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. 2019 Sep 11;10(9):672.
doi: 10.1038/s41419-019-1884-7.

SREBP-1 inhibitor Betulin enhances the antitumor effect of Sorafenib on hepatocellular carcinoma via restricting cellular glycolytic activity

Fan Yin  1 , Fan Feng  2 , Lei Wang  3 , Xiaoning Wang  4 , Zongwei Li  3 , Yu Cao  5
Affiliations

SREBP-1 inhibitor Betulin enhances the antitumor effect of Sorafenib on hepatocellular carcinoma via restricting cellular glycolytic activity

Fan Yin et al. Cell Death Dis. .

Abstract

Lipid metabolism that correlates tightly to the glucose metabolic regulation in malignant cells includes hepatocellular carcinoma (HCC) cells. The transcription factor Sterol Regulatory Element Binding Protein 1 (SREBP-1), a regulator of fatty acid synthesis, has been shown to pivotally regulate the proliferation and metastasis of HCC cells. However, the intrinsic mechanism by which SREBP-1 regulates the survival of HCC cells remains unclear. In this study, among HCC patients who had dismal responses to Sorafenib, a high SREBP-1 level was found in the tumors and correlated to poor survival. This observation suggested the negative role of SREBP-1 in clinical HCC prognosis. Our mechanistical studies reveal that the inhibition of SREBP-1 via its inhibitor Betulin suppresses cellular glucose metabolism. In addition to the reduced glycolytic activity, a thwarted metastatic potential was observed in HCC cells upon Betulin administration. Moreover, our data show that SREBP-1 inhibition facilitated the antitumor effects of Sorafenib on HCC cells and xenograft tumors.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Fig. 1
Fig. 1. High SREBP-1 level correlates to poor prognosis of advanced HCC patients who received Sorafenib treatment.
a Patients were divided into SREBP-1 high and SREBP-1 low groups based on the expression level. b PCR results from ten representative specimens (five high and five low). c Overall survival (OS) comparison of HCC patients in the SREBP-1 high group and the SREBP-1 low group. d Time to progress (TTP) of HCC patients in the SREBP-1 high group and the SREBP-1 low group. e, f The relative SREBP-1 expressions at both mRNA (e) and protein levels (f) in different cell lines. Survival analysis was performed by the Kaplan–Meier method and compared by the log-rank test. Paired samples were tested by the paired-sample t-test. Significant: *p < 0.05 in all figures
Fig. 2
Fig. 2. SREBP-1 regulates the glycolytic activity of HCC cells.
a Extracellular acidification rate (ECAR) measurement in high metastatic MHCC97-H cells transfected with control or SREBP-1 siRNAs. b ECAR measurement in low metastatic MHCC97-L cells transfected with empty or SREBP-1-expressing vectors. c Oxygen-consumption rate (OCR) measurement in MHCC97-H cells from a. d OCR measurement in MHCC97-L cells from c. e MHCC97-H cells were treated with the indicated concentrations of Betulin (100, 30, 10, 3, 1, 0.3, or 0.1 μmol/L). Next, the cells were harvested for quantitative RT-PCR. The inhibition rates of Betulin on gene expression were calculated and shown by a heatmap
Fig. 3
Fig. 3. SREBP-1 regulates the sensitivity of HCC cells to Sorafenib.
a MHCC97-H cells transfected with control or SREBP-1 siRNAs were treated with the indicated concentrations of Sorafenib for 48 h and then harvested for MTT experiments. All the results were shown as mean ± SD. b MHCC97-L cells transfected with empty or SREBP-1-expressing vectors were treated with the indicated concentrations of Sorafenib for 48 h and then harvested for MTT experiments. c MHCC97-H cells pretreated with 3 μM Betulin or vehicle control were treated with the indicated concentrations of Sorafenib for 48 h and then harvested for MTT experiments. d MHCC97-H cells pretreated with 3 μmol/L Betulin or vehicle control were treated with Sorafenib at the IC50 concentration for 48 h. Then, cells were harvested for transwell experiments
Fig. 4
Fig. 4. Betulin synergizes Sorafenib’s effect on HCC s.c. tumor growth.
a MHCC97-H cells were injected into nude mice subcutaneously. At day 6, mice started receiving vehicle control, or the indicated concentrations of Sorafenib, or 2 mg/kg Betulin or the indicated concentration of Sorafenib + 2 mg/kg Betulin orally every other day 10 times. At day 21 post treatment, mice were killed and tumors were obtained (N = 10). b Quantitative results of tumor volume and tumor weight from a
Fig. 5
Fig. 5. SREBP-1 knockdown synergizes Sorafenib’s effect on HCC s.c. tumor growth.
a MHCC97-H s.c. tumors were established by using MHCC97-H cells transfected with control or SREBP-1 siRNAs, or SREBP-1 siRNAs plus SREBP-1mut expressing vector. MHCC97-H s.c. tumor-bearing mice received vehicle control, or Sorafenib at the IC50 concentration, or 2 mg/kg Betulin or Sorafenib + Betulin orally every other day 10 times. At day 21 post treatment, mice were killed and tumors were obtained (N = 10). b Quantitative results of tumor volume and tumor weight from a. c Western blotting of SREBP-1 and apoptosis and proliferation-associated proteins in tumors from a. β-actin was used as internal control
Fig. 6
Fig. 6. Betulin synergizes Sorafenib’s effect on HCC in situ growth in the mouse liver.
a The in situ xenograft tumors were established by injected MHCC97-H cells directly into mouse livers. Tumor-bearing mice then received vehicle control, or the indicated concentrations of Sorafenib, or 2 mg/kg Betulin or the indicated concentration of Sorafenib + 2 mg/kg Betulin orally every other day 10 times. At day 21 post treatment, mice were scanned by in vivo small-animal MicroPET imaging (N = 10). The images show the in situ tumors. b The 18F-FDG intensity analysis and quantification results. c Relative inhibitory rate of tumor growth calculated from (b). d Livers from control or tumor-bearing mice from a. e Representative tumor nodules from each treatment group. f Quantification results from e
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