α‐Mangostin suppresses the de novo lipogenesis and enhances the chemotherapeutic response to gemcitabine in gallbladder carcinoma cells via targeting the AMPK/SREBP1 cascades

Abstract High rates of de novo lipid synthesis have been discovered in certain kinds of tumours, including gallbladder cancer (GBC). Unlike several other tumours, GBC is highly insensitive to standard adjuvant therapy, which makes its treatment even more challenging. Although several potential targets and signalling pathways underlying GBC chemoresistance have been revealed, the precise mechanisms are still elusive. In this study, we found that α‐Mangostin, as a dietary xanthone, repressed the proliferation and clone formation ability, induced cell cycle arrest and the apoptosis, and suppressed de novo lipogenesis of gallbladder cancer cells. The underlying mechanisms might involve the activation of AMPK and, therefore, the suppression of SREBP1 nuclear translocation to blunt de novo lipogenesis. Furthermore, SREBP1 silencing by siRNA or α‐mangostin enhanced the sensitivity of gemcitabine in gallbladder cancer cells. In vivo studies also displayed that MA or gemcitabine administration to nude mice harbouring NOZ tumours can reduce tumour growth, and moreover, MA administration can significantly potentiate gemcitabine‐induced inhibition of tumour growth. Corroborating in vitro findings, tumours from mice treated with MA or gemcitabine alone showed decreased levels of proliferation with reduced Ki‐67 expression and elevated apoptosis confirmed by TUNEL staining, furthermore, the proliferation inhibition and apoptosis up‐regulation were obviously observed in MA combined with gemcitabine treatment group. Therefore, inhibiting de novo lipogenesis via targeting the AMPK/SREBP1 signalling by MA might provide insights into a potential strategy for sensitizing GBC cells to chemotherapy.


| INTRODUC TI ON
Gallbladder cancer (GBC), the most common malignant biliary tumour, has the seventh highest mortality rate of all gastrointestinal cancers. 1 Although the incidence of gallbladder cancer is very low at approximately 2.5 cases in 1 × 10 5 people, GBC prognosis is very dismal with a 5-year survival rate of 5%. 2 Currently, gemcitabine/cisplatin is the recognized reference regimen for the first-line treatment of patients suffering from advanced biliary tract cancers, including gallbladder cancer. 3 However, patients undergoing the first-line chemotherapy often have a rapidly worsening performance status, and only a small number of patients are suitable for subsequent treatment. 3 Unlike other tumours, GBC is especially resistant to the currently available routine adjuvant therapy, thus making GBC management challenging. 4,5 A few potential targets and signalling pathways underlying GBC chemoresistance have been identified; however, the precise mechanism requires further investigation. [6][7][8] Thus, studies on the identification of agents that enhance the response to chemotherapeutic drugs and further elucidate the molecular basis of drug resistance are urgent and significant.
Metabolic reprogramming plays an essential role in tumourigenesis and malignant aggravation of cancer. 9 Elevated aerobic glycolysis and fatty acid synthesis are the most significant aspects of cancer cell metabolism. Newly synthesized lipids are utilized to form the cell membrane during cell proliferation and to supply energy for tumour development. Thus, high rates of de novo lipid synthesis have been detected in numerous types of tumour cells, such as hepatocellular carcinoma, 10 breast cancer, 11 pancreatic cancer, 12 gallbladder cancer, etc. 13 Sterol regulatory element-binding proteins (SREBPs) belong to an important family of transcription factors that control gene expression of core enzymes of lipogenesis. 14 Targeting the key enzymes of lipogenesis is an effective strategy to blunt tumour growth and impair tumour survival. 15 Notably, previous studies have demonstrated that targeting SREBP1 can abolish the cancer stemness and enhance the chemotherapeutic response to gemcitabine in pancreatic cancer cells. 16 5′-AMP-activated protein kinase (AMPK), a kinase directly targeting SREBP1, can stimulate phosphorylation at Ser372, inhibit SREBP-1c cleavage and intranuclear translocation, and suppress the expression of SREBP-1c target genes in hepatocytes exposed to high levels of glucose, thus decreasing lipogenesis. 17 Identification of agents that repress lipogenesis might help to provide insights into a promising strategy to enhance the treatment outcome of gallbladder cancer.
As a dietary xanthone, α-mangostin is mainly isolated from the pericarp of mangosteen or Garcinia mangostana L. Previous studies have demonstrated that α-mangostin has a number of biological activities, including antibacterial, 18 cardioprotective, 19 neuroprotective 20 and anticancer effects. 21,22 In addition, α-mangostin kills cancer cells by inducing cell cycle arrest, apoptosis and autophagic cell death; moreover, α-mangostin suppresses oxidation, invasion and metastasis of several types of cancer. 21 However, the molecular mechanisms of the effects of α-mangostin in GBC cells have not yet been reported. Interestingly, α-mangostin triggers the autophagy-related cell death of glioblastoma cells by activating AMPK (AMP-activated protein kinase). 23 AMPK is a canonical upstream regulator of SREBP1, which is the key transcriptional factor regulating de novo lipid synthesis.
Thus, in this study, we hypothesized that α-mangostin represses de novo lipogenesis and enhances the chemotherapeutic response to gemcitabine in gallbladder carcinoma cells by targeting the AMPK/SREBP1 cascades.

| MATERIAL S AND ME THODS
The present study was authorized by the Ethical Committee of the First Affiliated Hospital of Medical College, Xi'an Jiaotong University, China.

| Cell culture and reagents
The GBC cell lines, GBC-SD, and normal biliary epithelial cell line,

| Colony formation assay
After plating the GBC-SD and NOZ cells at 1000 cells/well into 35 mm petri dishes and allowing the cells to attach overnight, MA (5 μmol/L) or gemcitabine (10 μmol/L) was used to treat the cells for 24 hours, and the culture medium was replaced with the fresh medium. After 2 weeks of culture, cell colonies were formed. At expected time-points, the colonies were fixed with 4% paraformaldehyde, stained with 0.1% crystal violet solution, rinsed and imaged; the number of the colonies was counted and statistically evaluated.

| Ethynyl deoxyuridine (EdU) incorporation assay
The EdU incorporation assay was conducted using an EdU kit (Roche) according to the manufacturer's instructions. The results were visualized by a Zeiss confocal microscope at a magnification of 200×, and the signals were counted in at least five random fields.

| Flow cytometry analysis
We performed flow cytometry analysis to evaluate cell apoptosis and cell cycle progression. We used an Annexin V-FITC/7-AAD apoptosis detection kit from Becton Dickinson (BD) to assess apoptosis following the manufacturer's guidelines. In brief, NOZ and GBC-SD cells were plated into 6-well plates at a density of 1 × 10 5 cells/well.

| Oil Red O staining
We performed Oil Red O staining to visualize the lipid droplets in the GBC cells. After finishing the designated treatment, the cells were rinsed with PBS three times and fixed in 4% paraformaldehyde for 1 hour. Then, propylene glycol was used to rinse the cells; the cells were stained with pre-warmed 0.25% Oil Red O working solution in a 60°C oven for 30 minutes. After PBS rinsing, the cell nuclei were visualized by haematoxylin staining. Finally, we photographed the cells using a light microscope (Nikon Eclipse Ti-S) at 400× magnification.

| Western blotting analysis
RIPA lysis buffer was used to extract the total protein; the concentration of the total protein was measured using a BCA protein assay kit (Pierce) following the manufacturer's instructions. The WB assay was conducted as previously reported. 22 The expression of designated proteins was visualized by enhanced chemiluminescence (Millipore). Images were captured using a ChemiDoc XRS imaging system (Bio-Rad), and the ImageJ software was used for densitometry analysis of each band. β-Actin was used as an internal loading control.

| Immunofluorescence staining
With implementing the designated treatment, gallbladder cancer cells were fixed in 4% formaldehyde for 30 min, permeabilized with 0.3% Triton X-100, incubated with 5% BSA (blocking buffer) for 1 hour at room temperature and incubated overnight with a primary antibody at 4°C. The cells were then incubated with a red dye-conjugated secondary antibody from Jackson ImmunoResearch Laboratories for 1 hour at room temperature; then, the cells were stained with DAPI to visualize the nuclei. Finally, the slides were mounted and observed under a Zeiss confocal microscope at a magnification of 400×.

| RNA interference
Loss-of-function analysis was performed using siRNAs against AMPK and SREBP1. The sequences of siRNA for AMPK (sense: UUCUCCGAA CGUGUCACGUTT; antisense: ACGUGACACGUUCGGAGAATT) and a negative control siRNA (sense: UUCUCCGAACGUGUCACGUTT; antisense: ACGUGACACGUUCGGAGAATT) were designed, and siRNAs were synthesized by GenePharma Co., Ltd. The siRNA sequences for silencing SREBP1 were described previously. 16 The transfection was conducted as previously reported. 24 After 24 hour of transfection, the cells were utilized for further experiments.

| Statistical analysis
Every experiment was repeated at least three times. The results are shown as the means ± standard deviation. Student's t test was performed to compare two groups. One-way ANOVA followed by the LSD post hoc test was used for statistical analysis of multiple comparison using SPSS (SPSS 18.0; SPSS Inc). P < .05 was considered to be statistically significant.

| Depletion of SREBP1 potentiates gemcitabine sensitivity in GBC cells
Owing to the evidence supporting the impact of SREBP1 on the lipogenesis, de novo lipogenesis has been validated to be essential  Figure 5D). Moreover, colony formation was substantially reduced after depletion of SREBP1; SREBP1 depletion enhanced the inhibition of colony formation by gemcitabine ( Figure 5E). In addition, we found that silencing SREBP1 potentiated the GEM-induced apoptosis of NOZ cells (Figure 5F), demonstrating the importance of SREBP1 in the GEM-mediated promotion of apoptosis in GBC cells.

| Inhibition of SREBP1 by α-mangostin enhances the sensitivity of GBC to gemcitabine
To confirm whether α-mangostin increases the sensitivity of NOZ cells to gemcitabine, we examined colony formation of NOZ cells after treatment with MA and gemcitabine. As shown in Figure 6A Figure 6B). Furthermore, NOZ cells were treated with 5 μmol/L α-mangostin and 10 μmol/L gemcitabine. The results of the MTT assay indicated that the viability of cancer cells was remarkably lower in the group treated with the combination of α-mangostin and gemcitabine than that in the groups treated with α-mangostin or gemcitabine alone ( Figure 6C). In addition, the expression levels of the genes involved in lipid metabolism, including SREBP1, FASN and ACC, in NOZ cells exposed to MA and gemcitabine were assessed by WB analysis. As shown in Figure 6D, the expression of SREBP1, FASN and ACC was substantially reduced by MA treatment; interestingly, gemcitabine treatment tended to display higher expression of SREBP1, FASN and ACC, which was consistent with previously reported results. 16 Gemcitabine treatment enhanced the cancer stemness via elevated expression of SREBP1. 16 However, in the group treated with the combination of gemcitabine and MA, MA can abrogate the effects F I G U R E 5 SREBP1 depletion enhances gemcitabine sensitivity in the gallbladder cancer cells. A, B, GBC-SD and NOZ cells were incubated with a gradually increasing concentration of gemcitabine (0, 10 −3 , 10 −2 , 10 −1 , 10 0 , 10 1 , 10 2 and 10 3 μmol/L) for 24, 48 or 72 h; the MTT assay was used to analyse cancer cell viability. C, The efficacy of siRNAs silencing SREBP1 in NOZ cells was determined by WB analysis. D, After transfection with si-Control or si-SREBP1 for 48 h, cell viability was determined by the MTT assay. E, The effect of si-Control or si-SREBP1 combined with gemcitabine on colony formation in NOZ cells. Images are representative of three independent experiments. Scale bar: 1 cm. F, The effects of si-SREBP1 on apoptosis of NOZ cells after treatment with 10 μmol/L gemcitabine for 48 h was examined by flow cytometry. **P < .01. GEM, gemcitabine of gemcitabine on the expression of SREBP1, FASN and ACC ( Figure 6D). Collectively, these results illustrate that inhibition of SREBP1 activity by α-mangostin enhances the sensitivity of GBC cells to gemcitabine.  Gallbladder cancer is a relatively rare but highly lethal neoplasm, lacking effective strategies to conquer this disease. Patients suffering from GBC usually have a few signs or symptoms; however, their condition can deteriorate rapidly due to the development of F I G U R E 7 α-Mangostin enhances the chemosensitivity of gemcitabine in vivo. A, Representative images of subcutaneous xenografts in nude mice implanted with NOZ and treated with MA, GEM and MA combined with GEM (n = 6 per group). B, C, Xenograft weight (mg) and tumour sizes were monitored and quantified. n = 6 per group, **P < .01 or ## P < .01 by ANOVA for tumour weight; **P < .01 by repeatedmeasures ANOVA for tumour sizes. D, TUNEL staining, immunohistochemistry staining and semiquantification of the data of Ki-67 and FASN in the xenograft tissues from various groups. Magnification is ×400; the scale bar represents 20 μm. n = 6, **P < .01 or ***P < .001 by ANOVA metastasis. 33 Currently, gemcitabine/cisplatin has been recognized as the preferred regimen for the first-line treatment of patients suffering from advanced biliary tract cancers, including gallbladder cancer. 3 However, patients undergoing the first-line chemotherapy often have a rapidly worsening performance status; only a small number of patients will be suitable for subsequent treatment. 3 Here, we show that α-mangostin is effective in inhibiting lipogenesis and increasing sensitivity to gemcitabine via SREBP1 suppression in gallbladder cancer cells in vitro; then, we evaluated how α-mangostin alone or in combination with gemcitabine influences the subcutaneous GBC growth in nude mice. Our results demonstrated that MA or gemcitabine administration to nude mice harbouring NOZ tumours can reduce tumour growth; moreover, MA administration can significantly potentiate gemcitabine-induced inhibition of tumour growth.

| Inhibition of SREBP1 induced by α-mangostin enhances the chemosensitivity to gemcitabine in vivo
Hence, α-mangostin appears to be a good candidate for further de-

ACK N OWLED G EM ENTS
This study was supported by grants from the National Natural Science Foundation of China (No. 81803015) and the Fundamental Research Funds for the Central Universities (No. xjj2018092).

CO N FLI C T O F I NTE R E S T
No conflicts of interest exist.

AUTH O R CO NTR I B UTI O N S
EL and DD conceived and designed the experiments; YS, YF, YH, JJ and CW performed the experiments; YW and XD analysed the data; QG contributed reagents/materials/analysis tools; YS, EL and DD wrote the paper. All authors read and approved the final manuscript.

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.