Withaferin A decreases glycolytic reprogramming in breast cancer – PubMed Black Hawk Supplements

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Reprogrammed glucose metabolism is considered as the hallmark of cancer with therapeutic implications. Phytocompounds have potential to inhibit cancer metabolism. Here, we tested the ability of Withaferin A (WA), a withanolide derived from Withania somnifera, in modulating cancer metabolism. The assessed effect of WA on aerobic glycolysis in breast cancer cell lines showed that WA decreases the glucose uptake, lactate production and ATP generation by inhibiting the expression of key glycolytic…
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Withaferin A decreases glycolytic reprogramming in breast cancer - PubMed

Withaferin A decreases glycolytic reprogramming in breast cancer

Asifa Khan et al. Sci Rep. .

Abstract

Reprogrammed glucose metabolism is considered as the hallmark of cancer with therapeutic implications. Phytocompounds have potential to inhibit cancer metabolism. Here, we tested the ability of Withaferin A (WA), a withanolide derived from Withania somnifera, in modulating cancer metabolism. The assessed effect of WA on aerobic glycolysis in breast cancer cell lines showed that WA decreases the glucose uptake, lactate production and ATP generation by inhibiting the expression of key glycolytic enzymes i.e., GLUT1, HK2 and PKM2. We also identified that WA induced inhibition of cancer glycolysis by targeting c-myc as validated by silencing experiments followed by metabolic readouts. Decreased glycolysis resulted in reduced cell viability, biomass and colony forming ability of breast cancer cells. To further validate our in vitro findings in breast cancer patients, we analyzed 90 metabolic pathways in ~ 2000 breast tumors and observed that glycolysis is the most deregulated pathway in breast tumors. Deregulated glycolysis also predicted poor prognosis in breast cancer patients. In addition, patient data showed correlation between c-myc expression and glycolytic deregulation in breast cancer. Taken together, our results highlight the role of WA in inhibiting breast cancer metabolism via c-myc/glycolysis axis.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1

Effect of WA on the cell viability of breast cancer cell lines. (a–d) Breast cancer cells were treated with either WA at different conc. (1, 3, 5, and 10 µM) for 24 h or DMSO as vehicle control. Data was recorded and presented in mean ± SD from the three independent experiments and *P < 0.03, **P < 0.005, ***P < 0.0002, and ****P < 0.0001, ns = non-significant, calculated using ANOVA.

Fig. 2
Fig. 2

Treatment with WA reduces colonization in breast cancer cell lines. Colony formation assay performed using 5 µM WA in (a) MCF7, (b) MDA-MB-231, (c) MDA-MB-468 (d) MDA-MB-453.

Fig. 3
Fig. 3

WA decreases the glucose uptake, lactate release and ATP production in breast cancer. (a, b) WA (5 μM) decrease the glucose uptake and lactate release in T47D, MDA-MB-231, MDA-MB-468, and MCF-7 cells. Cells were treated with DMSO or 5 µM WA for 24 h, media were collected, and the rate of glucose uptake and lactate production was determined using commercial kits as described in the Materials and Methods section. (c) Intracellular ATP was extracted from cells treated with DMSO or 5 µM WA for 24 h, as described in Materials and Methods. The bars represent the mean ± SD of 3 independent biological replicates. P value calculated using ANOVA and represented as *P < 0.03; **P < 0.0021; ***P < 0.0002; and ****P < 0.0001, ns = non-significant.

Fig. 4
Fig. 4

WA decreases protein expression of key glycolytic enzymes and Warburg effect via c-myc inhibition. (a) Immunoblotting of protein extracts after WA treatment of MDA-MB-231 and MCF-7 cells, using anti-GLUT1, anti-HK2, anti-PKM2, anti-c-myc and anti-β-actin antibodies. Original and uncropped blots are presented in supplementary file (Fig. 4Aand 4B) (b) Silencing of c-myc in MDA-MB-231 and MCF-7 reduces the expression of key glycolytic enzymes, HK2 and GLUT1. (c,d) Densitometric analysis of the Western blot normalized with β-actin using alpha ease 4.0. Software. The groups of the blots were cropped from the different blots and the samples were derived from the same experiment and these blots were processed in parallel. (e) Silencing of c-myc reduces the glucose uptake, lactate secretion, and ATP production in MDA-MB-231. (f) Correlation of PDS score and mRNA of c-myc with PDS of glycolysis shows strong positive correlation (Spearman) between them. The bars represent the mean ± SD of 3 independent biological replicates. P value calculated using ANOVA or Student’s t-test, **P < 0.0021; ***P < 0.0002.

Fig. 5
Fig. 5

Evaluation of the effect of glycolysis on breast cancer patient dataset (a) Table showing the list of top 10 deregulated metabolic pathways in breast cancer patients (METABRIC) along with their pathway deregulation score (PDS). On the basis of PDS score, glycolysis found to be the top deregulated pathway. (b) Box and whisker plot represents the PDS of glycolysis in tumor vs normal tissue. (c) Bar graph showing the relative abundance of glycolytic metabolites in breast tumors as compared to normal samples (Terunuma et al.). (red: tumor; blue: normal) (d) Kaplan-Meier curve showing the effect of glycolysis alone and in-combination with myc on the survival of breast cancer patients.

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Withaferin A decreases glycolytic reprogramming in breast cancer – PubMed