Taraxasterol suppresses cell proliferation and boosts cell apoptosis via inhibiting GPD2-mediated glycolysis in gastric cancer

Abstract

Gastric cancer (GC) is the most common malignant tumor of digestive tract. Taraxasterol (TAX), a kind of phytosterol, has been proved to exert anti-tumor functions in GC. Herein, the current work was carried out to identify the biological role of TAX and molecular mechanisms underlying TAX in the progression of GC. In the present study, CCK-8 assay, Colony formation assay, EDU staining and TUNEL staining were performed to evaluate the malignant behaviors of GC cells. Levels of proliferation and apoptosis-associated proteins were assessed using western blotting analysis. Besides, GPD2 expression in GC cells was presented on CCLE database and the interaction between TAX and GPD2 was obtained from STRING database. The glucose uptake, lactate production, LDH activity, ATP and expressions of glycolysis-associated enzymes were measured to evaluate glycolysis level. Results of the present research revealed that TAX suppressed the proliferative and clone-forming abilities of GC cells and boosted the apoptosis of GC cells. TAX reduced GPD2 expression in GC cells. Furthermore, overexpression of GPD2 reversed the inhibitory effects of TAX on the proliferative and clone-forming abilities of GC cells as well as abolished the promoting effects of TAX on the apoptosis of GC cells. Besides, upregulation of GPD2 abrogated the inhibition of TAX on glycolysis. To conclude, TAX could suppress GC progression via inhibiting GPD2-mediated glycolysis, which helps to develop a promising molecular target for GC therapies.

Keywords: GPD2; Gastric cancer; Glycolysis; Taraxasterol.

Fig. 1

TAX suppressed the proliferative and clone-forming abilities of GC cells. a After treatment with 0, 5, 10, 15, 20 μM TAX for 48 h, viabilities of human gastric cancer cell line HGC-27 and human gastric mucosal epithelial cell line GES-1 were assessed using CCK-8 assay. b After treatment with 0, 15 μM TAX, the cloning formation of HGC-27 and GES-1 cells was examined by colony formation assay. c qRT-PCR was performed to detect the mRNA levels of Ki67 and PCNA in HGC-27 cells after treatment with 0, 15 μM TAX. d Western blotting analysis was applied to detect the expressions of proliferation-associated proteins (Ki67 and PCNA) in HGC-27 cells after treatment with 0, 15 μM TAX. e EDU staining was employed to detect the proliferation of HGC-27 cells after treatment with 0, 15 μM TAX. **p < 0.01, ***p < 0.001

Fig. 2

TAX boosted the apoptosis of GC cells. a and b TUNEL staining was employed to detect the apoptosis of HGC-27 cells after treatment with 0, 15 μM TAX. c Western blotting analysis was conducted to detect the expressions of apoptotic-associated proteins (Bcl-2, Bax, Cleaved caspase-3, Caspase-3) in HGC-27 cells after treatment with 0, 15 μM TAX. ***p < 0.001

Fig. 3

TAX reduced GPD2 expression in GC cells. a Broad Institute Cancer Cell Line Encyclopedia (CCLE) database presented GPD2 expression in GC cell lines. b Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database was employed to assess the interaction between TAX and GPD2. c qRT-PCR was performed to detect the mRNA level of GPD2 in HGC-27 cells after treatment with 0, 15 μM TAX. d Western blotting analysis was applied to detect the protein expression of GPD2 in HGC-27 cells after treatment with 0, 15 μM TAX. **p < 0.01, ***p < 0.001

Fig. 4

Overexpression of GPD2 reversed the inhibitory effects of TAX on the proliferative and clone-forming abilities of GC cells. a qRT-PCR was performed to detect the mRNA level of GPD2 in HGC-27 cells transfected with Ov-GPD2. b Colony formation assay was employed to examine the influence of GPD2 overexpression on the inhibitory effect of TAX on the clone-forming ability of HGC-27 cells. c qRT-PCR was performed to detect the influence of GPD2 overexpression on the regulatory effects of TAX on the mRNA levels of Ki67 and PCNA in HGC-27 cells. d Western blotting analysis was applied to detect the influence of GPD2 overexpression on the regulatory effects of TAX on the protein levels of Ki67 and PCNA in HGC-27 cells. e EDU staining was employed to assess the influence of GPD2 overexpression on the inhibitory effect of TAX on the proliferative ability of HGC-27 cells. ***p < 0.001

Fig. 5

Overexpression of GPD2 reversed the promoting effects of TAX on the apoptosis of GC cells. a and b TUNEL staining was employed to examine the influence of GPD2 overexpression on the promoting effect of TAX on the apoptosis of HGC-27 cells. c Western blotting analysis was conducted to detect the influence of GPD2 overexpression on the regulatory effects of TAX on the protein levels of Bcl-2, Bax, Cleaved caspase-3 and Caspase-3 in HGC-27 cells. ***p < 0.001

Fig. 6

TAX inhibited GPD2-mediated glycolysis in GC cells. a–d The glucose uptake, lactate production, LDH activity and ATP level in HGC-27 cells were measured to evaluate the effects of TAX and GPD2 on glycolysis in GC cells. e Western blotting analysis was conducted to detect the effects of TAX and GPD2 on the expression levels of glycolysis-associated enzymes (HK2, LDHA and PFKM) in HGC-27 cells. *p < 0.05, **p < 0.01, ***p < 0.001