Effect of gold-conjugated resveratrol nanoparticles on glioma cells and its underlying mechanism

Abstract

BACKGROUND:

Glioblastoma is the most aggressive brain tumor with poor prognosis. Although Resveratrol (Rsv) is known to have therapeutic effects on glioma, the effects of gold-conjugated resveratrol nanoparticles (Rsv-AuNPs) on glioma cells are rarely reported.

OBJECTIVE:

We aimed to investigate the effects of Rsv-AuNPs on glioma cells and its underlying mechanism.

METHOD:

Human glioma cell line U87 was treated with different concentrations of Rsv-AuNPs. CCK-8, transwell, and wound healing assay were performed to measure the effects of Rsv-AuNPs on cell proliferation, invasion, and migration ability, respectively. Flow cytometry assay was used to detect the effects of Rsv-AuNPs on apoptosis. Changes of protein expressions related to proliferation, invasion, migration, and apoptosis were measured by Western blot assay. In addition, the inhibitory role of Rsv-AuNPs in the PI3K/AKT/mTOR signaling pathway was verified by using PI3K inhibitor LY294002.

RESULTS:

Rsv-AuNPs treatment significantly suppressed proliferation, migration, and invasion of U87 cells (all P < 0.05) and increased the apoptosis rate (P < 0.05). The changes of proteins related to proliferation, migration, invasion and apoptosis were consistent (all P < 0.05). Moreover, Rsv-AuNPs treatment significantly inhibited the phosphorylation of PI3K, AKT and mTOR proteins in U87 cells (P < 0.05).

CONCLUSION:

The present study found that Rsv-AuNPs inhibited the proliferation, migration, and invasion of U87 cells and induced apoptosis by inhibiting the activation of PI3K/AKT/mTOR signaling pathway. In the future, Rsv-AuNPs might be applied to the clinical treatment of glioma through more in-depth animal and clinical research.

Keywords

Gold-conjugated resveratrol nanoparticles glioma cell mechanism

1. Introduction

Glioma, which is characterized by rapid proliferation and invasiveness of tumor cells, commonly develops in the central nervous system and accounts for about 80% of malignant brain tumors [1–4]. According to the World Health Organization, glioma is divided into four grades from grade I to grade IV, with the latter being the most invasive with dismal prognosis [5,6]. Currently, surgery, radiotherapy and chemotherapy are the main strategies for treating glioma, but its 5-year survival rate is still only about 5% [7].

Resveratrol (Rsv) is a phenol ubiquitously present in many plants such as grapes, mulberry and peanut [8]. Previous studies have shown that Rsv possesses various therapeutic effects, such as anti-oxidation, anti-inflammatory and heart protection [9–11]. It has also been found that Rsv could play a protective role in Alzheimer’s disease, Parkinson’s disease, and other neurodegenerative diseases [12–14]. In addition, Rsv has also been shown to have anti-cancer activities for breast, lung, and liver cancers [15]. In the treatment of glioma, prior preclinical investigations revealed that Rsv inhibited the proliferation of glioma cells and suppressed glioma angiogenesis and tumor growth [17]. These preliminary findings suggested that Rsv could be an adjuvant for the treatment of glioblastoma [18].

The advent of nanotechnology has revolutionized and opened a new horizon for cancer diagnosis and treatment. The gold nanoparticles (AuNPs) are widely used as nanomaterials for theranostic and biomedical applications for a host of medical conditions, including cancer, neurodegenerative diseases and hepatitis [19,20]. Compared with simple metallic nanoparticles, gold nanoparticles combined with phytochemicals have enhanced cell absorption, bioavailability, and anti-cancer activities [21]. Previous studies that used Rsv as a reducing agent and stabilizer to biosynthesize AuNPs found that the synthetic Rsv-AuNPs could dramatically suppress the migration and invasion of breast cancer cells [22]. Rsv-AuNPs has also been noted to suppress pancreatic cancer cell proliferation by downregulating the expressions of cyclin A, cyclin B, promoting oxidative stress by enhancing cytochrome C expression, and inducing apoptosis by decreasing BcL-2 expression, and increasing Bax expression [23]. However, studies that assessed the effects of Rsv-AuNPs on glioma cells remain scarce.

Therefore, this study aimed to investigate the effects of Rsv-AuNPs on glioma cell functions and its potential mechanism to provide new ideas in the treatment of glioma. In order to ascertain whether the anticancer effects of Rsv-AuNPs were superior to Rsv or AuNPs alone, we also compared the impacts of Rsv, AuNPs and Rsv-AuNPs on the functions of glioma cells.

2. Materials and methods

2.1. Materials

Rsv, HAuCl4 and AuNPs were purchased from Sigma-Aldrich (USA, #34092, #484385, #900475). Dimethyl sulfoxide, phosphate buffered saline (PBS), trypsin digestion solution with or without EDTA and 100×penicillin-streptomycin mixture were obtained from Solarbio (China, #D8371, #P1022, #T1320, #T1350, #1400). CCK-8 kit was purchased from Dojindo (Japan, #CK04). RIPA lysis buffer, BCA protein assay kit, blocking solution, primary antibody diluent, second antibody diluent, TBST, ECL luminescent assay, secondary antibodies, paraformaldehyde, crystal violet solution were all purchased from Beyotime (China, #P0013B, #P0012S, #P0252, #P0256, #P0258, #P0231, #P0018FS, #A0201, #P0099, #C0121). Polyvinylidene difluoride (PVDF) membrane was obtained from Millipore (USA, #IPVH00010). Dulbecco’s modified eagle medium (DMEM), fetal bovine serum were purchased from GIBCO (USA, #12491015, #16140089). Pipette Tips were bought from Axygen (USA, #MR-200-RL, #MR-10XT-RL, #MR-1000XT-RL). Transwell upper chamber, cell culture dish and plates, and matrigel were all obtained from Corning (USA, #4395, #351029, #354721, #353072). Annexin V-FITC/PI double staining kit was purchased from BD (USA, #556547). The specific primary antibodies were obtained from Abcam: β-actin (1:3000; #8226, Abcam, UK), p-AKT (1:1000; #38449, Abcam, UK), AKT (1:2000; #ab8805, Abcam, UK), p-PI3K1:1000; #182651, Abcam, UK), PI3K (1:2000; #18416, Abcam, UK), p-mTOR (1:1000; #109268, Abcam, UK), mTOR( 1:2000; #32028, Abcam, UK), Cyclin E1 (CCNE1, 1:1000; #133266, Abcam, UK), Cyclin D1 (CCND1, 1:1000; #40754, Abcam, UK), matrix metalloproteinase 2 (MMP-2,1:2000; #92536, Abcam, UK), MMP-9 (1:2000; #76003, Abcam, UK), Bax (1:1000; #32503, Abcam, UK), Cleaved Caspase-3 (1:2000; #32042, Abcam, UK), Caspase-3 (1:2000; #32351, Abcam, UK).

2.2. Synthesis and identification of Rsv-AuNPs

Rsv-AuNPs were synthesized using the chloroauric acid reduction method, as previously reported [24]. In brief, Rsv (10 μg/mL) dissolved in dimethyl sulfoxide was added to a stirred HAuCl4 solution (1 mM) and kept under room temperature for 10 min till the color changed from yellow to deep rugby red, indicating the successful production of AuNPs. The solution was continuously mixed at room temperature for 2 h to obtain Rsv-AuNPs. The prepared solution was then centrifuged at 18,000 g for 1 h. Rsv-AuNPs were kept and washed with deionized water for 3 times, followed by centrifuging at 12,000 g for 10 min to ensure absence of unbound Rsv molecules in the NP dispersion. Rsv-AuNPs powder was obtained by freeze-drying. The particle powder was resuspended in deionized water, and the UV-Vis spectrum of Rsv-AuNPs solution was measured in the wavelength range of 300--800 nm using Ultrospec 6300 Pro. The average particle size and zeta potential of Rsv-AuNPs were measured according to the dynamic light scattering method [23]. The morphology and distribution of Rsv-AuNPs were observed by transmission electron microscope (Hitachi H-7650, Japan).

2.3. Cell culture

Human glioma cell line U87 was purchased from Shanghai Fuheng Biotechnology (China). The cells were cultured in a DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin. Once U87 cells reached 80–90% confluence, adherent cells were trypsinized, counted and replated.

2.4. CCK-8 test

U87 cells were seeded into 96-well plates at a density of 10⁴/well. After culture for 24 h, 10 μL CCK-8 solution was added to each well and the plates were cultured for 3 h. Then the cell plate was placed into a microplate reader (Multiskan MK3; Thermo Fisher Scientific) to measure the absorbance value at the wavelength of 450 nm.

2.5. Cell migration assay

U87 cells were seeded into 6-well plates at a density of 3 × 10⁵ cells/well. When the cells reached 70–90% confluency, sterilized 200 μL pipette tips were used to draw three straight lines parallel to each other at the same distance at the bottom of each hole. The debris was then washed with PBS, and treatment with Res, AuNPs or Res-AuNPs were carried out. Finally, a culture medium containing 5% fetal bovine serum was added, and then the plate was cultured for 24 h. At 0 and 24 h, cell migration was observed under an inverted microscope and analyzed by the Image J software (National Institutes of Health, USA).

2.6. Transwell assay

U87 cells (5 × 10⁴/well) and 200 μL serum-free medium was added to the transwell upper chamber coated with matrigel. 500 μL normal medium containing 10% fetal bovine serum was added to the lower chamber. After incubation for 24 h, the upper surfaces of the transwell chambers were scraped with cotton swabs, and the migrated and invaded cells were fixed with 4% paraformaldehyde and stained with crystal violet solution. The transmembrane cells were counted under an inverted optical microscope (Nikon, Japan).

2.7. Flow cytometry

Annexin V-FITC/PI double-staining assay was used to detect cell apoptosis. U87 cells were seeded into 6-well plates at a density of 10⁶/well and treated with Res, AuNPs or Res-AuNPs. The cells were then harvested, suspended and stained using Annexin V-FITC/PI. The cells were analyzed by flow cytometry (NovoCyte, ACEA Biosciences) and the apoptosis rate was calculated.

2.8. Western blot

Proteins were extracted from whole-cell lysate and separated by 10% SDS-PAGE electrophoresis, followed by transferring to the PVDF membrane. Then, the PVDF membrane was cultured with blocking solution at room temperature for 1 h and incubated with the corresponding primary antibodies at 4 °C overnight. The membrane was then incubated with corresponding secondary antibody conjugated to horseradish peroxidase for 1 h at room temperature. The membrane was treated with chemiluminescence reagents according to the vendor’s instructions and analyzed by the Image J software.

2.9. Statistical analysis

All experiments were performed independently for 3 times. All results in this study were expressed as mean ± standard deviation. SPSS 18.0 (SPSS, USA) software was used for data analysis. Under the same treatment condition, one-way analysis of variance (ANOVA) and Student’s t-test were applied for comparisons among 3 groups and 2 groups, respectively. A statistically significant difference was considered at P < 0.05.

3. Results

3.1. Characterization of Rsv-AuNPs

As shown in Fig. 1, the maximal absorbance, diameter and the zeta potential of Rev-AuNPs was about 540 nm, 36.89 ± 18.95 nm and - 34.7 ± 2.14, respectively. Under transmission electron microscopy (Fig. 1E), Rev-AuNPs were predominantly spherical with a diameter of 37.66 ± 6.24 nm and equally distributed, indicating that Rsv is a reliable reducing agent and stabilizer.

Fig. 1.

Characterization of Rsv-AuNPs. (A, B) UV-vis spectra of Rsv and Rsv-AuNPs; (C, D) The particle size and zeta potential of Rsv-AuNPs were measured by dynamic light scattering. (E) Transmission electron microscopy images of Rsv-AuNPs.

3.2. Rsv-AuNPs suppressed glioma cell proliferation

To explore the effects of Rsv-AuNPs on the proliferation of glioma cells, U87 cells were treated with Rsv-AuNPs at a concentration of 0 (Control), 5 μg/mL, 10 μg/mL and 20 μg/mL for 12, 24, 36 and 48 h, respectively. We found that Rsv-AuNPs could inhibit the proliferation of U87 cell proliferation in a dose-dependent manner (Fig. 2A, P < 0.05). Moreover, Rsv-AuNPs treatment for 24 h significantly suppressed the expression of proliferation-related proteins, including CCNE1 and CCND1 (Fig. 2B, all P < 0.05).

Fig. 2.

Effects of Rsv-AuNPs on the proliferation of glioma cell line U87 cells. (A) Cell proliferation was measured by CCK-8 assay; (B) The expression levels of proliferation-related proteins CCNE1 and CCND1 were detected by Western blot assay. Compared with Control group (0 μg/mL Rsv-AuNPs), ^∗ P < 0.05, ^∗∗ P < 0.01, ^∗∗∗ P < 0.001.

In addition, we also explored whether treatment of Rsv-AuNPs for 24 h could have any cytotoxicity on normal glial cells or other glioma cell lines. As shown in Supplementary Fig. S1, the treatment of Rsv-AuNPs had no cytotoxicity on normal glial cell line HEB. Although 1 and 5 μg/mL Rsv-AuNPs could significantly inhibit the proliferation of both U87 and U251 cells (all P < 0.05), the cytotoxicity was more obvious in U87 cells. Thus, we used U87 cell lines in our study.

3.3. Rsv-AuNPs inhibited the invasion and migration of glioma cells

Transwell and wound healing assay was used to evaluate the effects of Rsv-AuNPs on the invasion and migration of U87 glioma cells, respectively. As shown in Figs 3A and 3B, Rsv-AuNPs significantly inhibited the invasion and migration of U87 cells (all P < 0.05) after treating with different concentrations of Rsv-AuNPs (1 and 5 μg/mL) for 24 h. Compatibly, the expressions of MMP-2 and MMP-9, both of which were indicators for cell invasion and migration, were noted to be decreased by Western Blot (Fig. 3C, all P < 0.05).

Fig. 3.

Effects of Rsv-AuNPs on invasion and migration of glioma U87 cells. (A) Transwell assay was used to detect the invasion ability of cells; (B) The changes of cell migration ability were observed by wound healing assay; (C) The expression MMP2 and MMP9 were measured by Western blot assay. Compared with Control group (0 μg/mL Rsv-AuNPs), ^∗∗ P < 0.01, ^∗∗∗ P < 0.001.

3.4. Rsv-AuNPs induced U87 apoptosis

Compared with the Control group, Res-AuNPs treatment could significantly induce U87 cells apoptosis (Fig. 4, all P < 0.05). In addition, Western Blot also showed decreased expressions of Bax but increased expression of Cleaved Caspase-3 (all P < 0.05), further suggesting increased apoptosis with Rsv-AuNPs treatment.

Fig. 4.

Effects of Rsv-AuNPs on U87 cell apoptosis. (A) Apoptosis was detected by flow cytometry; (B) The expressions of apoptosis-related proteins MMP2 and MMP9 were measured by Western blot assay. Compared with Control group, ^∗∗ P < 0.01, ^∗∗∗ P < 0.001.

3.5. Rsv-AuNPs suppressed the activation of PI3K/AKT/mTOR signaling pathway

Compared with the Control group, Res-AuNPs treatment could significantly inhibit the activation of PI3K/AKT/mTOR signal pathway in U87 cells (Fig. 5, all P < 0.05).

Fig. 5.

Effects of Rsv-AuNPs on the activation of PI3K/AKT/mTOR signal pathway in U87 cells. Compared with Control group, ^∗ P < 0.05, ^∗∗ P < 0.01.

3.6. Validation of inhibitory effects of Rsv-AuNPs on the activation of PI3K/AKT/mTOR signaling pathway

We then used PI3K inhibitor LY294002 to further verify the inhibitory effect of Rsv-AuNPs on the PI3K/AKT/mTOR signaling pathway. As shown in Fig. 6A–E, Rsv-AuNPs or LY294002 treatment alone could inhibit U87 cells proliferation (all P < 0.05), invasion and migration ability (all P < 0.05), and induce apoptosis (all P < 0.05). Meanwhile, the changes of function-related proteins were consistent with them (all P < 0.05). In addition, the activation of the PI3K/AKT/mTOR signaling pathway was significantly suppressed with Rsv-AuNPs or LY294002 treatment alone (all P < 0.05). The inhibitory effects were more obvious with combined treatment of Rsv-AuNPs and LY294002. These results suggested that Rsv-AuNPs played an anti-tumor role by suppressing the activation of PI3K/AKT/mTOR signaling pathway.

Fig. 6.

Validation of the inhibitory effects of Rsv-AuNPs on PI3K/AKT/mTOR signaling pathway. (A) Cell proliferation was measured by CCK-8 assay; (B) Transwell assay was used to detect the invasion ability of cells; (C) The changes of cell migration ability were observed by wound healing assay; (D) Apoptosis was detected by flow cytometry; (E, F) The expression of cell function-related proteins, including CCNE1, CCND1, MMP2, MMP9, Bax, Cleaved Caspase-3 and PI3K/AKT/mTOR signaling pathway, were measured by Western blot assay. Compared with Control group, ^∗ P < 0.05, ^∗∗ P < 0.01, ^∗∗∗ P < 0.001; Compared with Rsv-AuNPs (1 μg/mL), ^# P < 0.05,^## P < 0.01, ^### P < 0.001.

3.7. Comparison of the effects of Rsv, AuNPs and Rsv-AuNPs on the functions of glioma cells

To determine whether Rsv alone or in combination with AuNPs could have such anti-glioma effects, we compared the effects of Rsv, AuNPs and Rsv-AuNPs on the proliferation, migration, invasion and apoptosis of U87 cells. The selection of Rsv and AuNPs concentrations were based on previous studies [25,26]. As shown in Fig. 7A–F, compared with the Control group, Rsv, AuNPs and Rsv-AuNPs group significantly inhibited the cell proliferation (all P < 0.05), invasion and migration ability (all P < 0.05), and induced apoptosis (all P < 0.05). The changes of function-related proteins were consistent with them (all P < 0.05). Although the activation of the PI3K/AKT/mTOR signaling pathway was significantly inhibited in all groups (all P < 0.05), it was more obvious with combined treatment of Rsv and AuNPs (all P < 0.05).

Fig. 7.

Comparison of the effects of Rsv (10 μg/mL), AuNPs (30 μg/mL) and Rsv-AuNPs (1 μg/mL) on the functions of glioma cells. (A) Cell proliferation was measured by CCK-8 assay; (B) Transwell assay was used to detect the invasion ability of cells; (C) The changes of cell migration ability were observed by wound healing assay; (D) Apoptosis was detected by flow cytometry; (E, F) The expression of cell function-related proteins, including CCNE1, CCND1, MMP2, MMP9, Bax, Bad, Bcl-2 and PI3K/AKT/mTOR signaling pathway, were measured by Western blot assay. Compared with Control group, ^∗ P < 0.05, ^∗∗ P < 0.01, ^∗∗∗ P < 0.001; Compared with Rsv group, ^$ P < 0.05, ^$$ P < 0.01, ^$$$ P < 0.001.

4. Discussion

Glioma is a common intracranial malignancy. Although considerable progress has been made in the surgical and medical treatment of glioma, the prognosis of patients with glioma is still poor [3,4]. Previous studies have shown that Rsv has significant neuroprotective and anti-cancer effects [12–15]. Moreover, the current application of nanomaterials as new and effective anti-cancer drugs in humans has shed light on novel treatments for glioma. In line with this, AuNPs are one of the most important nanoparticles used extensively for medical applications [19]. Our study found that Rsv-AuNPs showed significant anti-glioma effects by inhibiting glioma cell proliferation, invasion, migration and inducing apoptosis. In addition, suppression of the PI3K/AKT/mTOR signaling pathway might also be involved in the inhibitory effect of Rsv-AuNPs on glioma cells.

In congruent with our study results, prior studies have also demonstrated that Rsv could inhibit glioma cell proliferation, invasion, and migration and induce apoptosis [27–29]. Similarly, Babaei et al. found that AuNPs could significantly induce apoptosis of U87 cells, and inhibit cell proliferation and migration ability in a time-dependent manner [30]. Based on previous reports, we demonstrated that Rsv-AuNPs could suppress glioma cell proliferation in a time- and dose-dependent manner. In addition, Rsv-AuNP treatment could also inhibit cell invasion, migration ability and induce apoptosis.

Next, we explored the underlying molecular mechanism of Rsv-AuNPs in regulating glioma cell functions. Earlier investigations have consistently noted that the PI3K/AKT/mTOR signaling pathway played a crucial role in tumor cell proliferation, migration, invasion and apoptosis [31]. Furthermore, aberrant activation of the PI3K/AKT/mTOR signaling pathway in human glioma have been corroborated [32,33]. mTOR is a downstream molecule in the PI3K/AKT signaling pathway that functions to regulate the expression of cyclins, such as CCNE1 and CCND1 [34]. In this study, the phosphorylation of AKT, PI3K and mTOR were significantly decreased in the Rsv-AuNPs treated group as compared with the control group. These results suggested that Rsv-AuNPs could inhibit the activation PI3K/AKT/mTOR signaling pathway in U87 cells. To validate this hypothesis, we used LY294002, a PI3K inhibitor, to further confirm the inhibitory effects of Rsv-AuNPs on U87 cells. We noted that the inhibitory effects of Rsv-AuNPs on U87 cells proliferation, invasion, migration and pro-apoptotic effects were similar to that of LY294002 alone. In addition, combined treatment of Rsv-AuNPs and LY294002 showed enhanced anti-cancer effects than Rsv-AuNPs or LY294002 treatment alone. These results suggested that Rsv-AuNPs indeed exerted anti-cancer effects by inhibiting the activation of PI3K/AKT/mTOR signaling pathway.

5. Conclusion

In this study, Rsv-AuNPs were found to suppress the proliferation, migration, and invasion of U87 cells and induce apoptosis by suppressing PI3K/AKT/mTOR signaling pathway activation. In the future, it is expected to be applied to the clinical treatment of glioma through more in-depth animal and clinical research.

Footnotes

Acknowledgements

The authors have no acknowledgments.

Ethics statement

This article does not contain any studies with human participants or animals performed by any of the authors.

Conflict of interest

The authors declare that they have no conflict of interest.

Data availability statement

The datasets generated and/or analysed during the current study are available from the corresponding author on reasonable request.

Funding

This study was supported by Nantong Municipal Health Commission Scientific Research (No. MB20210800).

Supplementary materials

The supplementary materials are available from .

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