti-CRC activity by inhibiting the STAT3 pathway. Gracillin should be further explored as a unique STAT3 inhibitor for the treatment of CRC.

ACK N OWLED G EM ENTS
This work was financially supported by the Natural Science

CO N FLI C T O F I NTE R E S T
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.