Alpha-Lipoic Acid, Auraptene, and Particularly Their Combination Prevent the Metastasis of U87 Human Glioblastoma Cells

Background The primary malignant brain tumor glioblastoma multiforme (GBM) is most commonly detected in individuals over 60 years old. The standard therapeutic approach for GBM is radiotherapy combined with temozolomide. Recently, herbal products, such as alpha-lipoic acid (ALA) and auraptene (AUR), have shown promising anticancer effects on various cancer cells and animal models. However, it is not well understood how ALA, AUR, and their combination in GBM work to combat cancer. Thus, the purpose of this study was to investigate the antimetastatic effects of the ALA-AUR combination on U87 human glioblastoma cells. Methods The inhibitory effects of ALA, AUR, and the ALA/AUR combination on the migration and metastasis of U87 cells were evaluated using a wound healing test and gelatin zymography. The expression levels of matrix metalloproteinase MMP-2 and MMP-9 were assessed at the transcriptional and translational levels using quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting, respectively. Results Our findings revealed that combination therapy reduced cell migration and metastasis, which was indicated by the reduction in MMP-2/-9 expression both at mRNA and protein levels, as well as their enzymatic activity in U87 cells. Conclusion This study demonstrated that the combination of ALA and AUR effectively inhibited the migration and metastasis of U87 cells. Thus, given their safety and favorable specifications, the combination of these drugs can be a promising candidate for GBM treatment as primary or adjuvant therapy.


Introduction
Glioblastoma multiforme (GBM) is a malignant and progressive brain tumor that arises from glial cells called astrocytes. GBM is classifed into four stages based on the staging system developed by the World Health Organization (WHO) [1]. GBM mainly afects individuals over 60 years old and those with a family history of GBM and has a higher incidence rate in men compared to women [2]. Te main molecular characteristics of GBM are the high proliferation rate and distant metastasis, which explain the low survival rates among GBM patients (14-16 months) [3]. Complete resection of the lesions followed by radiotherapy and chemotherapy with temozolomide is the current standard of care for managing GBM [4]. GBM tumors often grow quickly and recur either as a result of incomplete removal or resistance to radiation and chemotherapy [5,6]. In recent decades, herbal and natural products have been highlighted as novel anticancer agents due to their specifc properties [7,8]. Terefore, in the current study, we examined the inhibition of the migration of glioblastoma cancer cells by the combination of alpha-lipoic acid (ALA) and auraptene (AUR).
1.1. ALA. ALA and its reduced form known as dihydrolipoic acid (DHLA) (Figure 1) are herb-derived compounds with notable antitumor efects [10]. Regarding its favorable properties and scant toxicity, ALA seems to be a desirable candidate for alternative therapy in cancer [11][12][13]. ALA is a pleiotropic compound with a variety of pharmacological activities, such as antidiabetic, antiobesity, antiinfammatory, and hypotensive efects. ALA is also centrally involved in glutathione regeneration and vitamins C and E metabolism in addition to its ability to chelate heavy metals [14][15][16][17]. Furthermore, previous studies have shown that ALA efectively alleviates cancer cell proliferation and angiogenesis and stimulates the apoptosis of these cells; thus, it is considered a suitable candidate for cancer therapy [18,19]. Recent studies have indicated that ALA attenuates cell proliferation by triggering the AMP-activated protein kinase (AMPK) signaling cascade and the consequent inhibition of the v-Akt murine thymoma viral oncogene/protein kinase-B (Akt/PKB) signaling [20,21]. Te proteolytic breakdown of the extracellular matrix components by gelatinases, such as matrix metalloproteinase MMP-2 and MMP-9, is a critical step in cancer development and metastasis [22,23]. Previous studies have collectively suggested ALA as a potent inhibitor of MMP-2 and MMP-9 activities, which reduces the invasion of various cancer cells [24][25][26]. ALA also successfully induces cancer cell death by increasing the expression of proapoptotic genes such as Bcl-2-associated X protein (Bax) and decreasing the expression of the primary antiapoptotic gene, B-cell lymphoma 2 (Bcl2) [27,28].
1.2. AUR. AUR, also known as 7-geranyloxycoumarin, is a bioactive monoterpene coumarin ether ( Figure 2) that is derived from the members of the Rutaceae and Apiaceae families [29]. AUR lacks toxicity and exhibits efciency against various cancers; therefore, it can be proposed as a novel alternative anticancer agent [30,31]. AUR is a pleiotropic compound with distinct pharmacological effects in the body including antidiabetic, antigenotoxic, antiinfammatory, antioxidant, and immunomodulatory properties [32,33]. AUR potently inhibits tumorigenesis and tumor proliferation by targeting various cytokines, growth factors, and transcription factors as well as their downstream signaling pathways. It also exerts regulatory efects on apoptosis and genes modulating cellular proliferation [34]. Consistently, AUR alleviates the proliferation of MCF-7 cells and suppresses cyclin D1, which is pivotal for cell cycle promotion and its dysregulation is widely observed in cancers [35,36]. Similar to ALA, previous studies have proposed AUR to be a metastasis inhibitor indicated by the suppression of MMP-2 and MMP-9 enzymatic activities. Moreover, AUR efciently decreased angiogenesis in several cancers by diminishing the expression of vascular endothelial growth factor (VEGFR)-1/2 [37][38][39]. In addition, AUR has been previously shown to suppress the Bcl-2 family expression and stimulate the Bax expression gene, thus acting as an apoptosis inducer in cancer [40,41].
It is crucial to suggest new therapeutic techniques with higher success rates and fewer side efects because GBM is one of the most aggressive and malignant types of tumors and current medications occasionally fail or have undesirable side efects. In recent decades, herbal compounds have been widely used for cancer treatment due to their vast range of pharmacological efects, availability, and safety. Terefore, we examined the efects of the ALA/AUR combination on cell migration and metastasis of U87 cancer cells in the current study for the frst time.

Cell
Culture. Te U87 cells were kept in a humidifed incubator at 37°C and a 5% CO 2 concentration. Te cells were cultured in high-glucose DMEM supplemented with 10% FBS and 1% penicillin-streptomycin antibiotics, and the culture media were changed daily.

Scratch Wound-Healing Assay.
Te inhibitory efects of the ALA/AUR combination on the migratory properties of U87 cells were evaluated using the wound-healing scratch assay. A total of 7 × 10 5 cells/well were seeded into 6-well  plates. After 24 h of incubation at 37°C, when the confuency of the attached cells reached 90%, the cell monolayer was scratched using a sterile pipette tip. Ten, the detached cells were cautiously washed with phosphate-bufered saline (PBS), and the attached cells were treated with 1/4 of the half-maximal inhibitory concentration (IC50) of each compound (AUR: 0.05 mM, ALA: 3.125 mM) and their combination. Images of the wound gap were captured using an inverted microscope at 0, 24, and 48 h posttreatment and analyzed with ImageJ software.

Gelatin Zymography.
Te enzymatic activity of MMP-2 and MMP-9 in U87 cells was determined using gelatin zymography [42]. For this purpose, U87 cells were treated with AUR (0, 0.2, and 0.4 mM), ALA (0 and 12.5 mM), and a combination of both (0.2 mM AUR-12.5 mM ALA). After 24 h of incubation, each sample's culture medium was collected and centrifuged at 13000 rpm at 4°C for 12 min, and the protein concentration of the supernatant was determined using the BCA technique. Equal amounts of the samples (20 μg per lane) were loaded on a 10% separating SDS-PAGE gel with 0.1% gelatin. Every 20 min after electrophoresis, the gel was washed three times with washing bufer containing 2.5% Triton X-100 (to completely remove the SDS), and then it was kept at 37°C for 24 h in the developing bufer (containing 2.5% Triton X-100 in 50 mM Tris (PH 7.4), CaCl 2 (5 mM), and ZnCl 2 (1 μM)). Te gel was then stained with a staining bufer (0.5% Coomassie brilliant blue R-250 in distilled water containing 25% methanol and 10% acetic acid) at room temperature for 90 min. In the next step, the gel was destained in a 10% acetic acid solution until white bands appeared. Te gelatinolytic activity of the samples (the density of the gelatine degradation zones) was captured by a GS-800 ™ calibrated densitometer (Bio-RAD, USA) and quantifed using Image J 1.52a software.

Gene Expression Analysis.
Using an RNA extraction kit (Pars Tous, Iran), the total RNA of the cells was extracted in order to further explore the expression of MMP-2 and MMP-9 after the ALA/AUR treatment of U87 cells. Ten, using a NanoDrop spectrophotometer, the purity and quantity of RNAs were determined. In the next step, complementary DNAs (cDNAs) were created from RNAs by following the instructions of the Easy cDNA Synthesis Kit (Pars Tous Co., Iran). Quantitative real-time polymerase chain reaction (qRT-PCR) was conducted using SYBR Green PCR master mix (SMOBIO, Taiwan) on a Roche realtime thermal cycler. Table 1 lists the specifc primers for MMP-2 and MMP-9. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a housekeeping gene to normalize gene expression data, and fold changes were calculated using the 2 −∆∆CT method [43].

Western
Blotting. For this purpose, 7 × 10 5 U87 cells/ well were seeded into a 6-well plate and incubated overnight. Ten cells were treated with AUR (0 and 0.2 mM), ALA (0 and 12.5 mM), and ALA/AUR combination (0.2 mM AUR-12.5 mM ALA) for 24 h. Afterwards, radio-immuno precipitation assay (RIPA) lysis bufer was used to lyse the cells, and the lysates were centrifuged. Te obtained supernatants were kept at −70°C, and the BCA method was employed to measure the protein content of each sample, following procedures involving the separation of the proteins from the lysates using 7.5-15% SDS-PAGE and transferring them onto a polyvinylidene difuoride (PVDF) membrane. After skim milk blocking, the primary antibodies were diluted as instructed by the manufacturer (1 : 1,000 dilution), and then the membrane was incubated overnight with the primary antibodies at 4°C. In the next step, for 1 h, the membranes were treated with the horseradish peroxidase-conjugated secondary antibody (1 : 3000 diluted). Te SuperSignal ® West Femto kit (Termo Fisher Scientifc, Inc., USA) was used to detect the amounts of each protein. Te relative expression was quantifed using the Image J 1.52a software and was compared to that of the betaactin protein as the housekeeping control [44].

Statistical Analysis.
One-way analysis of variance (ANOVA) was used, followed by the Dunnett test, to compare the values and quantitative ratios obtained from the various groups. All experiments were run in triplicate, and the results are shown as mean (SD). P values less than 0.05 were considered to be statistically signifcant.  [45]. Tus, we performed a scratch assay to investigate the inhibitory efects of the ALA/AUR combination on cell migration. Our results are demonstrated in Figure 1. As observed, the ALA/AUR combination signifcantly diminished the migratory properties of U87 cells compared to monotherapy or the control group ( Figure 3).

Gelatinase Activity Was Diminished by ALA/AUR
Combination. Here, we used gelatin zymography to examine the efect of the ALA/AUR combination on MMP-2 and MMP-9 enzymatic activities. In comparison with the control group, AUR and ALA, as well as the ALA/AUR combination, signifcantly reduced MMP-2/9 enzymatic activity in U87 cells. Te combination signifcantly reduced MMP-2/9 activity compared to ALA and AUR monotherapy, as measured by the quantitative examination of gelatinolytic zones (Figure 4).

ALA/AUR Combination Decreased the mRNA Levels of MMP-2 and MMP-9.
To further investigate the efect of ALA, AUR, and their combination on the transcriptional expression of gelatinases MMP-2 and MMP-9, the qRT-PCR method was employed. Figure 5 indicates that the mRNA levels of MMP-2 and MMP-9 in U87 cells were signifcantly Evidence-Based Complementary and Alternative Medicine lowered after ALA, AUR, or ALA/AUR treatment compared to the control group. Te ALA/AUR combination also exhibited a stronger inhibitory efect on MMP-2 and MMP-9 expressions compared to ALA or AUR monotherapy (p < 0.0001).

ALA/AUR Combination Attenuated the Protein Levels of MMP-2 and MMP-9.
We employed western blotting to determine how the ALA/AUR combination afected the levels of MMP-2 and MMP-9 proteins in vitro. A signifcant reduction in MMP-2 levels was observed in the ALA/AUR  Evidence-Based Complementary and Alternative Medicine combination group compared to the monotherapy or control groups ( Figure 6).

Discussion
Tumor progression, which includes metastasis, is dependent on the ability to infltrate adjacent tissues and spread throughout the body. Te process of metastasis, which is responsible for malignancy and, as a result, the death of patients, is based on the migration and invasion of cancer cells [46]. Tus, this process has been particularly targeted for therapeutic interventions in numerous in vivo and in vitro studies. In the feld of cancer treatment, some monotherapies may not be adequately efective, and, therefore, it seems necessary to boost the favorable efects of these therapies, limit the extent of drug resistance, and shorten the length of treatments. According to the previous studies based on cell and animal models, ALA and AUR have shown distinct anticancer efects as well as fewer side efects [34,47]. However, no study has been conducted on the combination of both drugs regarding cell migration. Terefore, this study investigated the in vitro efects of the ALA/AUR combination on cell migration and metastasis of U87 cells for the frst time.
Te obtained data demonstrated that the ALA/AUR combination exerts more antimigratory and antimetastatic efects than monotherapy with each of these compounds. Te efects of the ALA/AUR combination on cell migration were frst assessed using a wound healing assay. In U87 cells, the ALA/AUR combination signifcantly suppressed cell migration relative to ALA and AUR monotherapies. Ji Jeon et al. reported that ALA inhibited cell migration and invasion by repressing the epithelialmesenchymal transition (EMT) in thyroid cancer cells [21]. A study conducted by Yamasaki et al. revealed that ALA reduced cell migration through inhibitory efects on integrin β1 expression, which plays an important role in cell adhesion in bladder cancer cells [48]. Te antimigration and antiangiogenic activities of AUR were also detected by Charmforoshan et al. and were found to be directly related to a reduction in cell migration and the mRNA expression levels of VEGFR-1 and VEGFR-2 [38]. Overall, ALA, AUR, and particularly the ALA/AUR combination can potentially be suitable candidates for hindering and controlling the metastasis of U87 cells.
Gelatinases, particularly MMP-2 and MMP-9, are efcient agents that trigger metastasis by causing the proteolytic destruction of extracellular matrix (ECM) components [22,46]. Tese enzymes are released in their inactive form and are subsequently activated by a mechanism referred to as the cysteine switch. Numerous studies have demonstrated that MMPs are crucial to the metastatic process [47][48][49]. To investigate the activity of these enzymes, the gelatin zymography analysis was performed. Te results revealed a remarkable inhibition of the gelatinase activity of U87 GBM cells after treatment with the ALA/AUR combination. In agreement with our results, it was reported that ALA suppresses the MMP-2 activity in mouse embryonic fbroblasts Balb/3T3 (3T3 cells) [49]. Furthermore, Lee et al. discovered that ALA signifcantly reduced the MMP-2/9 enzymatic activity while also suppressing metastasis and migration in the MDA-MB-231 breast cancer cell line [26]. Tripathy et al. highlighted ALA as an MMP-9 and TGF-β activity inhibitor, consequently attenuating the cell invasion and metastasis of 4T1 and MDA-MB-231 breast cancer cells [25]. Regarding the antimetastatic properties of AUR, Jamialahmadi et al. proved that AUR impedes the migration and metastasis of A2780 ovarian cancer cells by suppressing the MMP-2/9 activity [37]. In a model of dextran sulfate sodium (DSS)-induced ulcerative colitis, the DSS-induced increase in the enzymatic activity of MMP-2, -7, and -9 was diminished by AUR therapy [39]. Altogether, in addition to indicating the prominent contribution of MMP-2/9 to cell migration, these fndings highlight the notable modulatory efects of ALA and AUR treatment, particularly in combination with each other, on these enzymes.
To further investigate the efect of the drug combination on gelatinases MMP-2/9 and whether their enhanced activity is due to increased protein and expression levels, MMP-2/9 mRNA and protein levels were determined. Te results showed that monotherapy with ALA or AUR could lead to a reduction in transcriptional and translational expression levels of MMP-2 and MMP-9 while the combination of both drugs exhibited a more potent efect. In accordance with our results, it was reported that ALA decreases MMP-2/9 expression in breast cancer cells and vascular smooth muscle cells [24,25], thereby reducing cell migration. Similarly, Kawabata et al. showed that AUR diminished the expression of MMPs in colorectal adenocarcinoma cells through the modulation of the extracellular signal-regulated kinase (ERK) signaling pathway [50]. In addition, it was reported that AUR reduced MMP-9 secretion in lipopolysaccharide (LPS)-stimulated human macrophages [51]. Terefore, it can be concluded that the combination of AUR/ALA decreases gelatinase activity partly via downregulating MMP-2/9 expression.

Conclusion
According to our results, the combination of ALA and AUR showed promising potential in the treatment of GBM by reducing the expression and activity of proteins involved in metastasis and migration, such as MMP-2 and MMP-9. Tis combination is safe and exhibits remarkable antimetastatic efects against GBM, but its efcacy should be verifed in other cell lines and animal models. Furthermore, mechanism-based investigations are needed to fully elucidate the mechanism underlying the anticancer and antimetastatic efects of the ALA/AUR combination. Notably, there is still a dearth of data regarding the combination of these compounds, and the characterization and identifcation of the components are crucial to address all concerns; nonetheless, our data collectively suggest this combination as an alternative antimetastatic agent.

Data Availability
All data generated during this study are included within the article.

Ethical Approval
Te research was approved by the Research Ethics Committee of Kerman University of Medical Sciences (IR.KMU.AH.REC.1400.053).

Conflicts of Interest
Te authors declare that there no conficts of interest.

Authors' Contributions
AS and MS guided the study design, AI performed the experiments and wrote the draft of the manuscript, FM and MJ designed the experiments and contributed to the interpretation of the results, and AS supervised the project. Finally, the manuscript has been reviewed and approved by all the authors.