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ORIGINAL ARTICLE
Year : 2020  |  Volume : 16  |  Issue : 2  |  Page : 263-268

Luteolin-loading of Her-2-poly (lactic-co-glycolic acid) nanoparticles and proliferative inhibition of gastric cancer cells via targeted regulation of forkhead box protein O1


1 Digestive Department, The First Affiliated Hospital of Fujian Medical University, Taijiang, P.R. China
2 Digestive Department, Hospital of Fujian Normal University, Fujian Normal University, Shangsan, Cangshan, P.R. China
3 State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Shanghai Jiaotong University School of Medicine, Shanghai, P.R. China
4 Digestive Department, Union Hospital of Fujian Medical University, Gulou, Fuzhou, Fujian, P.R. China

Date of Submission04-Jul-2018
Date of Decision12-Nov-2019
Date of Acceptance15-Nov-2019
Date of Web Publication28-May-2020

Correspondence Address:
Dan Li
Department of Gastroenterology, Union Hospital, Fujian Medical University, 29 Xinquan Road, Fuzhou 350001, Fujian Province
P.R. China
Jian Ding
Department of Gastroenterology, First Affiliated Hospital, Fujian Medical University, 20 Chazhong Road, Fuzhou 350005, Fujian Province
P.R. China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jcrt.JCRT_438_18

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 > Abstract 


Background: Developing the natural medicine that allow for the specific targeting cytotoxicity is a very important research area in the development of anti-tumor drugs.
Aims and Objectives: This study was conducted to determine the targeted inhibitory effects of luteolin-loaded Her-2-poly (lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) on gastric cancer cells and to delineate the mechanism underlying the inhibition of tumors by luteolin.
Materials and Methods: Luteolin-loaded Her-2-PLGA NPs (Her-2-NPs) were prepared, physically and chemically characterized, and their effects on gastric cancer cells were investigated. The rate of NP uptake by cells and the cell morphology were observed using confocal microscopy; the rates of cell proliferation and apoptosis were identified using the 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide assay and flow cytometry, respectively; and the mRNA and protein expression levels of forkhead box protein O1 (FOXO1) were determined using quantitative polymerase chain reaction and Western blotting, respectively.
Results: Compared with nontargeted microspheres, Her-2-NPs led to significantly enhanced uptake of luteolin by SGC-7901 cells. Luteolin-loaded Her-2-NPs also significantly inhibited the proliferation of gastric cancer cells, weakened their migratory ability, and increased both the mRNA and protein expression levels of FOXO1.
Conclusion: Luteolin-loading of Her-2-NPs could potentially be used as a novel anti-cancer drugs for targeted cancer therapy.

Keywords: Drug therapy, Her-2 antibody, poly (lactic-co-glycolic acid) nanoparticles, targeting


How to cite this article:
Ding J, Li Q, He S, Xie J, Liang X, Wu T, Li D. Luteolin-loading of Her-2-poly (lactic-co-glycolic acid) nanoparticles and proliferative inhibition of gastric cancer cells via targeted regulation of forkhead box protein O1. J Can Res Ther 2020;16:263-8

How to cite this URL:
Ding J, Li Q, He S, Xie J, Liang X, Wu T, Li D. Luteolin-loading of Her-2-poly (lactic-co-glycolic acid) nanoparticles and proliferative inhibition of gastric cancer cells via targeted regulation of forkhead box protein O1. J Can Res Ther [serial online] 2020 [cited 2020 Sep 30];16:263-8. Available from: http://www.cancerjournal.net/text.asp?2020/16/2/263/285187




 > Introduction Top


Gastric cancer that originates in the gastric mucosal epithelium is the most common malignancy occurring in the digestive tract and has a complex etiology with a high incidence rate; it has been reported to be the second most common cause of mortality among all malignancies.[1],[2] Studies have reported that more than 30% of human malignancies exhibit Her-2 overexpression, with Her-2 expression in gastric cancer tissues reported to be higher than that in the adjacent normal tissues.[3],[4] On the other hand, since its discovery in 1990, more than 90 types of forkhead box genes (FOX gene, first derived from the “fork” mutation in fruit flies[5]) have been identified. There are reports of low expression of transcription factor FOX protein O1 (FOXO1) in tumor tissues of patients with Her-2-positive gastric cancer, which could lead to the progression of malignancies and a poor prognosis.[6],[7],[8]

Although chemotherapy is an important strategy for the treatment of tumors,[9] chemotherapeutic drugs have extremely severe cytotoxic effects and are predisposed to tumor resistance. Consequently, natural medicine has been focused on for the development of antitumor drugs. Studies on traditional Chinese medicine have identified and confirmed numerous herbal preparations that have yielded satisfactory clinical and therapeutic outcomes on tumors. Flavonoids have been reported to demonstrate a wide range of pharmacological effects,[10],[11] including antibacterial effects, protective effects on the heart and blood vessels, antispasmodic effects, expectorant effects, inhibitory effects on enzymatic activity, immunosuppressive effects, antioxidation effects, antiradiation effects, and diuretic effects. Luteolin is a natural flavonoid compound that has been shown to possess the diverse above-mentioned pharmacological effects.[12] A large number of studies have demonstrated that luteolin inhibits the proliferation of malignancies, primarily by inducing cell apoptosis and cell cycle arrest, thereby suggesting that it could be a potential new drug for the treatment of gastric cancer.[13],[14] We analyzed the biological efficacy of luteolin by incorporating it into poly (lactic-co-glycolic acid) (PLGA) nanoparticles (NPs)[15],[16] that were modified with Her-2 antibody, to develop immunotargeted microspheres with sustained release of the drug.


 > Materials and Methods Top


Experimental materials

The following experimental materials and equipment were used in this study: Gastric cancer SGC-7901 cells (ATCC), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), dimethyl sulfoxide (DMSO) (Shanghai Beyotime Biotechnology), RE52CS Rotary Evaporator (Beijing No. 6 Factory), SK5200H Ultrasonic Instrument (Shanghai Kudos Ultrasonic Instrument Co., Ltd.), Agilent 1100 High-Performance Liquid Chromatography (Agilent Technologies, Inc.), EmulsiFlex-C5 high-pressure homogenizer (Avestin, Canada), TEM-100CX II transmission electron microscope (Nippon Electronics Co., Ltd., Japan), Zetasizer 3000HS laser particle size analyzer (Malvern Corporation, UK), laser-focused microscope (Media Cybernetics Inc., Washington, USA), FACS Caliber flow cytometer (BD, New Jersey, USA), and polymerase chain reaction (PCR) amplifier and electrophoresis instrument (Beijing No. 61 Factory).

Preparation of Her-2-poly (lactic-co-glycolic acid) nanoparticles

First, 40 mg of PLGA-COOH and 20 mg of hexadecyl quaternized carboxymethyl chitosan[17],[18] were dissolved in 4.0 mL of CH2 Cl2 to afford the oil phase, and luteolin was dissolved in 12 mL of phosphate-buffered saline (PBS, pH = 7.4) to afford the aqueous phase. The aqueous and oil phases were then mixed and subsequently emulsified for 3 min using an ultrasonic probe system in an ice bath. After the formation of a uniform emulsion, the residual CH2 Cl2 was removed using a rotary evaporator, and drug-loaded PLGA-COOH microspheres were obtained. Next, 0.3 mg of anti-Her-2 antibody and 3.0 g of EDC and NHS were added to the above-mentioned drug-loaded PLGA NP solution and transferred into a 50 mL three-necked flask. The mixture was stirred in an ice bath for 6 h and then dialyzed for 6 h to remove the unreacted coupling agent and yield Her-2-antibody-modified PLGA-NPs (Her-2-NPs).

Cell culture

The SGC-7901 cells were cultured in Dulbecco's Modified Eagle Medium containing 10% fetal bovine serum in an incubator with 5% CO2 at 37°C. After culturing for 24 h, at 66% confluency, the culture medium was replaced for passaging. Cells from the logarithmic growth phase were selected for the experiment.

Cytotoxicity experiment

The gastric cancer SGC-7901 cells and BGC-823 cells culturedin vitro were seeded onto 96-well plates at a density of 8000 cells per well containing 100 μL of the culture medium. The cells were cultured overnight, and then NPs or Her-2-NPs were added at gradients such that their final concentrations reached 0, 10, 50, 100, 200, 500, and 1000 μg/mL. The cells were cultured at 37°C for 0.5, 2, and 24 h, and 10 μL of MTT reagent (5 mg/mL) was added to each well. The plates were then placed in an incubator for 3 h, and the formazan crystals were observed under a microscope. After removing the medium, 150 μL of DMSO solution was added to each well to dissolve the crystals, and the results were read using a microplate reader at a wavelength of 490 nm and were statistically analyzed.

Cell uptake experiment

SGC-7901 cells in the logarithmic growth phase were seeded onto 6-well plates at a density of 5 × 105 cells per well. After culturing the cells at 37°C for 24 h, an appropriate amount of PE-labeled NPs was added to each well such that the concentration of rhodamine reached 20 ng/mL. The cells were incubated at 37°C for 4 h and then rinsed thrice with PBS, and 2 μg/mL 4,6-diamino-2-phenyl indole (DAPI) solution was added. The cells were incubated for 15 min at room temperature, washed three times with precooled PBS, and fixed with 4% POM for 15 min. The excess paraformaldehyde was then removed, and the cells were stored in cooled PBS. The uptake profile of the cells was observed under a laser-focused microscope.

Proliferative inhibition of cells as evaluated via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay

SGC-7901 cells from a logarithmically growing culture were harvested, digested with trypsin, and single-cell suspensions were obtained. After mixing, cells were seeded into 96-well plates at a density of 5 × 103 cells per well. PBS was added to the marginal empty wells of the plates to prevent evaporation of the culture medium. The cell suspensions in the 96-well plates were cultured in an incubator with 2.5% CO2 at 37°C for 12 h, after which the excess liquid in the plates was removed. Drug-loaded NPs were added to each well such that the final concentrations of luteolin reached 0, 12.5, 25, 50, and 100 μg/mL. The cells were cultured in the incubator for 24, 48, and 72 h. The medium was then removed, and 200 μL of DMSO was added to each well. The mixture was shaken at 37°C for 15 min, and the absorbance value (OD) of each empty well was measured at 490 nm using a microplate reader. The cell viability was calculated from three duplicates for each well. The proliferative inhibition rate of the cells was calculated using the following formula: proliferative inhibition rate (%) = (1 − D experimental group/D control group) × 100%.

Clone formation inhibition test

SGC-7901 cells in the logarithmic growth phase were harvested, digested with trypsin, and prepared into single-cell suspensions. The cell density was adjusted to 500 cells/mL, and then 1 mL/well of suspension was added to 12-well plates. The cells were cultured in the incubator for 24 h, after which NPs were added until the final concentrations of luteolin reached 0, 1.25, 2.5, 5, and 10 μg/mL. The cells were then cultured in the incubator for another 7 days. The culture medium was then removed, and the cells were washed twice with PBS before the addition of fixation solution (methanol: acetic acid = 3:1) for 10 min followed by drying. The cells were subsequently stained with crystal violet for 15 min, following which the staining solution was removed by washing, the cells were dried, and the number of cell clones was recorded and photographed.

Detection of cell apoptosis by flow cytometry

SGC-7901 gastric cancer cells in the logarithmic growth phase were harvested, digested with trypsin, and prepared into single-cell suspensions. After mixing, cells were seeded into 6-well plates at a density of 2 × 105 cells per well and cultured in an incubator for 24 h. Different concentrations of drug-loaded NPs were subsequently added to the cells such that the final concentrations of luteolin reached 0, 1.25, 2.5, 5, and 10 μg/mL, and then the cells were cultured for another 48 h. The cells were then washed thrice with precooled PBS, resuspended in 200 μL of binding buffer, and mixed with 5 μL of propidium iodide and 10 μL of Annexin V-FITC. After staining in the dark, the specimens were immediately placed on ice, and the treated cells undergoing apoptosis were detected by flow cytometry.

Detection of mRNA and protein expression levels by reverse transcription polymerase chain reaction and Western blotting

At 48 h posttreatment with the luteolin-loaded microspheres, the cells were harvested, centrifuged at 950 × g for 10 min, and washed twice with PBS, with the excess PBS being removed. The gastric cancer cells were lysed using TRIzol, and total RNA was extracted. The cDNA template was obtained following the instructions of the Takara RT kit, and the relative mRNA expression level of FOXO1 was detected by the two-step SYBR Green assay method.[19],[20]

The primers for FOXO1 were 5'-TGTTCGACAGTCAGCCGC-3' (forward) and 5'-GGTGTCTGAGCGAT-3' (reverse). The primers for the internal reference GAPDH were 5'-TGTTCG ACAGTCAGCCGC-3' (forward) and R: 5'-GGTGTCTGAGCGAT-3' (reverse). The relative expression levels were calculated using the 2ΔΔCT method.

At 48 h posttreatment with luteolin-loaded microspheres, the cells were harvested, centrifuged at 950 ×g for 10 min, and washed twice with PBS, with the excess PBS being removed. The cells were then lysed using cell lysate for 15 min on ice, centrifuged at 1200 ×g for 15 min, and the supernatant was collected. After the quantification of protein using the bicinchoninic acid assay, equal amounts of specimens were separated by 10% SDS-PAGE electrophoresis, transferred onto the polyvinylidene difluoride membrane, blocked with 5% skim milk powder, and incubated with primary antibody overnight at 4°C and then secondary antibody for 2 h at room temperature. After incubation with enhanced chemiluminescence, the specimens were developed.

Statistical methods

The experimental data were processed using SPSS 18.0 software(International Business Machines Corporation,1 New Orchard Road, Armonk, New York 10504-1722, United States). The data were expressed as x̶ ± s, and P < 0.05 indicated a statistically significant difference. Completely randomly designed single-factor analysis of variance (one-way ANOVA) was used to compare intergroup differences, with a detection level (α) of 0.05.


 > Results and Discussion Top


Physical characterization of Her-2 nanoparticles

The high-pressure liquid chromatography results showed that the encapsulation efficiencies of luteolin by PLGA-NPs and Her-2-PLGA-NPs were 91.8% ± 6.2% and 90.4% ± 6.1%, respectively, demonstrating that the microspheres prepared in this study displayed a high efficiency of luteolin encapsulation. [Table 1] shows the results of particle size and potential measurements using nanosize, which shows that a slightly larger nano-microsphere size and slightly lower potential as result of the Her-2 antibody modification.
Table 1: Nanoparticle size and zeta potential

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The transmission microscopy results are shown in [Figure 1]. PLGA-NPs [Figure 1]a had regular morphology, spherical or spherical, and a size of around 110 nm in the dry state, slightly smaller than the DLS (Dynamic Light Scattering) results. This was probably because of the contraction of PLGA-NPs during specimen preparation and the higher surface roughness of the Her-2-antibody-modified NPs, which was not as regular as that of PLGA-NPs [Figure 1]b. This indicated that the antibody modification resulted in significant alterations to the surface structure of the NPs and facilitated the recognition and affinity by antigens on the cell surface.
Figure 1: Electron micrograph of nanospheres containing luteolin. (a) NPs-Lu, (b) Her-2-NPs-Lu. NPs: Nanoparticles

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Cytotoxicity experiments

The prerequisites for the clinical application of biological nanomaterials are good biocompatibility and low cytotoxicity.[21] As shown in [Figure 2], the Her-2-PLGA-NPs prepared in this study exhibited low inhibitory effects on gastric cancer cells at 100 μg/mL after 24 h, and the cytotoxic effect on the gastric cancer cell lines gradually increased when the concentration was >500 μg/mL for 24 h. The NPs prepared in this study demonstrated low toxicity on gastric cancer cells at the normal-use dose of ≤500 μg/mL, thus laying the basis for further studies on drug loading and targeted delivery.
Figure 2: Toxicity of no-load nanospheres to gastric cancer cells. (a) Toxicity of no-load nanospheres to SGC-7901 cells. (b) Toxicity of no-load nanospheres to BGC-823 cells

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Laser confocal microscopy

The gastric cancer cells were treated with PE-labeled targeted and nontargeted NPs at the same concentration for 30 min. Subsequently, sectioning was performed, and the phagocytosis of the two types of NPs by the gastric cancer cells was observed by laser confocal microscopy [Figure 3]. Compared with the nontargeted NPs, the Her-2-antibody-modified NPs were phagocytosed more rapidly by the gastric cancer cells. This result suggests that antibody modification significantly enhanced the efficiency of recognition and endocytosis of NPs by gastric cancer cells, thereby laying the foundation for targeted drug delivery.
Figure 3: Phagocytotic effect of gastric cancer cells on nanospheres

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Cell proliferation rate and invasive ability

As shown in [Figure 4], the luteolin-loaded NPs had a stronger inhibitory effect on the proliferation of gastric cancer cells in both the free drug group and the nonfree drug group at 48 h after the cells had been treated with NPs (P < 0.05). The proliferative inhibition of gastric cancer cells increased significantly with increasing concentration, and the inhibitory effects in both the targeted and nontargeted groups increased. The targeted delivery of Her-2 significantly enhances the inhibitory effects of luteolin on tumor cell growth.
Figure 4: Inhibition curve of luteolin-coated nanospheres on gastric cancer cells

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As shown in [Figure 5], luteolin displayed an inhibitory effect on the clonal formation of gastric cancer cells, with the inhibitory effects of luteolin in both the targeted and nontargeted groups being more pronounced than those in the free luteolin group. Within the NP groups, the inhibitory effects of luteolin in the targeted group were stronger than those of the nontargeted group. Targeted delivery significantly enhanced the inhibitory effects of luteolin on tumor cell growth.
Figure 5: Effect of nanospheres containing luteolin on the formation of gastric cancer cells

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Cell apoptosis

Luteolin was shown to induce apoptosis of gastric cancer cells. At 48 h after the treatment of gastric cancer cells with different concentrations of luteolin, both the early and late apoptotic rates of cells increased with increasing drug concentrations. As shown in [Figure 6], compared with the nontargeted group, the percentages of early and late apoptotic cell populations in the targeted group were significantly increased. The induced difference in the cell apoptotic rate in the targeted group and the nontargeted group at the same concentration was significant.
Figure 6: Flowchart of gastric cancer cells undergoing apoptosis induced by luteolin

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mRNA and protein expression levels of forkhead box protein O1

The literature reports a decreased expression of FOXO1 in gastric cancer cell lines.[22] In this study, changes in FOXO1 at both mRNA and protein levels were detected under the same concentration of luteolin. As shown in [Figure 7]a, the mRNA expression levels of FOXO1 in the nontargeted PLGA-NPs and the Her-2-PLGA-NPs changed by 2.7- and 3.85-fold, respectively, in the presence of free luteolin compared with those in the control group. Similarly, the protein expression levels of FOXO1 in the nontargeted PLGA-NPs and Her-2-PLGA-NPs detected by reverse transcription polymerase chain reaction changed by 3.2- and 6.7-fold, respectively, in the presence of free luteolin. [Figure 7]b and [Figure 7]c. These results suggest that luteolin may exert inhibitory effects on gastric cancer cells by increasing the expression of FOXO1.
Figure 7: Changes in the mRNA and protein expression levels of forkhead box protein O1 (a) The relative mRNA expression level of FOXO1 detected by the two-step SYBR Green assay method. (b) The relative expression of FOXO1 assessed by RT-PCR. (c) Western blot analysis for FOXO1 and GAPDH

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 > Conclusions Top


In this study, luteolin was used as the subject of drug delivery research. Due to its poor water solubility, luteolin was loaded onto antibody-modified microspheres, and targeted delivery and increased concentrations of luteolin were achieved using Her-2-NPs. Both targeted and nontargeted luteolin-loaded NPs were successfully prepared and their relevant biological characteristics were investigated. The results showed that compared with the nontargeted group, Her-2-NPs could target gastric cancer cells and were able to upregulate the expression of FOXO1, thereby inhibiting the proliferation and invasion of gastric cancer cells and inducing apoptosis. The results of this study could provide an important basis for the inhibition of tumor cell growth by luteolin. In addition, they could provide a theoretical basis for the mechanisms underlying the pathogenesis of gastric cancer and its treatment, by evaluating cancer-targeting microspheres for the enhancement of pharmacological values using the FOXO1 gene as the target.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]
 
 
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