**The Inhibitory Mechanisms of Tumor PD-L1 Expression by Natural Bioactive Gallic Acid in Non-Small-Cell Lung Cancer (NSCLC) Cells**

**Dong Young Kang 1,**†**, Nipin Sp 1,**†**, Eun Seong Jo 1, Alexis Rugamba 1, Dae Young Hong 2, Hong Ghi Lee 3, Ji-Seung Yoo 4, Qing Liu 5, Kyoung-Jin Jang 1,\* and Young Mok Yang 1,\***


Received: 9 March 2020; Accepted: 16 March 2020; Published: 19 March 2020

**Abstract:** Non-small-cell lung cancer (NSCLC) is the most common lung cancer subtype and accounts for more than 80% of all lung cancer cases. Epidermal growth factor receptor (EGFR) phosphorylation by binding growth factors such as EGF activates downstream prooncogenic signaling pathways including KRAS-ERK, JAK-STAT, and PI3K-AKT. These pathways promote the tumor progression of NSCLC by inducing uncontrolled cell cycle, proliferation, migration, and programmed death-ligand 1 (PD-L1) expression. New cytotoxic drugs have facilitated considerable progress in NSCLC treatment, but side effects are still a significant cause of mortality. Gallic acid (3,4,5-trihydroxybenzoic acid; GA) is a phenolic natural compound, isolated from plant derivatives, that has been reported to show anticancer effects. We demonstrated the tumor-suppressive effect of GA, which induced the decrease of PD-L1 expression through binding to EGFR in NSCLC. This binding inhibited the phosphorylation of EGFR, subsequently inducing the inhibition of PI3K and AKT phosphorylation, which triggered the activation of p53. The p53-dependent upregulation of miR-34a induced PD-L1 downregulation. Further, we revealed the combination effect of GA and anti-PD-1 monoclonal antibody in an NSCLC-cell and peripheral blood mononuclear–cell coculture system. We propose a novel therapeutic application of GA for immunotherapy and chemotherapy in NSCLC.

**Keywords:** natural bioactive compound; gallic acid; EGFR signaling; p53; PD-L1; immunotherapy

#### **1. Introduction**

Lung cancer, constituting 18% of all global cancer deaths, is one of the representative causes of death globally [1]. It is classified into two main groups: small-cell lung cancer (SCLC; 15% of all lung cancers) and non-SCLC (NSCLC; 85% of all lung cancers) [2,3]. NSCLC can be further subcategorized into three subtypes: adenocarcinoma, squamous cell carcinoma, and large cell carcinoma [4]. Despite many efforts to treat NSCLC, the overall survival rate is only 15.9% within five years [5]. Further, many patients receiving NSCLC chemotherapies struggle with adverse reactions, drug resistance, and the

necessary target specificity of some types of drugs. Thus, unsatisfactory outcomes in NSCLC treatment have motivated researchers to identify novel agents such as natural compounds [6–8]. Moreover, the main advantage of such drugs having fewer side effects relative to non-natural drugs has spurred scientists to reveal their molecular mechanisms.

Gallic acid (3,4,5-trihydroxybenzoic acid; GA), a natural phenolic compound, is widely distributed in natural plants, fruits, and green tea [9,10]. Many studies have reported that GA exhibits anti oxidative, anti mutagenic, anti carcinogenic, antibacterial, antiviral, and anti-inflammatory effects [11–15]. However, the main interest in GA and its derivatives surrounds its anticancer activity. Previous studies have revealed that GA effectively induces selective apoptosis in various cancer cells, including HeLa, HCT-15, SH-SY5Y, and NSCLC cells [16–19] and inhibits proliferation and migration via regulating fatty acid synthase in TSGH-8301 cells [20]. Recent studies also revealed potential anticancer effects of GA is due to its ability to inhibit cell proliferation and to induce apoptosis *in vivo* [21,22].

Programmed cell death ligand-1 (PD-L1), also known as CD274 and B7-H1, is a transmembrane protein expressed on the surface of antigen-presenting cells such as dendritic cells, macrophages, and B-cells. It is also overexpressed and found in various types of cancer [23–26]. PD-L1 specifically binds to programmed cell death-1 (PD-1), which is an important inhibitory receptor expressed on the surface of immune-related lymphocytes like T-cells, B-cells, and myeloid cells [27]. The binding of PD-L1 to PD-1 inhibits the proliferation, cytokine generation and release, and cytotoxicity of T-cells. Thus, the binding leads to an immunosuppressive effect and allows cancer cells to escape immune eradication via assistance from tumor-specific T-cells [28]. PD-L1 overexpression in cancer cells promotes cancer progression and leads cancer cells to malignancy. Moreover, the intrinsic signal transduction by PD-L1 enhances the survival of cancer cells through increasing the resistance toward proapoptotic stimuli such as interferons [29]. PD-L1 expression at the transcriptional level is regulated individually or cooperatively by many oncogenic transcription factors such as MYC, AP-1, STAT, IRF1, HIF, and NF-κB. Some studies have demonstrated that there is a tendency toward higher PD-L1 expression in *TP53*-mutated and low p53-expression cancer cells, which imply that PD-L1 expression is considerably related to p53 status in cancer cells [30–33]. In addition, tumor suppressor gene *PTEN*, one of the most frequently mutated genes in human cancers, downregulates PD-L1 expression, which signifies that tumor suppressors play an important role in controlling PD-L1 expression [34–36].

Previous research has demonstrated that apigenin, a kind of flavonoid, induces growth-suppressive and proapoptotic effects in melanoma cells. Additionally, such significantly inhibits the expression of interferon (IFN)-γ-induced PD-L1, which may indicate the existence of an immunosuppressive effect shown by a natural compound [37]. In this study, we examined the anticancer and immunosuppressive activities of natural bioactive GA in NSCLC A549 and H292 cells (wild-type p53 and epidermal growth factor receptor (EGFR)), which are kinds of NSCLC cells. The binding of GA to EGFR inhibited the EGFR phosphorylation, leading to the promotion of p53 expression in both A549 and H292 cells. Furthermore, highly expressed p53 decreased PD-L1 protein expression through enhancing the miR-34a related to PD-L1 downregulation at the transcriptional level. As such, our results suggest an immunosuppressive effect of GA toward NSCLC cells, which might imply a potential possibility for clinical application in NSCLC treatment.

#### **2. Results**

#### *2.1. GA Downregulates the PD-L1 Expression in NSCLC Cells*

To determine whether GA inhibits the cell proliferation of A549 and H292 cell lines, GA-treated cells were compared with non-treated control cells. The results of MTT assay showed that the cell growth of GA-treated cells is significantly inhibited in a time- and concentration-dependent manner (Figure S1A). This result was confirmed with crystal violet assay by treating GA in A549 and H292 cells for 48 h (Figure S1B). From this data, we respectively identified an IC50 dosage of 400 μM in A549 and 100 μM in H292 cell lines at 48 hours, respectively, information which was used for further studies. We checked the same concentration in non-cancerous cells (HUVEC cell line) and found that 400 μM GA inducing around 8% cell death which indicated that this concentration does not make much toxicity in normal cells (Figure S2). Recently, cancer immunotherapy based on PD-1/PD-L1 blockade has shown clinical efficacy in the treatment of multiple cancers [26,28]. In addition, a study of the drug-induced inhibition of PD-L1 expression in cancer cells has been conducted [37]. To investigate whether GA influences PD-L1 expression, we assessed the expression levels of PD-L1 by the impact of GA in NSCLC cells. As shown in Figure 1A, results from western blotting suggested that GA strongly decreases the expression levels of PD-L1 protein in A549 and H292 NSCLC cells. In addition, GA showed a greater than 70% inhibitory effect as compared with in non-treated control cells among A549 cells (Figure 1B). Subsequently, we performed a real time PCR experiment to examine the influence of GA on the messenger RNA (mRNA) expression of PD-L1 in A549 and H292 cell lines. In accordance with data from real time PCR, GA also downregulated the expression of PD-L1 mRNA in a concentration-dependent manner in both A549 and H292 cells (Figure 1C). These results suggest that bioactive natural GA has a significant inhibitory effect on PD-L1 expression (both protein and mRNA) in A549 and H292 cell lines, which imply the potential of using it as an immune anticancer agent.

**Figure 1.** Gallic acid (GA) reduces the programmed death-ligand 1 (PD-L1) expression in non-small-cell lung cancer (NSCLC) cells. (**A**) The expression levels of PD-L1 protein in A549 and H292 cells were detected after GA treatment in concentrations indicated for 48 hours. (**B**) The relative expression levels of PD-L1 protein were determined by densitometry and normalized to β-actin. Data are representative of three independent experiments. \*\*\* *p* < 0.001 (*t-*test). (**C**) The expression levels of PD-L1 mRNA in A549 and H292 cells were detected after GA treatment in concentrations indicated for 48 hours. The relative expression levels of PD-L1 mRNA were determined by real time qPCR and normalized to GAPDH mRNA. Data are representative of three independent experiments. \*\*\* *p* < 0.001 (*t-*test).

#### *2.2. GA Binds to EGFR and Then Inhibits its Phosphorylation*

EGFR phosphorylation induces various oncogenic signaling pathways for cell proliferation, invasion, and metabolic reprogramming in many cancer cells [38]. Therefore, to inhibit EGFR phosphorylation in cancer cells, many clinical applications have been stimulated to develop EGFR tyrosine kinase inhibitors (TKIs) such as erlotinib, gefitinib, and lapatinib [39,40]. In previous studies, we found that the binding of natural compounds to EGFR, causing a decrease in EGFR phosphorylation, inhibited the proliferation, migration, invasion, and angiogenesis of human breast adenocarcinoma cells [41,42]. To understand the impact of GA for EGFR phosphorylation, we identified the binding ability of GA to EGFR. Molecular docking was performed with an AutoDock Vina platform (Oleg Trott, The Scripps Research Institute, La Jolla, CA, USA). We found that GA is docked in the ATP

binding site of EGFR, and this result may imply the direct binding of GA to EGFR (Figure 2A). Subsequently, we further performed an immunoblot analysis for understanding whether the GA/EGFR binding influences the phosphorylation of EGFR and found that, GA significantly downregulated the phosphorylation of EGFR in both A549 and H292 cells (EGFR wild-type NSCLC cells) (Figure 2B,C). However, this treatment did not affect the expression levels of total EGFR mRNA (Figure 2D). These results may imply that GA could influence the inhibition of EGFR signal transduction in two NSCLC cells. Moreover, these results led us to investigate the binding specificity of GA to EGFR, where we conducted a competitive binding experiment of GA and EGF (25 ng/mL pre-treatment for 15 min) versus EGFR. Here, GA significantly inhibited EGF-induced EGFR phosphorylation in both A549 and H292 cells (Figure 2E,F). This result suggests that GA binds to EGFR as compared with the natural ligand (EGF) for EGFR, and this act of binding clearly induces the inhibition of EGFR phosphorylation.

**Figure 2.** GA binding to epidermal growth factor receptor (EGFR) inhibits the phosphorylation of EGFR. (**A**) Binding of GA (PubChem CID: 370) to the ATP-binding domain of EGFR (Protein Data Bank ID: 4LQM) as determined by molecular docking using AutoDock Vina. (**B**) The expression levels of total EGFR and phosphorylated EGFR (pEGFR) protein in A549 and H292 cells were detected after GA treatment in concentrations indicated for 48 hours. (**C**) The relative levels of pEGFR protein were determined by densitometry and normalized to β-actin. Data are representative of three independent experiments. \*\*\* *p* < 0.001 (*t-*test). (**D**) The expression levels of EGFR mRNA in A549 and H292 cells were detected by real time PCR after GA treatment in concentrations indicated for 48 hours. The relative levels of EGFR mRNA were determined and normalized to GAPDH mRNA. Data are representative of three independent experiments. (**E**) A549 and H292 cells for detecting the expression levels of total EGFR and pEGFR protein were treated with or without 25 ng/mL EGF for 15 minutes and then further treated with GA (A549: 400 μM; H292: 100 μM) for 48 hours. (**F**) The relative levels of pEGFR protein were determined by densitometry and normalized to β-actin. Data are representative of three independent experiments. \*\*\* *p* < 0.001 (*t-*test). # *p* < 0.001 vs. control.

#### *2.3. GA Reduces the Phosphorylation of PI3K*/*AKT That is One of the Downstream Targets of EGFR Signaling*

EGF/EGFR signal transduction has been known to lead to the constitutive activation of downstream signaling pathways associated with MAPKs, STAT3, and PI3K for regulating PD-L1 expression in various cancer cells [43]. A previous study found that the PD-L1 expression of EGFR–mutated PC-9 cells was significantly higher than those of EGFR wild-type LU-99, A549, and PC-14 cells. In EGFR inhibitor experiments, the EGFR TKI gefitinib induced a lower expression of phosphorylated AKT and STAT3, which prompted the downregulation of PD-L1 expression [44]. To determine a key EGFR-downstream pathway associated with PD-L1 expression, we used an immunoblot analysis. As shown in Figure S3, GA treatment did not inhibit the phosphorylation of JAK2/STAT3, which is one of the main pathways. However, GA efficiently controlled the PI3K/AKT pathway by inhibiting their phosphorylation but not total protein level (Figure 3A,B). These results clearly show that the regulation of PI3K/AKT phosphorylation by GA could be responsible for PD-L1 expression in both A549 and H292 cells. Moreover, the downregulation of PI3K/AKT phosphorylation by GA may indicate a beneficial effect in terms of controlling various oncogenic signals, such as cellular proliferation, invasion, angiogenesis, and metastasis.

**Figure 3.** GA inhibits the phosphorylation of AKT and PI3K protein in a GA concentration-dependent manner. (**A**) The expression levels of pAKT and pPI3K protein in A549 and H292 cells were detected after GA treatment in concentrations indicated for 48 hours. (**B**) The relative expression levels of pAKT and pPI3K protein were determined by densitometry and normalized to β-actin. Data are representative of three independent experiments. \*\*\* *p* < 0.001 (*t-*test).

#### *2.4. GA Activates the Expression of Tumor Suppressor Factor p53 for Inhibiting the Expression of PD-L1*

The tumor suppressor factor p53 plays an important role in cell-cycle arrest and apoptosis induction in response to oncogenic or other stresses for the prevention of cancer development. However, it is downregulated or mutated in an inactive form in almost all human cancer cells. A previous study found that p53 is led into Mdm2-mediated ubiquitination and degradation by PI3K/AKT signal transduction in breast cancer MCF-7 cells but not p53 mRNA [45]. Furthermore, p53-regulated IFN-γ induced PD-L1 expression in melanoma cells [32]. To investigate the effect on p53 by GA, we checked the protein levels of p53 with or without GA treatment in A549 and H292 cells and found that GA upregulates the expression levels of p53 protein in a concentration-dependent manner (Figure 4A,B). In addition, the expression levels of p53 protein were nearly doubled in A549 cells. Further, the mRNA levels of p53 identified by real time PCR showed a significant increase in a GA concentration-dependent manner in H292 cells as well as in A549 cells (Figure 4C). From these data, although a previous study revealed that PI3K/AKT signaling induced by their phosphorylation regulates only p53 protein levels, the PI3K/AKT signaling controlled by GA plays a key role in regulating both protein and mRNA levels of p53. These results additional imply that GA regulates p53 from mRNA levels through the downregulation of PI3K/AKT phosphorylation. To further understand the role of p53 in PD-L1 regulation, we used GA with or without p53 siRNA and determined whether specific gene silencing influences PD-L1 expression in A549 and H292 cells. As shown in Figure 4D,E, the gene silencing of p53 significantly affected the increase in PD-L1 proteins compared to non-treated control, which was decreased by GA treatment. In contrast, the effect by GA regulated the protein levels of p53 and PD-L1 in two NSCLC cells. These results imply that the regulation of PD-L1 by GA is indirectly controlled by way of inducing an increase in p53 protein level. In addition, the upregulation of p53 by GA may induce various p53-mediated anti-oncogenic factors such as the regulation of miRNA.

**Figure 4.** GA increased p53 expression. (**A**) The expression levels of p53 protein in A549 and H292 cells were detected after GA treatment in concentrations indicated for 48 hours. (**B**) The relative expression levels of p53 protein were determined by densitometry and normalized to β-actin. Data are representative of three independent experiments. \* *p* < 0.05 and \*\* *p* < 0.01 (*t-*test). (**C**) The expression levels of p53 mRNA in A549 and H292 cells were detected by real time qPCR after GA treatment in concentrations indicated for 48 hours. The relative expression levels of p53 mRNA were determined and normalized to GAPDH mRNA. Data are representative of three independent experiments. \* *p* < 0.05 and \*\*\* *p* < 0.001 (*t-*test). (**D**) The expression levels of p53 and PD-L1 protein in A549 and H292 cells, treated with GA (A549: 400 μM; H292: 100 μM) or 30 pM p53 siRNA, were detected by western blotting at 48 hours. (**E**) The relative expression levels of p53 and PD-L1 protein were determined using densitometry and normalized to β-actin. Data are representative of three independent experiments. \*\*\* *p* < 0.001 (ANOVA); # *p* < 0.001 vs. control.

#### *2.5. GA Upregulates p53-Dependent MiR-34a for Inhibiting the Expression of PD-L1*

miRNAs, a family of small noncoding RNAs, regulate wide biological processes including carcinogenesis, which severely is dysregulated in many cancer cells. Some miRNAs such as miR-513 and miR-570 directly target PD-L1 [46,47]. However, p53 indirectly regulates the expression levels of PD-L1 through inducing miR-34a in cancer cells [33]. Although many studies have shown results for the regulation of PD-L1 expression directly by miRNA, detailed studies of the actions brought on indirectly by p53 via

drugs including natural compounds is poorly understood. To understand the expression level of miR-34a by GA, we performed a real time PCR experiment because miR-34a is a well-known molecule transcriptionally induced by p53. As shown in Figure 5A, we found that it was significantly increased in a time- and GA concentration-dependent manner in both A549 and H292 cells. To further investigate miR-34a regulation by GA via p53, we additionally used p53 siRNA. The expression levels of miR-34a were decreased by p53 siRNA, but their expression levels were slightly increased by additional GA (Figure 5B). These results clearly suggest that miR-34a expression is regulated by GA-dependent p53. Additionally, we used a miR-34a inhibitor with or without GA to determine a more detailed interrelation analysis in the regulation of PD-L1 expression. In this experiment, we demonstrated that the inhibition of miR-34a function by its inhibitor is induced into an increase of PD-L1 protein which reversed by GA, but not p53 (Figure 5C,D). These results support that the expression of PD-L1 is regulated via miR-34a-induction through GA-dependent p53 in A549 and H292 cells.

**Figure 5.** GA upregulates the PD-L1 expression by p53 via miR-34a. (**A**) Relative expression levels of miR34a after treatment of GA (A549: 400 μM; H292: 100 μM) for 24 and 48 hours. Data are representative of three independent experiments. \*\* *p* < 0.01 (ANOVA); & *p* < 0.01 vs. control. (**B**) The expression levels of miR-34a in A549 and H292 cells, treated with GA (A549: 400 μM; H292: 100 μM), p53 siRNA (60 pM) or GA plus p53 siRNA were detected by RT-PCR at 48 hours. The relative expression levels of miR-34a were determined using densitometry and normalized to U6. Data are representative of three independent experiments. \*\*\* *p* < 0.001 (ANOVA); # *p* < 0.001 vs. control. (**C**) The expression levels of p53 and PD-L1 protein in A549 and H292 cells, treated with GA (A549: 400 μM; H292: 100 μM) or 30 pM miR-34a inhibitor, were detected by western blotting at 48 hours. (**D**) The relative expression levels of p53 and PD-L1 protein were determined by densitometry and normalized to β-actin. Data are representative of three independent experiments. \*\*\* *p* < 0.001 (ANOVA); # *p* < 0.001 vs. control.

#### *2.6. The Downregulation of PD-L1 Expression by GA Induces the Combination E*ff*ect with PD-1 Blockade*

To test the combination effect of PD-1 blockade and GA on antitumor activity, we evaluated cytotoxicity in an NSCLC-cell and peripheral blood mononuclear-cell (PBMC) coculture system in the presence of the anti-PD-1 monoclonal antibody (mAb) nivolumab, GA, or both. We observed a considerable apoptotic effect in the presence of both PD-1 mAb and GA in A549 and H292 cells (Figure 6A). Further, GA reduced the viability of cancer cells more effectively in comparison with a single blockade of PD-1 with PD-1 mAb. These results may indicate that the decrease of PD-L1 expression by GA regulates not only reducing survival signals of PD-L1 downstream but also activates the T-cell-mediated immune response. To further investigate the combination effect on PBMC

cytokine expression, we performed an IFN-γ analysis by enzyme-linked immunosorbent assay (ELISA). As shown in Figure 6B, GA treatment was observed to slightly increase the IFN-γ level more so than a single blockade of PD-1 with PD-1 mAb. In addition, treatment with both GA and PD-1 mAb considerably enhanced the IFN-γ production in the supernatant of the NSCLC-cell and PBMC coculture system. These results suggest that the decrease in PD-L1 expression brought about by GA enhances the effect observed with PD-1 mAb in the production of IFN-γ. Figure 7 is a graphical abstract which gave the conclusion of all these results. We checked the effect of this combination in a non-cancerous cell (HUVEC cell line) and found that these combination does not induce much cell toxicity in non-cancerous cells (Figure S3).

**Figure 6.** GA enhances the combination effect with anti-PD-1 mAb in an NSCLC-cell and peripheral blood mononuclear-cell (PBMC) coculture system. (**A**) The cell survival rates of NSCLC cells cocultured with PBMCs were examined after treatment with mock, GA (A549: 200 μM; H292: 50 μM), anti-PD-1 mAb (5 μg/mL), or both GA and anti-PD-1 mAb for 48 hours. \*\*\* *p* < 0.001 (ANOVA); # *p* < 0.001 vs. control. (**B**) The levels of interferon (IFN)-γ from the supernatants of the coculture system were measured by ELISA also following treatment with mock, GA (A549: 200 μM; H292: 50 μM), anti-PD-1 mAb (5 μg/mL), or both GA and anti-PD-1 mAb for 48 hours. \*\*\* *p* < 0.001 (ANOVA); # *p* < 0.001 vs. control.

**Figure 7.** Molecular regulatory mechanism of programmed death-ligand 1 (PD-L1) by natural bioactive gallic acid in NSCLC cells and proposed combination effect for NSCLC immunotherapy.

#### **3. Discussion**

An important concept in cancer treatment is that the cancerous cells should ideally be removed without influencing normal cells. Chemotherapy is the most common type of treatment, where chemicals or drugs to destroy cancer cells and cancer microenvironments are applied. Genomic studies such as those on *TP53, BCL2,* and *c-MYC* have accelerated the effective application of chemotherapy for developing anticancer drugs and reagents in cancer treatment [48–50]. Anticancer drugs, according to their mechanisms of action, are generally classified as either alkylating agents for damaging cancer cell DNA, antimetabolites for replacing the normal building blocks of RNA and DNA, or antibiotics for interfering with the enzymes involved in DNA replication [51–53].

Although observed therapeutic issues for NSCLC are still deemed to be unsatisfactory because of multidrug resistance and adverse effects [54,55], chemical drugs such as vinorelbine and cisplatin have been tested in NSCLC treatment [56,57]. To overcome these problems, the combined effects of two chemotherapy drugs including cisplatin or carboplatin plus one other drug have often been deployed to treat early-stage NSCLC. Despite many efforts, these chemotherapy-based regimens seem to have reached a therapeutic limit. Recently, many studies have reported the potential possibility of applying natural compounds in the treatment or control of various cancerous diseases. In previous studies, we demonstrated various anticancer effects of natural compounds [41,58,59]. Moreover, combination treatment using a chemotherapy drug and naturally derived drugs showed more effective anticancer effects, which imply that such might reduce the burden of adverse effects brought on by chemotherapy drugs alone [60]. However, a therapeutic strategy using natural compounds is difficult to apply without knowing the specific targets, which is one of the disadvantages of use. Thus, a targeted study focused on using natural compounds is essential to achieve more effective anticancer treatment. Many studies have investigated a phenolic natural compound, gallic acid, that acts as an anticancer agent against various cancers [16,17,19,20]. Nevertheless, these studies did not identify where the target position of GA is against various cancer cells or did they reveal detailed molecular mechanisms underlying the anticancer effects of GA in cancer cell death. In this study, we demonstrated that GA influences cancer cell viability and specifically binds to the tyrosine kinase receptor, EGFR in NSCLC cell lines.

EGFR is a cell-surface protein that binds with epidermal growth factor (EGF) [61]. EGFR often is mutated and/or overexpressed in several types of human cancers, including lung, ovary, breast, head, and neck cancer, and it serves to modulate the growth, differentiation, signaling, adhesion, migration, and survival of cancer cells. Usually, EGF-mediated EGFR phosphorylation induces three main signal transductions including JAK-STAT, KRAS-ERK, and PI3K-AKT-mTOR. These pathways are known to be involved in the growth, proliferation, inhibition of apoptosis, angiogenesis, and invasion of cancer cells [62,63]. For this reason, EGFR has been regarded as an attractive candidate for anticancer treatment because of its multifunctional role in tumorigenesis [38]. To date, two monoclonal antibodies, cetuximab and panitumumab, capable of inhibiting EGF or growth factor-mediated signaling pathways have been used for cancer therapy [64]. In addition, several TKIs such as erlotinib and gefitinib have been employed for the inhibition of EGFR phosphorylation. In this study, we demonstrated that GA inhibits EGFR phosphorylation by binding to EGFR in two NSCLC cells. Moreover, GA showed binding specificity and inhibited EGFR phosphorylation despite EGF-binding. These results may imply that GA is a selective and potent inhibitor against EGFR phosphorylation. Furthermore, the inhibition of EGFR phosphorylation by GA induced the downregulation of phosphorylated PI3K and AKT. Previous studies revealed that the inhibition of EGFR TKI-mediated EGFR phosphorylation induces the downregulation of phospho-PI3K and AKT [65,66]. Therefore, GA, which showed a similar effect to that of TKIs, may be a useful drug candidate for NSCLC treatment.

The tumor suppressor p53 is a transcription factor and plays a pivotal role in cell-cycle, DNA repair, senescence, and apoptosis [67–69]. Under various stresses such as DNA damage, p53 is phosphorylated and acetylated via posttranslational modification and then it is translocated to the nucleus for trans-activating numerous target genes that direct processes including cell-cycle arrest and/or apoptosis. Mutations of *TP53* have been discovered in more than 50% of human

cancers and p*53* mutation leads to not only the loss of cancer suppressive functions but also the acquisition of additional oncogenic functions such as growth and survival [70]. Wild-type p53 proteins (WTp53) are frequently downregulated because of their function of tumor suppression in many cancer cells. Previous studies have identified that the downregulation of WTp53 is associated with EGFR signal-mediated PI3K/AKT pathway activation in cancer cells [65,66,71,72]. As mentioned above, we demonstrated that the downregulation of EGFR phosphorylation by GA leads to the inhibition of PI3K and AKT phosphorylation. The decrease in their phosphorylation by GA induced the upregulation of WTp53 protein and mRNA in A549 and H292 cells. Furthermore, the competitive activity of GA in an EGF-dependent condition suggested that binding of GA to EGFR, may associated with the upregulation of p53 through inhibiting EGFR/PI3K/AKT4 phosphorylation. These results also suggest that natural bioactive GA may have a potential role as a chemotherapeutic drug for NSCLC treatment. Although many studies have revealed that p53 is related to some immune responses including IFN signaling [73,74], the expression of inflammatory cytokines and Toll-like receptors [75–77], and the activation of T- and natural killer cells [78], the correlation of p53 and tumor immune evasion is poorly understood. Recently, some studies reported that p53 interacts with the apoptotic pathway by regulating miRNAs in cancer cells [79,80]. Furthermore, the effect on p53 was augmented in miR-34a expression, which leads to decreased expression levels of PD-L1 in NSCLC cells [33]. Interestingly, we found that bioactive GA decreased the protein and mRNA levels of PD-L1 as compared with the control experiment, and the expression levels of p53 and miR-34a were upregulated by GA in NSCLC cells. These results propose that GA controls the expression of PD-L1 by regulating the p53–miR-34a pathway.

Finally, studies have revealed that PD-L1 expression in cancer cells enhances cell proliferation and resistance toward pro-apoptotic stimuli [29,81]. Furthermore, PD-L1 expression in cancer also enhances PD-L1-mediated tumor immune resistance from cytotoxic cluster of differentiation (CD)8 T-cells through the PD-1/PD-L1 blockade [82]. Thus, inhibition of PD-L1 expression will activate cytotoxic CD8 T-cell responses to various cancers. This approach has been labeled as PD-1/PD-L1 based-immunotherapy. Recently, many clinical approaches and successes are emerging through PD-1/PD-L1 blockade therapy. As mentioned above, we found that GA decreases PD-L1 expression in A549 and H292 cells. In combination experiments with a human monoclonal anti-PD-1 mAb (nivolumab), GA exhibited a more effective effect regarding cancer cell viability. In accordance with the decreased expression levels of PD-L1 by GA, the experimental condition involving anti-PD-1 mAb decreased NSCLC cell viability and oppositely increased the level of IFN-γ in the NSCLC-cell and PBMC coculture system.

#### **4. Materials and Methods**

#### *4.1. Cell Lines and Cell Culture*

Roswell Park Memorial Institute (RPMI) 1640 media and a penicillin–streptomycin solution was purchased from Gibco (Gaithersburg, MD, USA). Fetal bovine serum (FBS) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Trypsin ethylenediaminetetraacetic acid (0.05%) was obtained from Gibco (Gaithersburg, MD, USA). The human NSCLC cell lines H292 (no. 21848; Korean Cell Line Bank, Seoul, South Korea) and A549 (CCL-185; American Type Culture Collection, Manassas, VA, USA) were cultured in RPMI-1640 supplemented with 10% FBS and antibiotics (1% penicillin–streptomycin) at 37 ◦C with 5% CO2. For each experiment, at 70% to 80% confluence, cells were gently washed twice with phosphate-buffered saline. Unless otherwise specified, cells were treated with 100 μM of GA (in H292 cells) or 400 μM of GA (in A549 cells) for 48 hours at 37◦C under an atmosphere of 5% CO2.

#### *4.2. Immunoblotting*

Whole-cell lysates were prepared by radioimmunoprecipitation assay buffer (EMD Millipore, Burlington, MA, USA) containing phosphatase and protease inhibitors. Antibodies specific for β-actin

(sc-47778), p53 (sc-126) and secondary antibodies (antimouse (sc-516102), and antirabbit (sc-2357) antibody) were obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). pEGFR (#2234), EGFR (#3776s), pAKT (#4060), AKT (#4691), pPI3K (#4228), and PI3K (#4257) antibodies were purchased from Cell Signaling Technology Inc. (Danvers, MA, USA). PD-L1 (R30949) antibody was purchased from NSJ Bioreagents (San Diego, CA, USA). Recombinant human EGF (AF-100-15) was purchased from PeproTech Inc. (Rocky Hill, NJ, USA).

#### *4.3. Real Time Quantitative PCR (qPCR)*

Total RNA was isolated with the RNeasy Mini Kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer's protocol. The extracted RNA was quantified spectrophotometrically at 260 nm, and cDNA was synthesized at 42 ◦C for 1 h and 95 ◦C for 5 min with a first-strand cDNA synthesis kit (K-2041; Bioneer Corporation, Daejeon, Korea) and oligo d(T) primers. Real-time qPCR was conducted in a thermal cycler (C1000 Thermal Cycler, Bio-Rad, Hercules, CA) as follows: 2 μL diluted cDNA was added to diluted forward and reverse primers (1 μL each, 100 pM) and 10 μL TB Green Advantage Premix (Takara Bio, Japan) according to the manufacturer's instructions. We used the following primers for EGFR, sense 5'- TGGCAGTGTCTTAGCTGGTTGT -3' and anti-sense 5'- TGCACTCAGAGAGCTCAGGA -3', for PD-L1, sense 5'- TGCCAGGCATTGAATCTACA -3' and anti-sense 5'- GGCCTATTTCCTCCTCTTGG -3', for p53, sense 5'- AGGCCTTGGAACTCAAGGAT -3' and anti-sense 5'- CAGTCTGAGTCAGGCCCTTC -3', and for miR34a analysis: for miR34a, sense 5'- TGGCAGTGTCTTAGCTGGTTGT -3'. The measurement was carried out in triplicate. The relative expression of target genes was normalized to GAPDH or U6 snRNA.

#### *4.4. Transfections of siRNA and miRNA*

Lung cancer cells (1 <sup>×</sup> 10<sup>5</sup> cells) were seeded in six-well plates and grown to 60% confluence. The cells were then transfected with p53 siRNA (sc-29435; Santa Cruz Biotechnology, Dallas, TX, USA) or miR-34a inhibitor (AM 17000; Thermo Fisher Scientific, Inc., Waltham, MA, USA) using Lipofectamine transfection reagent (Thermo Fisher Scientific, Inc., Waltham, MA, USA). After 48 hours, transfected cells additionally were cultured with/without GA for an additional 48 hours under a cell culture condition.

#### *4.5. NSCLC-Cell and PBMC Co-Culture Experiments*

Lung cancer cells (5 <sup>×</sup> 104 cells) were seeded in 24-well plate until 70% to 80% confluence under a cell culture condition. Human PBMCs were isolated by Ficoll Paque density centrifugation from peripheral blood donated by healthy volunteers using Lymphoprep™ and SepMate™-50 (Stemcell Technologies, Vancouver, Canada). Then, the acquired PBMCs were added into each coculture system at a PBMCs/attached NSCLC cells ratio of 5:1. Some cocultured wells were treated with 5 μg/mL of anti-PD-1 mAb (nivolumab, #A1307; BioVision, Milpitas, CA, USA) and/or GA (A549: 200 μM and H292: 50 μM) and cultured for 48 hours. After 48 hours of co-culture, the culture supernatant was used to analyze the human IFN-γ level, while the viability of attached NSCLC cells was analyzed by MTT assay. The human IFN-γ level in cell-free supernatant was determined using an ELISA kit (#430104; BioLegend, San Diego, CA, USA) according to the manufacturer's protocol.

#### *4.6. Statistical Analyses*

All experiments were performed at least three times. Results are expressed as means ± standard errors of the mean. Statistical analyses were conducted using a one-way analysis of variance (ANOVA) or the Student's t-test. The one-way ANOVA was performed with Duncan's multiple-range test as a post-hoc test. Analyses were performed using the SAS 9.3 program (SAS Institute, Inc., Cary, NC, USA). A *p*-value of less than 0.05 was taken to indicate a statistically significant difference.

#### **5. Conclusions**

In summary, our results constitute the first study to disclose the detailed mechanism of PD-L1 downregulation, which could be mediated by bioactive natural GA in NSCLC cells. Moreover, we demonstrated that GA might not only directly inhibit cancer cell survival through the upregulation of tumor suppressor p53 but also indirectly enhance antitumor immunity through the downregulation of PD-L1. Thus, our findings additionally pave the way for further research on bioactive natural compounds to study its efficiency in combinations with immune checkpoint-based therapies and chemotherapeutic agents.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2072-6694/12/3/727/s1, Figure S1: Effects of GA on A549 and H292 cell viability. (A) MTT-based evaluation of A549 and H292 cell viability in different time- and GA concentration-dependent manners. Data are representative of three independent experiments. \*\*\* *p* < 0.001 (*t-*test). (B) A549 and H292 cell lines were treated with increasing concentration of GA for 48 h and the viability was checked using the crystal violet method. Data are representative of three independent experiments. \*\*\* *p* < 0.001 (*t-*test). Figure S2: Effects of GA on Huvec cell viability. The cell survival rates of Huvec cells were examined after treatment with 400 μM GA, anti-PD-1 mAb (5 μg/mL), or both GA and anti-PD-1 mAb for 48 hours. Figure S3: Effect of GA in Jak2/STAT3 pathway. (A) The expression levels of pSTAT3 and pJAK2 protein in A549 and H292 cells were detected after GA treatment in concentrations indicated for 48 h. Data are representative of three independent experiments. \* *p* < 0.05 (*t-*test). (B) Relative protein levels pJak2 and pSTAT3 were determined by densitometry analysis and normalized to β-actin. Data are representative of three independent experiments.

**Author Contributions:** K.-J.J. and Y.M.Y. designed the experiments. D.Y.K. and N.S. performed the most of experiments. E.S.J., A.R., and D.Y.H. helped on some of the experiments. K.-J.J., Y.M.Y., D.Y.K., N.S., H.G.L., J.-S.Y., and Q.L. analyzed the data, and K.-J.J. wrote the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was funded by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (No. 2018R1C1B6006146) and by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (No. 2019R1I1A1A01060399 and 2019R1I1A1A01060537).

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

### *Article* **Cancer Glycolytic Dependence as a New Target of Olive Leaf Extract**

#### **Jessica Ruzzolini 1, Silvia Peppicelli 1, Francesca Bianchini 1, Elena Andreucci 1, Silvia Urciuoli 2, Annalisa Romani 2, Katia Tortora 3, Giovanna Caderni 3, Chiara Nediani 1,\* and Lido Calorini 1,4,\***


Received: 19 December 2019; Accepted: 26 January 2020; Published: 29 January 2020

**Abstract:** Oleuropein (Ole), the main bioactive phenolic component of *Olea europaea* L. has recently attracted the scientific attention for its several beneficial properties, including its anticancer effects. This study is intended to investigate whether an olive leaf extract enriched in Ole (OLEO) may counteract the aerobic glycolysis exploited by tumor cells. We found that OLEO decreased melanoma cell proliferation and motility. OLEO was also able to reduce the rate of glycolysis of human melanoma cells without affecting oxidative phosphorylation. This reduction was associated with a significant decrease of glucose transporter-1, protein kinase isoform M2 and monocarboxylate transporter-4 expression, possible drivers of such glycolysis inhibition. Extending the study to other tumor histotypes, we observed that the metabolic effects of OLEO are not confined to melanoma, but also confirmed in colon carcinoma, breast cancer and chronic myeloid leukemia. In conclusion, OLEO represents a natural product effective in reducing the glycolytic metabolism of different tumor types, revealing an extended metabolic inhibitory activity that may be well suited in a complementary anti-cancer therapy.

**Keywords:** olive leaf extract; oleuropein; Seahorse analysis; cancer metabolism; glycolytic markers

#### **1. Introduction**

The Pasteur effect describes the inhibition of glycolysis by oxygen, through the inhibition of phosphofructokinase-1, the most important controlling enzyme of glycolysis, by ATP and citrate [1]. Otto Warburg, for the first time, showed that cancer cells do not follow this principle, since even under normoxic condition they prefer to exploit the glycolytic pathway, producing lactic acid from glucose. Indeed, while normal cells in the presence of oxygen use the oxidative phosphorylation, most cancer cells prefer the glycolysis, a phenomenon termed "Warburg effect" or aerobic glycolysis [2]. This is of a special importance for proliferating cancer cells which may regenerate NAD+, increase the availability of glycolytic biosynthetic intermediates and lactate production. Lactate may contribute to sustain proliferation either by stimulating the production of vascular endothelial growth factor or by promoting cellular motility, two favorable aspects for proliferating cancer cells, e.g., generation of new blood

vessels and expansion in neighboring tissues [3]. Lactic acid production and its release into the tumor microenvironment helps reduce the local extracellular pH, which might be instrumental for tumor progression, by promoting the invasive abilities of cancer cells [4], their resistance to apoptotic stimuli as well as chemo- and target therapies [5], by inducing anoikis resistance thus favouring tumor cell survival into the circulatory system [6], and importantly, by inhibiting the immune response supporting tumor cell escape [7]. The Warburg phenotype is regulated by numerous oncogenes, e.g., MYC transcription factor has been found to activate lactate dehydrogenase (LDH)A [8], and promote the switch from pyruvate kinase muscle isozyme 1 (PKM1) to 2 (PKM2), a limiting glycolytic enzyme of the final step of glycolysis, involved in the pyruvate and ATP production from phosphoenolpyruvate [9]. PKM2, in its less active dimeric form, reduces ATP generation leading to the production of lactate and several glycolytic intermediates, used as building blocks for the biosynthesis of cellular macromolecules, such as amino acids, lipids and nucleotides. In addition, mammalian target of rapamycin (mTOR) was demonstrated to be a key activator of the Warburg effect, as it induces under normoxic conditions several glycolytic enzymes, including PKM2 [10].

Recently, plant-derived compounds have drawn the attention of the scientific community for their several beneficial properties. In particular, polyphenols have been subjected to numerous studies and they showed anti-oxidant, anti-inflammatory, cardio- and neuro-protective functions as well as anti-cancer activity [11–15]. Moreover, their anti-cancer activity has been proved in a broad range of cancer models, so that some of these natural compounds have been included in clinical trials [16,17], as they showed promising effects in terms of promoting the anti-cancer response and decreasing at the same time the toxicity of conventional therapies [18–22].

Oleuropein (Ole) is the main bioactive phenolic compound of *Olea europaea* L. that has attracted great interest in the prevention and therapy of several non–communicable diseases, including cancer [23]. As to its anti-cancer properties, Ole affects and modulates multiple different biochemical processes and pathways involved in carcinogenesis. Indeed, Ole exerts an inhibitory effect on cancer cell proliferation, tumor growth and angiogenesis; it reduces inflammation and induces apoptosis [23–25]. In our previous study we found that Ole affects both the proliferation and the viability of A375 BRAF melanoma cells and potentiates their therapy response through pAKT/mTOR pathway [26]. In addition, we observed that an olive leaf extract enriched in Ole (OLEO), used at equimolar Ole concentration, was more effective to potentiate the cytotoxic effect, co-administered with conventional chemotherapeutic agents, compared to Ole alone [26]. Following this line of research, we decided to investigate if OLEO could be able to inhibit the metabolism of BRAF melanoma cells, that are usually glycolysis-addicted. The existence of a strong link between tumor-specific signalling pathways and metabolic adaptations is well known. Therefore, interfering with metabolic processes and metabolic enzymes may be a key strategy for cancer therapy. In this context, significant efforts have been recently done to elucidate how plant-derived natural compounds may act as modulators of tumor cell metabolism and, in this way, exert their anti-cancer activity [27].

Gerhauser, revising the knowledge on tumor metabolism and epigenetic variation of glycolytic genes, discovered that several of these processes are influenced by natural compounds [28]. Then, Gao and Chen underlined how several natural compounds may regulate HIF-1α-dependent anaerobic glycolysis of tumor cells: this actually represents a great contribution underlining the ability of natural products to inhibit one of the most critical transcription factors, i.e., HIF-1α, in cancer progression [29]. In this study, we proved that OLEO is able to reduce the glycolytic rate of both primary and metastatic melanoma cells, reducing the expression levels of critical glucose and lactate transporters (glucose transporter-1 (GLUT1) and monocarboxylate transporter-4 (MCT4), respectively) and enzymes, such as PKM2. Extending the study to other tumor types, we observed that OLEO is able to inhibit the glycolytic metabolism also in colorectal, breast and chronic myeloid leukemia cancer cells.

#### **2. Results**

In a previous work, with the aim to verify whether Ole might potentiate drug efficiency on BRAF mutant melanoma cells, we decided to use a non-toxic 250 μM dose able to reduce cell proliferation rate without affecting cancer cell viability and apoptosis. We found that Ole potentiates the cytotoxic effect of everolimus against BRAF melanoma cells inhibiting pAKT/mTOR pathway, as measured by the decrease of pAKT/S6. This effect was also demonstrated using an olive leaf extract enriched in an equimolar concentration of Ole [26]. Here, we confirmed that a similar OLEO, at a 200 μM dose, reduces the viability of A375 melanoma cells in a very limited amount (see the 48 and 72 h of treatment), as cell proliferation without modifying cell cycle phase distribution (Figure 1A–C). The same concentration of the extract does not modify viability of human mesenchymal stem cells at each time point of the experiments (see Figure S1). Further, the OLEO, at a 200 μM dose, significantly reduced the closure of a wound (Figure 1D), which was used as an assay of cell motility. The reduced closure of wounds of OLEO-treated melanoma cells discloses the ability of this natural product to inhibit cell motility. These findings prompted to investigate effects of OLEO on melanoma metabolism. We know that V600E mutant BRAF melanoma cells are strictly addicted to glycolysis, the so-called Warburg effect, thus it was possible that a reduction of the glycolytic pathway may have a role in the decreased proliferation and motility of OLEO-treated melanoma cells.

**Figure 1.** Effects of Ole-enriched leaf extract (OLEO) on A375 melanoma cells. (**A**) Dose-time response evaluated by MTT assay. Significance is indicated with \*; (**B**) Cell growth of A375 human melanoma cells treated with OLEO 200 μM; (**C**) Cell cycle distribution analyzed using FACS; (**D**) Effect of OLEO 200 μM on the motility of A375 cells evaluated by scratch wound healing assay. Significance is indicated with \*.

We evaluated the metabolic profile of melanoma cells after 200 μM OLEO administration through Seahorse Bioanalyzer XF96 analysis, thereafter studying the metabolic markers through real time PCR and Western Blot analysis.

We first tested the effect of OLEO extract on the glycolytic activity of A375 melanoma cells using a glycostress standard assay. Overall, OLEO impairs glycolysis rate without modifying glycolytic capacity and reserve of melanoma cells (Figure 2A). On the other hand, Mito stress analysis indicates that OLEO does not modify the respiration of melanoma cells.

To add information on OLEO-driven glycolysis inhibition, we exploited the Seahorse Bioanalyzer XF96 to check the metabolism of A375 melanoma cells exposed for 24 h to an equimolar concentration of pure Oleuropein, and we found an equivalent reducing effect on the glycolysis rate (Figure 2B), without any modification of respiration of these cells.

Further, to sustain the inhibitory role on the glycolytic metabolism of melanoma cells by OLEO, we tested its glycolysis inhibitory effect on a metastatic clone of A375 melanoma cells, called A375-M6, isolated from the lung of immunodeficient animals. In line with the previous results, we observed that OLEO exerts the same inhibitory effect on glycolysis of these metastatic cells, as showed in the glycostress analysis (Figure 2C). Overall, OLEO is able to repress the aerobic glycolysis of primary and metastatic melanoma cells.

**Figure 2.** Representative results of a glucose stress test of A375 melanoma cells treated with 200 μM OLEO (**A**) or 200 μM Ole (**B**) for 24 h, and of A375-M6 melanoma cells treated with 200 μM OLEO for 24 h (**C**). Plots on the right represent glycolysis, glycolytic reserve and glycolytic capacity extracted from glycolysis stress assay results obtained using the Seahorse Analyzer. Significance is indicated with \*.

Along with the dynamic investigation of metabolism expressed by A375 and A375-M6 melanoma cells following OLEO treatment, we identified a series of glycolytic biomarkers down regulated by our nutraceutical product. Testing both mRNA and protein levels, we observed that glucose transporter isoform 1 (GLUT1), pyruvate kinase isozymes M2 (PKM2) and monocarboxylate transporter 4 (MCT4) of OLEO-treated A375 melanoma cells are reduced by a 50% compared to control (i.e., untreated melanoma cells) (Figure 3). To underline the importance of these three key glycolytic biomarkers inhibited by OLEO: (1) GLUT1 is the major glucose transporter in cancer cells; (2) PKM2 is a modulator of glucose metabolism sustaining building block generation needed for cell proliferation; (3) MCT4

exports lactate and protons produced by glycolysis, preventing the inhibition of glycolytic enzymes such as phosphofructokinase activity, that is reduced by intracellular acidification.

**Figure 3.** Change in metabolic markers of A375 melanoma cells treated with OLEO 200 μM for 24 h. (**A**) Evaluation by quantitative real-time PCR of genes involved in metabolism; (**B**) Representative Western blot panels of PKM2, GLUT1 and MCT4 protein levels. Each band in the Western blot was quantified by densitometric analysis and the corresponding histogram was constructed by normalizing the density of each band to that of β-tubulin. Values presented are means ± SEM of three independent experiments. Significance is indicated with \*.

To extend our investigation on OLEO metabolic inhibition in cancer cells, we also tested HCT116 (a human colorectal carcinoma cell line), MDA-MB-231 (an undifferentiated triple-negative breast cancer cell line) and K562 cells (a chronic myeloid leukemia cell line) through the Seahorse Bioanalyzer XF96. OLEO does not modify substantially number and viability of colorectal, breast and leukemia cancer cells (Figure 4A,C,E), but was effective in reducing glycolysis rate of all these type of cancer cells; a higher dose of OLEO was needed to inhibit glycolysis of K562 cells. Overall, these results demonstrate a metabolic inhibitory activity of OLEO on a wide array of cancer histotypes, including that of malignant cells of a clonal disorder of hematopoietic stem cells (Figure 4B,D,F).

**Figure 4.** Effect of OLEO on glycolytic metabolism of breast cancer, colon carcinoma and myeloid leukemia cells. Cell number (**A**,**C**,**E**) and representative results of a glucose stress test of MDA-MB-231 (**B**), HCT116 (**D**), and K562 (**F**) cells, treated with 200 or 400 μM OLEO for 48 h. Plots on the right represent glycolysis, glycolytic reserve and glycolytic capacity extracted from glycolysis stress assay results obtained using the Seahorse Analyzer. Significance is indicated with \*.

#### **3. Discussion**

It is well known that cancer cells, compared to normal tissues, are characterized by a high rate of glycolytic metabolism. They indeed prefer to use glycolysis even in the presence of enough oxygen to sustain the oxidative phosphorylation (the so-called "Warburg effect" or aerobic glycolysis, to be distinguished from the anaerobic glycolysis exploited under hypoxic conditions). The higher glycolytic rate of cancer cells ensures them an adequate amount of energy and an ample availability of intermediate macromolecules useful to sustain a rapid cell proliferation and tumor mass expansion [3]. Nowadays, the deregulated metabolism is considered a hallmark of cancer and the identification of new compounds able to modulate tumor metabolism is under intense investigation. For this reason, natural agents can be a great importance, in particular because they demonstrated to interfere with most of the activities of cancer cells, at the same time showing, very low toxic effects on normal cells [11,18,30]. To sum up, several authors have underlined how plant-derived natural products interfere with tumor metabolism [22].

Here we show that OLEO is able to exert a significant inhibitory effect on cancer cell glycolysis. In particular, by a dynamic evaluation of cancer cell metabolism through the Seahorse Bioanalyzer XF96 platform, we observed that OLEO reduces the glycolytic rate of primary and metastatic melanoma cells, but also of colorectal, breast and chronic myeloid leukemia cancer cells. In line with our results, Sharma and colleagues showed that morin and/or esculetin impaired glycolysis and glutaminolysis preventing colon carcinogenesis [31]. Moreover, Gomez de Cedron and colleagues identified a new series of polyphenols characterized by a galloyl based "head" and a hydrophobic N-acyl "tail", able to inhibit glycolysis and mitochondrial respiration in colon cancer cells [32].

We observed that the glycolytic reduction exerted by our OLEO in melanoma cells is associated with a decreased GLUT1 expression, at both the mRNA and protein levels. GLUT1 is an important target in cancer treatment, being over-expressed by a wide range of tumor cells. Cancer cells may indeed take great advantages of the GLUT1 rapid response and its high affinity for glucose, in order to overcome the several stress conditions encountered in the host microenvironment and continue the progression towards malignancy. The *K*<sup>M</sup> value of GLUT1 for glucose is near 1 mM, a significantly less amount compared to the normal glucose level found in serum, allowing a relentless glucose transport into the cells. Of interest, GLUT1 represents the predominant glucose transporter isoform of fetus tissues, which exhibit a higher growth rate than adult ones, at comparable levels to those observed in tumor cells, requiring an increased supply of energy-producing substrates [32]. After birth, GLUT1 expression levels decrease and, even though the reasons behind its decline are not yet clear, it could occur a possible switch form a carbohydrate to a fat source of fuel that may induce this change in some organs [33]. For all these reasons, the development of new clinical strategies involving natural GLUT1 inhibitors such as OLEO, in combination with conventional anticancer agents, deserves the attention of the scientific community, sounding as promising combined therapeutic strategy, as recently reported for other natural compounds [34,35].

Along with GLUT1, we showed that OLEO is also able to down regulate PKM2, one of the four pyruvate kinase isoforms which is highly expressed in rapidly proliferating tissues including cancer. This metabolic enzyme is regulated by oncogenic tyrosine kinases which usually lead to an increase glycolytic rate in tumor cells. Despite, tyrosine phosphorylation of glycolytic enzymes usually increases the activities of a majority of glycolytic enzymes, the tyrosine phosphorylation of PKM2 paradoxically results in a decreased PKM2 activity that in turn promotes the Warburg effect [36]. It is possible that the OLEO-driven PKM2 reduction may reduce its glycolytic promotion [37]. PKM2 overexpression was observed in melanoma human samples compared to naevi, showing a gradient of increased expression from radial growth phase to metastatic melanoma. Furthermore, recent studies have shown that PKM2 is also able to act as a protein kinase using phosphoenolpyruvate as a substrate to promote tumorigenesis [36]. Then, Zhang and colleagues found that miR-625-5p regulates PKM2 expression at both mRNA and protein levels in melanoma cells, disclosing a miR/PKM2 role in glucose metabolism of melanoma cells [38]. PKM2 expression has been shown to be also reduced by other natural products such as resveratrol and curcumin. In particular, resveratrol inhibits aerobic glycolysis and PKM2 enzyme in HeLa (human cervical cancer), HepG2 (human liver cancer) and MCF-7 (human breast cancer) cancer cells through the inhibition of mTOR signaling [39]. Resveratrol was also demonstrated to impair hexokinase-2 enzyme in human non-small cell lung cancer cells inhibiting Akt signaling pathway [40], and pyruvate dehydrogenase complex in colon cancer cells [41]. Curcumin, a further well-known phytopolyphenolic compound, has been shown to decrease glucose uptake and lactate production in several cancer cells (lung, breast, cervical, prostate and embryonic kidney cancer cell lines) down-regulating PKM2 expression, interfering with the mTOR-HIF-1α axis [42].

MCT4 is the other glycolytic marker that we found to be inhibited by OLEO in melanoma cells. This monocarboxylate transporter acquires a key role in the metabolic activity of glycolytic cells through the proton-coupled transport of monocarboxylates, such as L-lactate, ketone bodies and pyruvate. An immunohistochemical study of the expression of MCT4 in 356 melanoma-bearing patients revealed

that this glycolytic marker is significantly increased in metastatic lesions and associated with a poor prognosis [43].

To conclude, in this study we demonstrated that OLEO is able to reduce the high glycolytic activity of various solid tumors, like melanoma, colorectal and breast cancer, but also of chronic myeloid leukemia cells, suggesting a possible usage of this natural product in combination with conventional therapy for a wide range of malignancy.

#### **4. Materials and Methods**

#### *4.1. Olive Leaf Extract's Preparation and Toxicity*

The OLEO used to treat normal and cancer cells was prepared and characterized as previously described ([26], see Figures S3 and S4). *Olea europaea* L. (cultivar Leccino), organic green leaves, were collected in April 2018 in Tuscany (Vinci, Florence, Italy) and immediately processed. The extraction using 15% of *Olea* leaves (45 g leaves/300 g double-distilled and purified water), was performed in water at a temperature of 50 ◦C for 60 min and at room temperature over the night (12 h) [44]. The final powder is obtained by lyophilization with a LYOVAC GT 2 system (Leybold GmbH, Cologne, Germany), freeze-drying yield 1.85%. The identity of the phenolic compounds of *Olea* dry extract powder and the composition of the solution used for the test in vitro, enriched in oleuropein, was ascertained using data from the HPLC/DAD and HPLC/mass spectrometry analyses, in accordance with a previous paper [45].

All the solvents (HPLC grade) and formic acid (ACS reagent) were purchased from Aldrich Chemical Company Inc. (Milwaukee, WI, USA). Tyrosol, luteolin 7-O-glucoside, chlorogenic and Ole were obtained from Extrasynthese S.A. (Genay, France). The HPLC-grade water was obtained via double-distillation and purification with a Labconco Water Pro PS polishing station (Labconco Corporation, Kansas City, MO, USA).

The OLEO used in our study has been tested in a sub-acute test of toxicity (7 days) on female F344 rats fed a diet containing 2.7 g of extract/kg of diet (corresponding to a dosage of 100 mg of extract/kg of b.w.) without inducing any change of body weight.

This lack of toxicity is in agreement with a recent study of Guex et al., which showed that an olive leaf extract, in vivo tested up to 2000 mg/kg, had no toxic or unwanted effects on rats [46]. Moreover, Sepporta et al. [47]'s paper demonstrated absence of toxicity in mice administered 125 mg of Ole/kg (b.w.). The standard dose of Ole used in vivo animal model, from 10 to 125 mg/kg, did not induce toxic effects, evaluated in terms of viability of the animals [48] or liver biomarkers, such as alanine and aspartate aminotransferase activities [49].

#### *4.2. Cell Lines and Culture Conditions*

In this study we used A375 human melanoma cell lines, obtained from American Type Culture Collection (ATCC, Rockville, MD, USA), A375M6, isolated in our laboratory from lung metastasis of SCID bg/bg mice i.v. injected with A375 [5]; human colorectal carcinoma cell line HT116, a kind gift of Dr. Matteo Lulli (Department of Clinical and Experimental Biomedical Sciences, University of Florence, Italy); human breast carcinoma cells MDA-MB-231, obtained from American Type Culture Collection (ATCC); human leukemia cells K562, a kind gift of Prof. Persio Dello Sbarba (Department of Clinical and Experimental Biomedical Sciences, University of Florence) and human mesenchymal stem cells (MSC) obtained from bone marrow aspirates of donors which signed informed consent [50]. A375, A375M6, HCT116 and MDA-MB-231 were cultured in Dulbecco's Modified Eagle Medium high glucose (DMEM 4500, EuroClone, Milan, Italy) supplemented with 10% fetal bovine serum (FBS, EuroClone); K562 were cultured in Roswell Park Memorial Institute 1640 medium (RPMI, EuroClone) supplemented with 10% FBS; MSC were expanded in Dulbecco's modified Eagle's medium with low glucose (DMEM 1000; Gibco, Life Technologies, Monza, Italy) supplemented with 20% FBS. Cells were maintained at 37 ◦C in humidified atmosphere containing 90% air and 10% CO2 and they were

harvested from subconfluent cultures by incubation with a trypsin-EDTA solution (EuroClone), and propagated every three days. Viability of the cells was determined by trypan blue exclusion test. Cultures were periodically monitored for mycoplasma contamination using Chen's fluorochrome test. Cells were treated with OLEO for 24–72 h.

#### *4.3. MTT Assay*

Cell viability was assessed using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) tetrazolium reduction assay (Sigma Aldrich, Milan, Italy) as described in [26]. Cells were plated into 96-multiwell plates in complete medium without red phenol. The treatment was added to the medium colture at different dose and times, according to the experiment. Then the MTT reagent was added to the medium and plates were incubated at 37 ◦C. After 2 h, MTT was removed and the blue MTT–formazan product was solubilized with dimethyl sulfoxide (DMSO, Sigma Aldrich). The absorbance of the formazan solution was read at 595 nm using the microplate reader (Bio-Rad, Milan, Italy).

#### *4.4. Cell Cycle Analysis*

Cell cycle distribution was analyzed by the DNA content using propidium iodide (PI) staining method. Cells were centrifugated and stained with a mixture of 50 μg/mL PI (Sigma-Aldrich, St. Louis, MO, USA), 0.1% trisodium citrate and 0.1% NP40 (or triton x-100) in the dark at 4◦C for 30 min. The stained cells were analyzed by flow cytometry (BD-FACS Canto, BD-Biosciences, San Jose, CA, USA) using red propidium-DNA fluorescence as previously described [5].

#### *4.5. Wound Healing Assay*

Cell migration was evaluated by an in vitro wound healing assay as previously described [6]. Cells were treated for 24 h with the extract, then cells have been detached and sown in 35 mm dishes at high confluence; cell monolayer was wounded with a sterile 200 mL pipette tip, washed with PBS and incubated in 1% FBS culture medium. Wound was analyzed following a 24-h incubation and photographed using phase contrast microscopy.

#### *4.6. Seahorse Analysis*

Seahorse analysis has been performed as previously described [51]. The extracellular acidification rate (ECAR) and the Oxygen Consumption Rate (OCR) were determined using the Seahorse XF96 Extracellular Flux Analyzer (Seahorse Bioscience, Billerica, MA, USA) through Seahorse XF Glycolysis Stress Test Kit (Agilent Technologies, Santa Clara, CA, USA), measuring preferentially the glycolytic function in cells, or Seahorse XF Mito Stress Test Kit (Agilent Technologies), measuring the dependence of cells on the oxidative metabolism. Cells were counted and seeded in XF96 Seahorse® microplates precoated with poly-D-lysine (ThermoFisher Scientific, Waltham, MA, USA). Cells were suspended in XF Assay Medium supplemented with 1 mM glutamine (from EuroClone, Paington, UK) in order to assess ECAR, in XF Assay Medium supplemented with 2 mM glutamine in order to assess OCR. Cells were left to adhere for a minimum of 30 min at 37 ◦C. The plate was left to equilibrate in a CO2-free incubator before being transferred to the Seahorse XF96 analyzer. The pre-hydrated cartridge was filled with the indicated compounds and calibrated for 30 min in the Seahorse Analyzer. All the experiments were performed at 37 ◦C. Normalization to protein content was performed after each experiment. The Seahorse XF Report Generator automatically calculated the parameters from Wave data that have been exported to Excel or Graphpad.

#### *4.7. RNA Isolation and Quantitative PCR (qPCR)*

Total RNA was isolated from cells by using TRI Reagent (Sigma, Milan, Italy). The amount and purity of RNA were determined spectrophotometrically. cDNAwas obtained by incubating 2 μg of total RNA with 4 U/μL of M-MLV reverse transcriptase (Promega, San Luis Obispo, CA, USA) according to the manufacturer's instructions. Quantitative real time PCR (qPCR) was performed as reported in [51] using the GoTaq® Probe Systems (Promega). The qPCR analysis was carried out in triplicate using an Applied Biosystems 7500 Sequence Detector with the default PCR setting: 40 cycles at 95 ◦C for 15 s, 60 ◦C for 60 s. mRNA was quantified with the DDCt method as described [52]. mRNA levels were normalized to β2 microglobulin as an endogenous control. The primer sequences used are listed in Table 1.

**Table 1.** Primer sequences for PCR.


#### *4.8. Western Blotting Analysis*

Cells were lysed and separated using electrophoresis as previously described [5]. Cells were washed with ice cold PBS containing 1 mM Na4VO3, and lysed in 100 mL of cell RIPA lysis buffer (Merck Millipore, Vimodrone, Milan, Italy) containing PMSF (Sigma-Aldrich), sodium orthovanadate (Sigma-Aldrich) and protease inhibitor cocktail (Calbiochem). Aliquots of supernatants containing equal amounts of protein (40 mg) in Laemmli buffer were separated on Bolt® Bis-Tris Plus gels 4e12% precast polyacrylamide gels (Life Technologies, Monza, Italy). Fractionated proteins were transferred from the gel to a PVDF (polyvinylidene difluoride) membrane using iBlot 2 system (Life Technologies, Monza, Italy). Blots were stained with Ponceau red to ensure equal loading and complete transfer of proteins, and then they were blocked for 1 h, at room temperature, with Odyssey blocking buffer (Dasit Science, Cornaredo, Milan, Italy). Subsequently, the membrane was probed at 4 ◦C overnight with primary antibodies diluted in a solution of 1:1 Odyssey blocking buffer/T-PBS buffer. The primary antibodies were: rabbit anti-PKM2 (1:1000, Cell Signaling Technology, Danvers, MA, USA), rabbit anti-MCT-4 (1:1000, Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-GLUT1 (1:1000, Cell Signaling Technology). The membrane was washed in T-PBS buffer, incubated for 1 h at room temperature with goat anti-rabbit IgG Alexa Flour 750 antibody or with goat antimouse IgG Alexa Fluor 680 antibody (Invitrogen, Monza, Italy), and then visualized by an Odyssey Infrared Imaging System (LI-COR® Bioscience, Lincoln, NE, USA). Mouse anti-β tubulin monoclonal antibody (1:1000, Cell Signaling Technology) was used to assess equal amount of protein loaded in each lane.

#### *4.9. Statistical Analysis*

Densitometric data are expressed as means ± standard errors of the mean (SEM) depicted by vertical bars from representative experiment of at least three independent experiments. Statistical analysis of the data was performed by ANOVA and Tukey's multiple comparisons test, and *p* ≤ 0.05 was considered statistically significant.

#### **5. Conclusions**

The rapid growth of cancer cells mainly depends on their high glycolytic metabolism. Indeed, tumor cells, compared to normal tissues, prefer to exploit the glycolytic pathway even in the presence of sufficient oxygen to sustain the oxidative phosphorylation. This is likely due to the fact that glycolysis guarantees a rapid availability of metabolic intermediates, assuring not only sufficient energy for their survival, but also an efficient production of nucleotides, amino acids and lipids needed to duplicate cell content before mitosis. In this study we show that the natural product OLEO is able to reduce the glycolytic rate of a wide range of solid and liquid tumor cells, without affecting their basal respiration but rather down-regulating the expression of three key effectors of the glycolytic pathway, i.e., GLUT-1, PKM2 and MCT4, likely resulting in a decreased glucose entrance and biomass production. Thus, the inhibition of glycolysis by OLEO acquires a great significance for the targeting of cancer cell growth and expansion. Our findings, together with previous evidence showing the anti-cancer effects exerted by OLEO, pure Ole and analogs [23,53,54], reinforce the hypothesis to propose the use of these natural compounds in combination with conventional therapy used in the treatment of cancer.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2072-6694/12/2/317/s1, Figure S1: Cell growth of human MSC treated with OLEO 200 μM, Figure S2: Detailed information of protein expression analysis by Western blot: (A) Original blot for the Figure 3B, (B) Densitometry and intensity ratio of each band.

**Author Contributions:** Conceptualization, J.R., C.N. and L.C.; validation, S.P., E.A., F.B.; formal analysis, J.R., S.P., E.A.; investigation, J.R., S.P., F.B., G.C., K.T.; resources, S.U., A.R., C.N., L.C.; writing—original draft preparation, J.R., S.P., L.C.; writing—review and editing, F.B., E.A., C.N., A.R., S.U., G.C.; visualization, J.R., S.P.; supervision, L.C., J.R.; project administration, L.C., C.N.; funding acquisition, L.C., C.N., A.R. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by Istituto Toscano Tumori and Ente Cassa di Risparmio di Firenze.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


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