Salvianolic Acid B Suppresses Non-Small-Cell Lung Cancer Metastasis through PKM2-Independent Metabolic Reprogramming

Abstract

Objective: Salvianolic acid B (Sal B) has been demonstrated to be a potential chemoprevention agent for several cancers. Herein, we investigated the pharmacological function of Sal B on non-small-cell lung cancer (NSCLC) metastasis.

Methods: Two NSCLC cell lines (NCI-H2030 and NCI-H1650) were disposed of by 200 μM Sal B or 10 μM PKM2 agonist TEPP-46. Wound healing and transwell experiments were implemented for analyzing migratory and invasive capacities. Epithelial-to-mesenchymal transition (EMT) markers β-catenin and E-cadherin were measured via western blotting. Cellular bioenergetics were evaluated with glucose uptake, lactate production, enolase activity, cellular ATP levels, as well as seahorse-based oxygen consumption rate (OCR), extracellular acidification rate (ECAR) analysis. Metabolic reprogramming markers PKM2, LDHA, and GLUT1 were detected via western blotting and immunofluorescence.

Results: The results showed that Sal B disposal weakened the migration and invasion of NCI-H2030 and NCI-H1650 cells and inactivated the EMT process according to downregulation of β-catenin and upregulation of E-cadherin. Sal B-treated NSCLC cells displayed decreased glucose uptake, lactate production, enolase activity, cellular ATP levels, OCR, and ECAR, indicating a reduction in metabolic reprogramming. Additionally, Sal B downregulated the expression of PKM2, LDHA, and GLUT1. TEPP-46 may reverse the inhibitory effect of Sal B on metastasis as well as metabolic reprogramming.

Conclusion: Our findings provide evidence that Sal B enables to weaken NSCLC metastasis through PKM2-independent metabolic reprogramming, which sheds light on the promising therapeutic usage of Sal B in treating NSCLC.

Figure 1

Sal B suppresses migration and invasion of NSCLC cells. (a, b) Cell viability of NCI-H2030 and NCI-H1650 cells that were exposed to a series of concentrations of Sal B lasting 24 or 48 hours through CCK-8. (c–e) Wound healing for assessing migration of 200 μM Sal B-treated NCI-H2030 and NCI-H1650 cells. Bar = 50 μM. (f–h) Evaluation of invasion of 200 μM Sal B-treated NCI-H2030 and NCI-H1650 cells through transwell. Bar = 50 μM. ^∗P < 0.05; ^∗∗P < 0.01; ^∗∗∗∗P < 0.0001.

Figure 2

Sal B weakens EMT process of NSCLC cells. (a–c) Western blotting of the expression of β-catenin and E-cadherin in 200 μM Sal B-treated NCI-H2030 cells. (d–f) Western blotting of the expression of β-catenin and E-cadherin in 200 μM Sal B-treated NCI-H1650 cells. ^∗∗P < 0.01; ^∗∗∗∗P < 0.0001.

Figure 3

Sal B weakens metabolic reprogramming of NSCLC cells. (a–d) Glucose uptake, lactate production, enolase activity, as well as cellular ATP production of 200 μM Sal B-treated NCI-H2030 and NCI-H1650 cells. (e, f) Measurement of OCR of 200 μM Sal B-treated two NSCLC cells when exposure to oligomycin, FCCP, and Rot/antiA. (g, h) Quantification of basal and maximal OCR of 200 μM Sal B-treated two NSCLC cells. (i, j) Measurement of ECAR of 200 μM Sal B-treated two NSCLC cells after exposing to glucose, oligomycin, and 2-DG. (k, l) Quantification of glycolysis ECAR of above two NSCLC cells. ^∗P < 0.05; ^∗∗P < 0.01; ^∗∗∗P < 0.001; ^∗∗∗∗P < 0.0001.

Figure 4

Sal B inhibits PKM2-mediated metabolic reprogramming of NSCLC cells. (a–c) Western blots for PKM2 as well as p-PKM2 expressions in 200 μM Sal B-treated NCI-H2030 cells. (d–f) Western blots for PKM2 as well as p-PKM2 expressions in 200 μM Sal B-exposed NCI-H1650 cells. (g, h) Immunofluorescence for investigating PKM2 expression in 200 μM Sal B-exposed NCI-H2030 and NCI-H1650 cells. Bar = 10 μM. ^∗P < 0.05; ^∗∗P < 0.01; ^∗∗∗P < 0.001.

Figure 5

Sal B restrains metabolic reprogramming-relevant genes LDHA and GLUT1 in NSCLC cells. (a–f) Western blots for measuring LDHA and GLUT1 expressions in 200 μM Sal B-treated NCI-H2030 as well as NCI-H2030 cells. (g, h) Immunofluorescence for investigating the expression of LDHA and GLUT1 in 200 μM Sal B-exposed NCI-H2030 and NCI-H1650 cells. Bar = 10 μM. ^∗∗P < 0.01; ^∗∗∗P < 0.001.

Figure 6

Sal B suppresses migration and invasion of NSCLC cells via PKM2-independent metabolic reprogramming. (a–d) Wound healing of the migration of NCI-H2030 and NCI-H1650 cells when exposure to 200 μM Sal B or 10 μM TEPP-46. Bar = 50 μM. (e–g) Transwell for evaluating the invasion of NCI-H2030 and NCI-H1650 cells following exposure to 200 μM Sal B or 10 μM TEPP-46. Bar = 50 μM. Compared with control group, ^∗∗P < 0.01; ^∗∗∗P < 0.001; ^∗∗∗∗P < 0.0001. Compared with Sal B group, ^#P < 0.05; ^##P < 0.01; ^###P < 0.001.

Figure 7

Sal B weakens EMT process of NSCLC cells through PKM2-independent metabolic reprogramming. (a–e) Western blotting for evaluating PKM2, GLUT1, β-catenin, and E-cadherin expressions in NCI-H2030 cells when exposure to 200 μM Sal B or 10 μM TEPP-46. (f–j) Western blotting of PKM2, GLUT1, β-catenin, and E-cadherin expressions in NCI-H1650 cells following exposure to 200 μM Sal B or 10 μM TEPP-46. Compared with control group, ^∗∗P < 0.01; ^∗∗∗P < 0.001; ^∗∗∗∗P < 0.0001. Compared with Sal B group, ^##P < 0.01; ^###P < 0.001; ^####P < 0.0001.