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

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.


Introduction
Non-small-cell lung cancer (NSCLC) is the most frequent and deadly type of primary lung malignancy, occupying 85% of all cases [1,2]. It is histologically classified as adenocarcinoma, squamous cell carcinoma as well as large-cell carcinoma [3,4]. Distant metastasis represents the dominating cause of mortality in NSCLC [5]. For resectable NSCLC tumors, primary treatment includes surgery with or without neoadjuvant therapy [6]. e mainstay of treatment for nonresectable tumors is radiotherapy and systemic treatments that are also offered for palliative treatment [7]. Systemic treatment contains conventional chemotherapy that is usually linked to remarkable side effects, targeted molecular treatment (such as tyrosine kinase inhibitors), as well as immunotherapies (such as immune-checkpoint inhibitors) [8]. Nevertheless, clinical drug resistance or nonresponse remains a challenge, which greatly hinders treatment success [9].
Many advances have been made in the comprehending of the pathophysiology of NSCLC metastasis. NSCLC is characterized by increased glucose and lactate utilization, and widespread heterogeneity in metabolic signaling [10]. Metabolic reprogramming represents a hallmark of cancer, and targeting metabolism has become a promising therapeutic strategy against NSCLC [11]. Pyruvate kinase may physiologically irreversibly catalyze phosphoenolpyruvate to pyruvate as a kinase in the final step of glycolysis [12]. Only tumor cells express the embryonic M2 isoform of pyruvate kinase (PKM2). As a crucial rate-limiting enzyme in glycolysis, PKM2 exerts an important function in the metabolic reprogramming of tumor cells [13]. e upregulation has been demonstrated in NSCLC, and secreted PKM2 facilitates NSCLC metastasis via activation of integrin β1/FAK signaling [14]. us, targeting PKM2 represents a potential therapeutic regimen of NSCLC.
Chemoprevention is considered a reasonable and attractive strategy to prevent or delay the development of NSCLC [15]. Chinese herbal medicine Salviae miltiorrhizae has been broadly applied in traditional Chinese medicine practice in treating cardiovascular and cerebrovascular diseases with minimal side effects [16]. Salvianolic acid B (Sal-B), extracted from the root of Salviae miltiorrhizae, is a major bioactive hydrophilic ingredient [17]. Several studies have demonstrated the anticancer pharmacological properties of Sal-B. For instance, Sal-B restrains glioma cells that are sensitive to radiotherapy through Fis-1-independent mitochondrial dysfunction [18]. Sal-B suppresses the growth of head and neck squamous cell carcinoma through cyclooxygenase-2 and apoptotic signaling [19]. Nevertheless, the pharmacological properties of Sal-B on anti-NSCLC remain indistinct. Here, we aimed to investigate the therapeutic effect of Sal-B on NSCLC metastasis. Our findings demonstrated that Sal B may weaken NSCLC metastasis with PKM2-independent metabolic reprogramming.

Wound Healing
Assay. NCI-H2030 and NCI-H1650 cells were inoculated onto a 24-well plate. When subconfluence was reached, wound healing was made to the cell monolayers utilizing 200 μL pipette tips. At 48 hours, wound healing was captured under light microscopy (×200; Leica, Germany), and the relative migration level was measured.
2.6. Glucose Uptake and Lactate Production. Glucose uptake was determined utilizing the glucose uptake assay kit (ab136955; Abcam) in accordance with the manufacturer's specification. Cell lysate was harvested as well as measured for lactate level utilizing the lactate assay kit (ab65330; Abcam) in accordance with the manufacturer's specification. e optical density value of cell lysate was measured after 24 hours. Lactate production was tested according to standard curves, followed by normalization to the cell lysate concentration.

Measurement of Enolase Activity.
Enolase activity was measured with an enolase activity assay kit (ab241024; Abcam). In brief, cell lysate was mixed with the reaction buffer. After being incubated for 10 minutes at 25°C, the optical density value was quantified at 570 nm. ereafter, each subsequent assay was carried out for 2 minutes until the optical density value of the most active specimens exceeded the highest standard. Enolase activity was finally calculated.

Enhanced ATP
Assay. An enhanced ATP assay was carried out utilizing an enhanced ATP assay kit (S0027; Beyotime). e supernatant was added to 100 μl of ATP detection solution. ereafter, the RLU values were recorded through a Promega Glomax 20/20 luminometer. A standard curve was drawn according to the RLU values of ATP at concentrations of 0, 0.01, 0.05, 0.1, 0.5, 1, 5, and 10 nmol/L. e protein concentrations were utilized for standardizing the results and are shown as ATP/protein (nmol/mg).

Seahorse Analysis.
Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were detected utilizing XF24 extracellular analyzer (Seahorse Bioscience, USA). e 24-well cell culture microplate coated with Corning ® Cell-Tak ™ Cell as well as tissue adhesive was used for allowing adhesion of suspended cells.

Statistical Analysis.
Statistical analysis was implemented via GraphPad Prism 8 software (San Diego, USA). e statistical comparison was carried out utilizing Student's t-test, one-way analysis of variance (ANOVA) or two-way ANOVA.
e data are displayed as the mean ± standard deviation from at least three independent experiments. Pvalues <0.05 were considered statistical significance.

Sal B Suppresses Migration and Invasion of NSCLC Cells.
For investigation of the therapeutic function of Sal B, two NSCLC cells (NCI-H2030 and NCI-H1650) were exposed to distinct concentrations of Sal B for 24 or 48 hours. As shown in CCK-8, at 24 hours of exposure, when the concentration of Sal B reached 400 μM, the cell viability of NCI-H2030 and NCI-H1650 cells displayed a significant reduction compared with controls (Figures 1(a) and 1(b)). At 48 hours of exposure, cell viability was remarkably decreased until the concentration reached 300 μM which was higher than controls. is demonstrated that Sal B exposure enabled to weaken the proliferative capacity of NSCLC cells. We further evaluated the therapeutic function of 200 μM Sal B on NSCLC metastasis. e data from the wound healing assay showed that the migration capacity of NCI-H2030 and NCI-H1650 cells was remarkably weakened by 200 μM Sal B (Figures 1(c)-1(e)). Additionally, 200 μM Sal B significantly reduced the invasion capacity of NCI-H2030 and NCI-H1650 cells (Figures 1(f )-1(h)). Hence, Sal B treatment enabled to suppress NSCLC metastasis.

Sal B Weakens Epithelial-Mesenchymal Transition (EMT)
Process in NSCLC Cells. EMT, a reversible developmental genetic program for transdifferentiating polarized epithelial cells to mesenchymal cells, enables to trigger metastasis during the progression of NSCLC [20]. Further analysis showed that 200 μM Sal B markedly reduced β-catenin expression as well as elevated E-cadherin expression in NCI-H2030 as well as NCI-H1650 cell lines (Figures 2(a)-2(f )), indicating that Sal B enabled to inactivate EMT process of NSCLC cells.

Sal B Weakens Metabolic Reprogramming of NSCLC
Cells. Metabolic reprogramming has been widely recognized as a cancer hallmark [13]. Further analysis was implemented for evaluating the therapeutic function of Sal B on the metabolic reprogramming of NSCLC cells. Compared with controls, 200 μM Sal B-treated NCI-H2030 and NCI-H1650 cells presented remarkably reduced glucose uptake, lactate production, enolase activity as well as cellular ATP production (Figures 3(a)-3(d)). To evaluate mitochondrial respiration, we measured OCR after adding sequential compounds containing 1 μM oligomycin, 0.5 μM FCCP, and 0.5 μM Rot/antiA. As a result, 200 μM Sal B prominently decreased OCR both in NCI-H2030 and NCI-H1650 cells than controls (Figures 3(e)-3(h)). We also evaluated glycolytic activity through measuring ECAR following sequential compounds containing 10 mM glucose, 1 μM oligomycin, and 50 mM 2-DG. e data showed that 200 μM Sal B-treated NCI-H2030 and NCI-H1650 cells displayed significantly decreased ECAR than controls (Figures 3(i)-3(l)). Altogether, Sal B enabled to weaken the metabolic reprogramming of NSCLC cells.

Sal B Inhibits PKM2-Mediated Metabolic
Reprogramming of NSCLC Cells. PKM2 is a central regulator of aerobic glycolysis of cancer cells, which accelerates lactate production as well as metabolic reprogramming [21]. Our western blotting showed that 200 μM Sal B-treated NCI-H2030 and NCI-H1650 cells displayed remarkably reduced expression of PKM2 and p-PKM2 in comparison to controls (Figures 4(a)-4(f)). Immunohistochemistry also demonstrated that Sal B was able to decrease PKM2 expression in NCI-H2030 and NCI-H1650 cells (Figures 4(g) and 4(h)).

Discussion
NSCLC is a fatal malignancy with a hallmark of abnormal metabolism [22]. Sal B represents a dominating water-soluble component extracted from Salvia miltiorrhiza [23]. Our data demonstrated that Sal B was capable of weakening NSCLC metastasis with PKM2-independent metabolic reprogramming. Metastasis represents an extremely complex multistage process [24]. Tumor cells are detached from the extracellular matrix and then colonize surrounding environment, demonstrating that benign nodules transform to aggressive malignancies [25][26][27]. Sal B-treated NCI-H2030 and NCI-H1650 cells displayed reduced migration and invasion capacities, indicating the inhibitory effect of Sal B on NSCLC metastasis. Previous research has demonstrated that Sal B may restrain the migration and invasion of hepatocellular carcinoma through RECK/STAT3 signaling [28]. For crossing the basement membrane along with the basal layer, tumor cells are functionally and morphologically altered through activating EMT that has the features of upregulation of β-cadherin and downregulation of E-cadherin [29]. Sal B administration enabled the decrease β-cadherin expression as well as the enhancement of E-cadherin expression of NCI-H2030 and NCI-H1650 cells, demonstrating the inactivation of EMT in NSCLC cells. Previous evidence has proposed that Sal B is capable of suppressing EMT to alleviate drug resistance via AKT/mTOR signaling in gastric cancer [30].
Tumor cells usually reprogram the metabolism to effectively support cell proliferation and survival [31]. Herein, Sal B treatment markedly reduced glucose uptake, lactate Evidence-Based Complementary and Alternative Medicine production, enolase activity, cellular ATP production, OCR, and ECAR of NCI-H2030 and NCI-H1650 cells, demonstrating that Sal B weakened the metabolic reprogramming of NSCLC cells. Limited evidence has proposed the suppression effect of Sal B on metabolic reprogramming. For instance, Sal B may weaken glycolysis in oral squamous cell carcinoma through PI3K/AKT/HIF-1α signaling [15]. Additionally, Sal B reduces M1-polarized macrophages in myocardial ischemia/ reperfusion damage through weakening mTORC1-dependent glycolysis [32][33][34]. PKM2 is a crucial regulator of metabolic reprogramming, which may catalyze the synthesis of pyruvate from phosphoenolpyruvate and facilitate glycolysis, thereby allowing tumor cells to thrive [35,36]. Hypoxia-triggered exosomes transmit cisplatin resistance to sensitive NSCLC cells through delivery of PKM2 [22]. PKM2 facilitates HSP90-independent stability of IGF-1R precursor protein as well as enhances growth of cancer cells in a hypoxic environment [36]. eEF2 kinase-triggered inactivated STAT3 may inhibit the proliferative capacity of NSCLC cells through phosphorylation of PKM2 [37]. TEAD4 enables to enhance NSCLC progression with PKM2-mediated glycolysis [38]. Silencing PKM2 may retrain tumor growth as well as invasion in NSCLC [39]. Altogether, accumulated pieces of evidence have provided the basic data required for PKM2-targeted gene therapy. Sal B may   Evidence-Based Complementary and Alternative Medicine lower PKM2 expression and its phosphorylation in NCI-H2030 and NCI-H1650 cells. LDHA and GLUT1 are crucial components of the glycolytic signaling [40]. LDHA [41] and GLUT1 [42] both display upregulation in NSCLC than normal tissues, which enable to independently predict undesirable clinical outcomes. LDHA activation may enhance the growth of NSCLC [43], and miR-449a weakens LDHA-independent glycolysis  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.
to strengthen the sensitivity of NSCLC cells to ionizing radiotherapy [44]. NSCLC growth and radiotherapy resistance depend upon GLUT1-independent glucose uptake in tumor-associated neutrophils [45]. LDHA and GLUT1 expression was decreased by Sal B in NCI-H2030 and NCI-H1650 cells, demonstrating the inhibitory function of Sal B in glycolytic signaling of NSCLC cells. Metabolic reprogramming represents a hallmark of cancer metastases [46,47], and tumor cells manipulate the metabolic profiles to meet the dynamic energy demands of the tumor microenvironment [48][49][50]. e data showed that Sal B enabled to weaken NSCLC metastasis with PKM2-independent metabolic reprogramming. In future studies, in vivo experiments are required for demonstrating the therapeutic efficacy of Sal B in NSCLC metastasis.

Conclusion
Altogether, this study provided evidence that Sal B was capable of weakening NSCLC metastasis via PKM2-independent metabolic reprogramming, shedding light on the promising therapeutic usage of Sal B in NSCLC therapy. Data Availability e datasets analyzed during the current study are available from the corresponding author on reasonable request.

Conflicts of Interest
e authors declare no conflicts of interest.