Polydatin down-regulates the phosphorylation level of STAT3 and induces pyroptosis in triple-negative breast cancer mice with a high-fat diet

Abstract

Background: To explore the impact of polydatin on mice with triple-negative breast cancer (TNBC) receiving a high-fat diet, as well as the underlying processes.

Methods: A total of 40 female Balb/c mice were randomly separated into 4 groups (4T1 + polydatin + fat diet group, 4T1 + high-fat diet group, 4T1 + polydatin group, and 4T1 group). To establish the obese TNBC mouse model, TNBC was xenografted 1×10⁵ 4T1 cells/50 µL per mouse at the right fourth mammary fat pad under anesthesia and the mice were fed a high fat diet. When the experiment was completed, total plasma cholesterol (TC) and cancer antigen (CA)15-3 were measured. The enzyme-linked immunosorbent assay (ELISA) method was used detect CA15-3. Oil red O staining was used to observe the morphological changes. Western blot analysis and reverse transcription polymerase chain reaction (RT-PCR) were used to detect the corresponding protein expression and the messenger RNA (mRNA) level.

Results: Polydatin decreased the degree of fatty liver, as determined by oil red O staining. The TC level in the 4T1 + fat diet group was significantly higher, and it was decreased in the 4T1 + polydatin group. The results of ELISA showed that compared with the 4T1 group, CA15-3 was significantly increased in the 4T1 + fat diet group, and polydatin was shown to significantly reduce the expression of CA15-3. Polydatin inhibited p-JAK2 and p-STAT3 mRNA and protein levels. Polydatin increased pyroptosis-related gene mRNA and protein level.

Conclusions: We believe that polydatin can effectively reduce blood lipid levels in TNBC mice with a high-fat diet, and play an anticancer role in TNBC. The underlying mechanism may be related to the JAK2/STAT3 signaling pathway and pyroptosis in TNBC. Our results contribute to validating the traditional use of polydatin in the treatment of TNBC with hyperlipidemia.

Keywords: JAK2/STAT3 signaling pathway; Polydatin; high-fat diet; pyroptosis; triple-negative breast cancer (TNBC).

Figure 1

Using an orthotopic Balb/c mouse xenograft model, we investigated whether receiving a high-fat diet or eating regularly would have any influence on the occurrence and progression of TNBC. (A) Comparison of the average tumor volume of 4T1 cells. The mean tumor volumes for each experimental group are shown by the points on the graph. (B) Weight comparisons of 4T1 cell tumors on an average basis. The SEM of each experimental group is shown by error bars. *P<0.05 and **P<0.01 were used to indicate statistical significance when compared to the regular chow diet-fed control group. TNBC, triple-negative breast cancer; SEM, standard error of the mean.

Figure 2

Polydatin therapy has an effect on the development of orthotopically xenografted TNBC cells 4T1 in high-fat Balb/c mice. (A) Comparative monitoring of the mean 4T1 cells tumor volume for the 4T1 + polydatin group vs. the 4T1 group during the course of the trial. The points denote the mean tumor volumes, while the error bars denote the standard error of the mean for each of the experimental groups. ^#P<0.05 indicates statistical significance in comparison with the 4T1 + polydatin control group. **P<0.01 indicates statistical significance in comparison with the 4T1 group. (B) At the later stage of the trial, the mean value of tumor weights was compared between the 4T1 + polydatin and 4T1 groups. Error bars represent the SEM. *P<0.05 is considered statistically significant. TNBC, triple-negative breast cancer; SEM, standard error of the mean.

Figure 3

Polydatin therapy effects on lung weight, CA15-3 level, fatty liver, and plasma TC in 4T1 tumors in Balb/c mice receiving a high-fat diet. (A) At the later stage of the experiment, we compared the mean lung weight of the 4T1 + polydatin group with that of the 4T1 group in an orthotopic Balb/c mice xenograft model. (B) The plasma levels of the breast cancer recurrence marker CA15-3 in the 4T1 + polydatin group were compared to those in the 4T1 group in an orthotopic Balb/c mice xenograft model. (C) The degree of fatty liver (arrow) was compared between the 4T1 + polydatin and 4T1 groups in an orthotopic Balb/c mouse xenograft model. The liver tissues were stained with Oil Red O. Magnification in its original state is ×400. (D) The mean plasma TC levels in the T1 + polydatin group were compared to those in the 4T1 group in 4T1 tumors. *P<0.05 and **P<0.01 are considered statistically significant. CA15-3, cancer antigen 15-3; TC, total cholesterol.

Figure 4

The impacts of polydatin treatments on JAK2 and STAT3 expression levels. (A) STAT3 and JAK2 mRNA expressions in breast cancer tissues treated with 4T1, 4T1 + polydatin, 4T1 + polydatin + fat diet, and 4T1 + fat diet. (B) STAT3, JAK2, phospho-STAT3, and phospho-JAK2 protein expressions in BC tissues treated with 4T1, 4T1 + polydatin, 4T1 + polydatin + fat diet, and 4T1 + fat diet. All blots were subjected to scanning densitometry in triplicate, and the integrated optical density of each band was standardized to that of GAPDH in the same blot. *P<0.05, **P<0.01 was considered statistically significant. mRNA, messenger RNA; BC, breast cancer.

Figure 5

Effects of polydatin treatments on the expression levels regarding pyroptosis-related genes. (A) Caspase-1, NLRP3, Caspase-3, IL-1β, GSDMD, and IL-18 mRNA expression in BC tissues treated with 4T1, 4T1 + polydatin, 4T1 + polydatin + fat diet, and 4T1 + fat diet. (B) Expression of NLRP3, GSDMD, pro-Caspase-1, Caspase-1, Caspase-3, Cleaved Caspase-3, IL-18, and IL-1β protein in BC tissues treated with 4T1, 4T1 + polydatin, 4T1 + polydatin + fat diet, and 4T1 + fat diet. For all the blots, scanning densitometry was performed in triplicate, and the integrated optical density of each band was normalized to the equivalent density observed for β-actin in the same blot. *P<0.05, **P<0.01 was considered statistically significant. BC, breast cancer; mRNA, messenger RNA; IL-18, interleukin 18; IL-1β, interleukin-1β.