Immunomodulatory effects of flavonoids: An experimental study on natural-killer-cell-mediated cytotoxicity against lung cancer and cytotoxic granule secretion profile

Abstract

Background:

A new approach involving immune-cell-mediated cancer therapy has been adopted extensively for the sake of lung cancer treatments by utilizing natural killer (NK) cells. NK cell activity can be enhanced with certain agents, and among them are flavonoids. Thus, this study was conducted to investigate the immunomodulatory roles of apigenin, luteolin and quercetin on NK cell activity against lung cancer cells and on the secretions of perforin and granulysin profile.

Methods:

The NK-92 cells were grown in complete α-Minimum Essential Medium (MEM). NCI-H460 lung cancer cells were cultured in Roswell Park Memorial Institute 1640 media. NK cell activity against lung cancer cells were done using MTT(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The secretions of perforin and granulysin profiles were then analysed using enzyme-linked immunosorbent assay.

Results:

Apigenin, luteolin and quercetin significantly increased the NK-cell-mediated cytotoxic activity against lung cancer cells at concentrations 12.5 µg/ml and 25µg/ml (P < 0.001). The secretion levels of perforin and granulysin from NK cells were also significantly enhanced with apigenin and luteolin treatment but not with quercetin.

Conclusions:

All three flavonoid compounds possessed some significant immunomodulatory actions on NK cell cytotoxic activity and granule secretion profiles towards lung cancer therapy.

Keywords

Flavonoids NK cells lung cancer cytotoxic granules

Introduction

Global lung cancer incidence is drastically increased and has recently reached the highest among all types of cancer. The World Health Organization estimated that the incidence of lung cancer was 11.6 % of nearly 18 million new cancer cases diagnosed in 2018 (highest among all cancer), with the highest mortality rate of 18.4%. In Malaysia, more than 4600 new lung cancer cases were diagnosed in 2018, and the majority died, despite being treated with radiotherapy, chemotherapy, surgery or combined methods.¹

Immune-cell-mediated cancer therapy is a new area of interest, and many scientists have been extensively studying the role of cytotoxic immune cells in the prevention and treatment of cancer. One of the promising cells to fight against the cancer cell is natural killer (NK) cells, which also kill various microbes and transformed cells of the body. The NK cell is also known as a cytotoxic lymphocyte of the innate immune system and comprises approximately 10–15% of the blood lymphocytes. They recognise and then spontaneously kill ‘stressed’ cells such as tumour- or viral-infected cells.² Interestingly, NK cells kill virus or cancer cells either by direct cytotoxicity or initiation of apoptosis or both.

Perforin, granulysin and granzyme are three cytolytic proteins stored inside NK cells. The destructive properties of these proteins to the targeted cells and leading to their lysis are the crucial role in the NK-cell-mediated cytotoxicity against cancer. Perforin destroys target cells by making pores and allows the entry of other proteases to induce lysis and apoptosis.^3–5 Granulysin is a membrane-destructing protein released by innate lymphocytes such as NK and cytotoxic T cells (CD8) when they are activated. It destroys target cells by creating a hole in their membranes and then it kills them.⁶

Long ago, researchers had continuously studied various botanical plants and their effects on the activity of NK cells against malignant diseases. Many scientists have endorsed the role of flavonoids in health and diseases. Flavonoids are the abundant phytonutrients found naturally in various fruits and vegetables. Based on their chemical structure, flavonoids are sub-classed into flavone, flavonol, flavonone, flavonolol, isoflavonoid and anthocyanin.⁷ The two flavones apigenin and luteolin are well known for their immunomodulatory activity.⁸ Quercetin, a subclass of the flavonol group, showed significant impact on cytotoxic immune cells.⁹ However, there have been limited studies done on the effect of apigenin, luteolin and quercetin of flavonoid subclasses.

Despite having various therapeutic measures to manage lung cancer, its incidence and mortality rates are still extremely high. It is time to consider alternative measures such as plant-derived bioactive compounds to fight against cancer and its mortality rate. We first reported the role of three flavonoids in NK cell activity against lung cancer cells by using cell viability assays. In addition, the effects of these three flavonoids on NK cell cytotoxic granule secretion were also examined.

Methods

The in vitro, cell-line-based experimental study was carried out in the ‘Tissue Culture Lab’, Medical Faculty, University Sultan Zainal Abidin, Malaysia. NK-92 (NK-92^® MI (ATCC^® CRL-2408™) and NCIH460 lung cancer (NCI-H460 [H460] (ATCC^® HTB-177™) cell lines were purchased from the American Type Culture Collection (ATCC). The cells were frozen in cryotubes and stored in liquid nitrogen at –196°C before being thawed and cultured. NK-92 cells were grown as monolayer cultures in α-Minimum Essential Medium (MEM) (Nacalai Tesque Inc., USA), which comprises 0.2 mM myoinositol, 0.1 mM 2-mercaptoethanol, 0.02 mM folic acid, 100–200 U/mL human recombinant Interleukin-2 (IL-2) (Elabscience, USA), 12.5% foetal bovine serum (FBS) (ATCC) and 12.5% horse serum (ATCC). NCI-H460 lung cancer cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 media consisting of RPMI, 10% FBS and 1% Pen-Strept antibiotic. The cells were incubated at 37°C in a humidified 5% carbon dioxide (CO₂) incubator (Galaxy 170 R, New Brunswick, Scotland).

Preparation of flavonoids

Apigenin, luteolin and quercetin (Sigma-Aldrich, Saint-Quentin-Fallavier) were dissolved in 100% Dimethylsulfoxide (DMSO) (Merck, Germany) to make up a stock concentration of 1 mg/ml. Prior to use, the samples were diluted with α-MEM medium up to a concentration of 400 μg/ml. All samples were prepared and diluted freshly prior to the experiment. The five working solutions of flavonoids were prepared (6.25, 12.5, 25, 50 and 100 µg/ml) based on our previous research findings where we observed that the percentage of NK cell proliferation was enhanced with serially increasing concentrations of apigenin, luteolin and quercetin from 3.12 to 100 µg/ml. However, the percentage of cell proliferation declined after 100 µg/ml concentration of all three flavonoids.

Cell viability assay

The experiments were conducted under strict aseptic conditions using a biological safety cabinet (ESC II series, Erla Technologies). The killing activity of NK cells treated by apigenin, luteolin and quercetin were evaluated using the colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, as previously described.¹⁰ Briefly, 100 μl of 5 × 10⁶ cells/ml NK cells were pre-incubated for 24 hours and subsequently treated with different concentrations of serially diluted flavonoids (6.25, 12.5, 25, 50 and 100 µg/ml, respectively). After 24 hours, NK cells were washed with RPMI media and seeded in a 96-well plate. Then, 100 μl aliquot of target NCIH-460 lung cancer cells (5 × 10⁶ cells/ml) (effector:target ratio 1:1) were added to each well in a 96-well plate and incubated for four hours at 37°C in a 5% CO₂ humidified incubator (New Brunswick, Scotland). Subsequently, 20 μl of MTT (5 mg/mL) were added to the cells in the dark and incubated for three hours, covered with aluminium foil. After incubation and removal of media, 50 μl of DMSO was added to each well to dissolve the formazan crystals formed and incubated for another 15 minutes in the incubator. The absorbance was read at a wavelength of 570 nm as measurement wavelength using the Tecan enzyme-linked immunosorbent assay (ELISA) micro-plate reader (infinite M200PRO, TECAN, Switzerland). NK cell activity was calculated according to a formula described previously¹⁰:

N K a c t i v i t y (%) = 100 % \times (O D T - (O D S - O D E)) / O D T

where ODT is the optical density value of the target cells (NCIH-460 lung cancer cells) control, ODS is the optical density value of the test samples and ODE is the optical density value of the effector cells (NK-92 cells) control. target cells’ control (NCIH-460 lung cancer cells).

Assessment of perforin and granulysin secretion

The level of perforin and granulysin secretion from the treated NK cells was determined using pre-coated ELISA according to a previous method.¹¹ In brief, NK cells (1 × 10⁷ cells/ml) were incubated with three flavonoid compounds in non-toxic concentrations (12.5 and 25 µg/ml for apigenin and luteolin, and 25 and 50 µg/ml for quercetin) over 24 hours at 37°C in a humidified incubator. After incubation, plates were centrifuged, and supernatant was collected. The perforin and granulysin (Human Perforin ELISA Kit-ab46068, Human Granulysin ELISA Kit-ab213787, Abcam, USA) levels were measured using an ELISA reader at 450 nm wavelength according to the manufacturer’s protocol.

Statistical analysis

The results were expressed as mean ± standard deviation (SD) of three independent experiments in triplicate samples. Statistical differences between the control and the flavonoid compounds with different concentrations to the cell line were analysed using one-way analysis of variance, followed by Dunett’s multiple comparison tests using the GraphPad Prism 6.0 program. Differences were considered to be statistically significant at P < 0.05. Significance levels were reported as follows: *p < 0.05, **p < 0.01, ***p < 0.001.

Results

The effect of apigenin, luteolin and quercetin concentrations on NK-cell-mediated cytotoxicity against lung cancer cells

The results revealed that all three flavonoids induce marked elevation of NK-cell-mediated cytotoxicity against lung cancer cells (Figure 1). The cytotoxic activity of NK cells was stimulated by all three flavonoids in a dose-dependent manner. The data illustrated that each compound imparted significant effect at all tested concentrations except 6.25 µg/ml for quercetin and 100 µg/ml for apigenin. The highest activity was achieved at 12.5 and 25 µg/ml of apigenin and luteolin (150 and 140%, respectively) compared to the control value of 48%; however, quercetin required 50 µg/ml to obtain the maximal activity. It is also observed that the NK cell killing effect declined as the doses of the three flavonoids were increased to a higher concentration (100 µg/ml).

Figure 1.

The effects of various concentrations of apigenin, luteolin and quercetin on NK-cell-mediated cytotoxicity against lung cancer in vitro. NK cells were cultured with different doses of apigenin, luteolin and quercetin for 24 hours before NCI-H460 target cells were added (at expected 1:1 effector:target ratio, NK cells act as effector cells, whereas lung cancer cells are known as target cells). NK cell activity was then measured using an MTT assay. Values shown are mean (±SD) percentage cytotoxic activity from three different observations.

The effects of apigenin, luteolin and quercetin on NK-92 perforin secretion profile

As shown in Figure 2, the perforin secretion increased with all tested concentrations of the three flavonoids compared to the untreated control. However, for apigenin and luteolin, especially at 12.5 µg/ml concentration, both flavonoids imparted significant effect. No significant differences were detected with 25 µg/ml tested doses of both apigenin and luteolin as well as with all tested doses of quercetin.

Figure 2.

The effect of apigenin, luteolin and quercetin on NK-92 perforin secretion profile. NK cells were pre-treated with different doses of three flavonoids (12.5 and 25 μg/ml for apigenin and luteolin, and 25 and 50 μg/ml for quercetin) overnight. Then, supernatant was collected, and perforin analysis was done using the sandwich ELISA method. Values shown are mean (±SD) from three different observations.

The effects of apigenin, luteolin and quercetin on NK-92 granulysin secretion profile

The ELISA results demonstrated that the NK cell protein granulysin secretion was obtained after treated with different doses of three flavonoids. In particular, the significant production of granulysin was found, especially with a 25 µg/ml dose of apigenin and both 12.5 and 25 µg/ml concentrations of luteolin (Figure 3). On the other hand, quercetin exerted no significant difference in NK cell granulysin secretion at all tested concentrations compared to the untreated control. The maximal NK cell granulysin secretion was achieved with 12.5 µg/ml of luteolin (28 pg/ml) compared to the control group (10 pg/ml).

Figure 3.

The effect of apigenin, luteolin and quercetin on NK-92 granulysin secretion profile. NK cells were pre-treated with different doses of three flavonoids (12.5 and 25 μg/ml for apigenin and luteolin, and 25 and 50 μg/ml for quercetin) overnight. Then, supernatant was collected and granulysin analysis was done using the sandwich ELISA method. Values shown are mean (±SD) from three different observations.

Discussion

Lung cancer is recently considered as the deadliest among all types of cancer. NK-cell-based immunotherapy is one of the promising ways of treating various types of cancer. Naturally occurring plant-based flavonoids have been extensively studied for their role in immunity. There are several studies that have shown the ability to increase the activity of NK cells by naturally occurring compounds.¹² Phenolic compounds such as apigenin, luteolin and quercetin are potential therapeutic agents for immune system modulation and cancer prevention.¹³ With reference to these studies, the present research was conducted to investigate the roles of apigenin, luteolin and quercetin in the cytotoxic activity of NK cells against lung cancer cells.

In this study, the cytotoxic activity of NK cells against lung cancer was significantly enhanced after being treated with a series of concentrations of apigenin, luteolin and quercetin as compared to the untreated control. Apigenin and luteolin showed a marked increase in the killing of cancer cells, especially at low concentrations (12.5 and 25 µg/ml), whereas quercetin required a higher dose (50 µg/ml) to achieve the optimal killing effect. This is likely due to the nature of flavonoid, since apigenin and luteolin belong to the flavone group, whereas quercetin is flavonol. The alteration in chemical structure and the number of hydroxyl groups present in each flavonoid as shown in figure 4 react differently in the activation of NK cells. In fact, the functional differences in biological effects of flavonoids are critically related with their chemical constitution fragment, side-chain variation and substituent groups, although they act on the same target.¹⁴

Figure 4.

Structures of apigenin, luteolin and quercetin. Adapted from https://link.springer.com/article/10.1007/s10565-019-09481-9.

Surprisingly, the gradual decline in the killing effect of pre-treated NK cells was noted in all three flavonoids when increasing their concentrations to a higher dose. In other words, the higher the dose of flavonoids, the lesser the NK cell killing effect was observed. Assumedly, a higher dose of flavonoids might disturb the proliferation, activity and metabolism of NK cells or their killing mechanisms. The cell activity and metabolism in vitro become slower when proliferation is excessive, or cells are packed in the cultured plate, which leads to contact inhibition.¹⁵ A study conducted in Tunisia also reported similar findings, where the authors documented that the higher killing activity of NK cells was found in low doses, but the effect declined when the flavone concentrations were increased.⁸ Similarly, another flavonoid class – namely, resveratrol – also favoured NK cell cytotoxicity at low doses; however, a dose-related inhibition of lytic activity was endorsed at high resveratrol concentrations.¹⁶ In the case of luteolin, it is documented that the killing of NK cells against human myeloid leukaemia K562 cells was significantly increased after treated with luteolin. The authors also demonstrated that the enhanced killing was due to the increased secretion of cytokine IL-2 and interferon.¹⁷

Arguably, the results obtained for quercetin treatment on NK cell activity were reported equivocally. Yu et al. reported that quercetin enhanced NK cell activity in BALB/c mice administered with WEHI-3 leukaemia cells and subsequently treated with quercetin. NK cell activity of leukocytes was increased and resulted in enhanced killing activity, which was demonstrated with YAC-1 target cells.¹⁸ In contrast, the flavonol quercetin was found to inhibit the killing activity of NK cells in circulating lymphocytes from human donors at high concentrations (100 μmol/L). Decreased cytolysis was observed and suggested to be caused by the inhibition of Ca²⁺ channels and Na⁺/K⁺ ATPase activity by quercetin.¹⁹ In addition, daily quercetin supplementation for 12 weeks significantly increased plasma quercetin levels but had no influence on measures of innate immune function in adult females.²⁰ However, we observed that NK cell killing activity towards lung cancer was significantly elevated after quercetin treatment compared to the untreated control. The nature of flavonoid compound, different cancer cell lines used in the study and the source of NK cells obtained might have contributed to the ambivalent findings.

The killing mechanisms of NK cells to destroy their target include cytotoxic granule secretion, cytokine production and antibody-dependent cellular toxicity. We examined the effects of all three flavonoids on NK cell perforin and granulysin secretion using the sandwich ELISA method. The perforin secretion by NK cells was significantly elevated after being treated with apigenin and luteolin in this study. This cytotoxic protein release is more significant when treating with a low dose of apigenin and luteolin (12.5 µg/ml). However, quercetin-treated NK cells have no significant impact on perforin release compared to normal control. Previous studies endorsed the effect of flavonoids on NK cell perforin secretion. Hou et al. reported that total flavonoids of Hippophae rhamnoides-activated NK cells enhance cytotoxic killing against K562 cancer cell line. The authors explained that the mechanisms of the flavonoid on NK-cell-mediated toxicity are due to their favoured expression of perforin and granzyme cytolytic proteins as well as up-regulation of various cytokines.²¹ However, a study documented that quercetin treated T-cell-mediated killing towards human colon cancer cells by increasing the expression of perforin, granzyme and interferon.⁹ Their findings were not consistent with our observations since we found insignificant changes in quercetin-induced NK cell perforin secretion. This is likely due to the difference in the nature of the cells. Although both NK cells and T cells are cytotoxic lymphocytes, they differ in some phenotypical and chemical properties, as well as in the presence of receptors in their membranes.²² Quercetin may bind and modulate differently in these different lymphocytes.

In this study, we found that apigenin- and luteolin-treated NK cells enhanced the release of granulysin compared to the untreated control, but the same response was not found with quercetin. The two flavones showed a significant release of cytotoxic granule secretion compared to flavonol quercetin in this study. Luteolin increases NK cell granulysin secretion at both 12.5 and 25 µg/ml concentrations, while apigenin-induced secretion of this protein is significant only with 25 µg/ml. Interestingly, low doses of apigenin and luteolin (12.5 µg/ml) showed stronger effects towards perforin release; however, in the case of granulysin, they displayed different effects. Apigenin required a higher dose (25 µg/ml) to activate significant secretion of NK cell granulysin. Once activated, NK cells release cytoplasmic granules containing proteins such as perforin, the saposin-like family member granulysin and serin-proteases called granzymes, like granzyme B, to cleave, for example, several pro-caspases, which are then able to trigger apoptosis in the target cell.^23,24 The synthesis and the exocytosis of these cytotoxic granules are influenced by the activation of NK cell receptors, cytokine production and different signalling pathways, as well as the influence of various plant-derived nutrients.^9,21,25,26 It is postulated that apigenin and luteolin would modulate the synthesis and release of NK receptors and cytokine and cytotoxic granule release. Our ongoing experiments on NK cell receptor and cytokine secretion profile by apigenin, luteolin and quercetin would be able to link to NK-cell-mediated cytotoxicity against lung cancer.

In addition, the presence of double bond and the difference in the positions of the hydroxyl group may play a role in the binding to NK cell receptors and subsequent activation of NK cell cytotoxic granule expression as well as its release. Hydroxyl (-OH) groups are present by luteolin and apigenin in their backbone, and it was suggested that these -OH may be involved in immunomodulatory activities since luteolin, which contains two hydroxyl groups at ring B, demonstrated stronger immunomodulatory properties than apigenin, which has only one hydroxyl group.^8,12 NK cell cytotoxic granule secretion is enhanced by increasing exocytosis, which is due to increasing intracellular calcium secretion via tyrosine-kinase-mediated or other calcium-dependant kinase activation.^27,28 Most likely, these flavonoids may either directly increase cytosolic calcium content or activate the kinase or both.

Both perforin and granulysin secretion were not significantly increased after quercetin treatment. The molecular weight (MW) of quercetin (MW = 302) is the highest among the three flavonoids (MW = 270 for apigenin, MW = 286 for luteolin), which could possibly affect the NK cell granule secretion.²⁹ Systemic bioinformatics analysis of flavonoids carried out by researchers from Shanghai documented that different structural and physico-chemical properties as well as variation in MW of different flavonoids may alter their functional properties.¹⁴ Further experiments are currently in progress to determine the different intracellular mechanisms responsible for the activation of NK-cell-mediated cytotoxicity against lung cancer.

Conclusion

In conclusion, NK-92-cell-mediated cytotoxicity against lung cancer cells was observed with all three flavonoids: apigenin, luteolin and quercetin. Apigenin- and luteolin-treated NK cells potentiate the secretion of cytotoxic granules for their NK-cell-mediated cytotoxicity against lung cancer, whereas quercetin showed no similar effect. Quercetin might portray different intracellular mechanisms in activating NK cells against lung cancer. Based on our findings, it could be deduced that apigenin, luteolin and quercetin could facilitate in NK cell-based cancer therapy. It is meritorious to further investigate these three flavonoids and their effect on immunomodulation in vivo.

Footnotes

Acknowledgements

The authors would like to express their sincere gratitude to Dr Abdi Wira Septama and Miss Nik Nurul Najha for their expert opinions and guidance, and to all laboratory staff for their kind help throughout the laboratory procedures.

Authors’ contributions

Prof Nordin Simbak, Dr Liyana Hazwani and Dr Nasir Mat Nor designed and provided concepts for the research project, and contributed to manuscript preparation and editing. Laboratory work, data acquisition and data analysis were carried out by Dr Aung Myo Oo, Dr Ohn Mar Lwin and Ms Nor Zidah, who also contributed to the preparation of the draft manuscript, referencing and corrections. All lecturers contributed substantial amount on literature search.

Availability of data and materials

The data that support the findings of this study are available from the corresponding author, upon reasonable request.

Declaration of conflicting interests

The authors have no conflicts of interest to declare.

Ethical approval

Sultan Zainal Abidin University does not require ethical approval for reporting individual cases or case series.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: The research is funded by Fundamental Research Grant Scheme (FRGS), project code RR236 (FRGS/2017/SKK06/UNISZA/01/2) and approved by the Ministry of Education, Malaysia.

Informed consent

Not applicable.

ORCID iD

Aung Myo Oo

References

Bray

Ferlay

Soerjomataram

, et al. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin 2018; 68: 394–424.

Vivier

Tomasello

Baeatin

, et al. Function of natural killer cells. Nature Immunol 2008; 9: 503–510.

Chávez-Galán

Arenas-Del Angel

Zenteno

, et al. Cell death mechanisms induced by cytotoxic lymphocytes. Cell Mol Immunol 2009; 6: 15–25.

Voskoboinik

Dunstone

Baran

, et al. Perforin: structure, function, and role in human immunopathology. Immunol Rev 2010; 235: 35–54.

Kastelan

Cytolytic granules. In: Parnham

(ed) Encyclopedia of inflammatory diseases. Basel: Birkhäuser, 2015.

Krensky

Clayberg

Biology and clinical relevance of granulysin. Tissue Antigens 2009; 73: 193–198.

Panche

Diwan

Chandra

SR.

Flavonoids: an overview. J Nutr Sci 2016; 5: e47.

Kilani-Jaziri

Mustapha

Mokdad-Bzeouich

, et al. Flavones induce immunomodulatory and anti-inflammatory effects by activating cellular anti-oxidant activity: a structure-activity relationship study. Tumor Biol 2015; 37: 6571–6579.

Peng

, et al. Quercetin potentiates the effect of γδ T cells via modulating the expressions of granzyme B, perforin and IFN-γ and also regulates the Wnt/β-catenin signalling pathway in human colon cancer cells. Bangladesh J Pharmacol 2015; 10: 251–259.

10.

Sarangi

Ghosh

Bhutia

, et al. Anti-tumor and immunomodulating effects of Pleurotus ostreatus mycelia-derived proteoglycans. Int Immunopharmacol 2006; 6: 1287–1297.

11.

Muhammadi

Fazeli

Taheri

, et al. Modulatory effects of curcumin on apoptosis and cytotoxicity-related molecules in HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) patients. Biomed Pharmacother 2017; 85: 457–462.

12.

Grudzien

Rapak

Effect of natural compounds on NK cell activation. J Immunol Res 2018; 2018: 4868417.

13.

Grigore

Plant phenolic compounds as immunomodulatory agents. In: Soto-Hernández

Palma-Tenango

García-Mateos

(eds) Phenolic compounds: biological activity. London: IntechOpen, 2017, pp.75–98.

14.

Qiu

Yang

, et al. Exploring the mechanism of flavonoids through systematic bioinformatics analysis. Front Pharmacol 2018; 9: 918.

15.

Riss

Moravec

Niles

, et al. Cell viability assays. In: Markossian

Sittampalam

Grossman

, et al. (eds) Assay guidance manual. Bethesda, MD: Eli Lilly & Company and the National Center for Advancing Translational Sciences, 2013, https://www.ncbi.nlm.nih.gov/books/NBK144065/ (accessed March 2020).

16.

Huyan

, et al. Concentration-dependent biphasic effects of resveratrol on human natural killer cells in vitro. J Agric Food Chem 2014; 62: 10928–10935.

17.

Norzila

Hasmah

Veronique

, et al. Human natural killer (NK) cell activation by luteolin from Brucea javanica leaves. J Cancer Res Exp Oncol 2018; 10: 10–14.

18.

Lai

Yang

, et al. Quercetin inhibited murine leukemia WEHI-3 cells in vivo and promoted immune response. Phytother Res 2010; 24: 163–168.

19.

Middleton

Kandaswami

Theoharides

TC.

The effects of plant flavonoids on mammalian cells: implications for inflammation, heart disease, and cancer. Pharmacol Rev 2000; 52: 673–751.

20.

Heinz

Henson

Nieman

, et al. A 12-week supplementation with quercetin does not affect natural killer cell activity, granulocyte oxidative burst activity or granulocyte phagocytosis in female human subjects. Br J Nutr 2010; 104: 849–857.

21.

Hou

Wang

, et al. Effects of total flavonoids of sea buckthorn (Hippophae rhamnoides L.) on cytotoxicity of NK92-MI cells. Int J Immunopathol Pharmacol 2017; 30: 353–361.

22.

Sivori

Vitale

Morelli

, et al. p46, a novel natural killer cell-specific surface molecule that mediates cell activation. J Exp Med 1997; 186: 1129–1136.

23.

Trapani

Davis

Sutton

, et al. Proapoptotic functions of cytotoxic lymphocyte granule constituents in vitro and in vivo. Curr Opin Immunol 2000; 12: 323–329.

24.

Krzewski

Strominger

JL.

The killer’s kiss: the many functions of NK cell immunological synapses. Curr Opin Cell Biol 2008; 20: 597–605.

25.

Krzewski

Coligan

JE.

Human NK cell lytic granules and regulation of their exocytosis. Front Immunol 2012; 3: 335.

26.

Chang

Chen

YYM

, et al. Ganoderma lucidum stimulates NK cell cytotoxicity by inducing NKG2D/NCR activation and secretion of perforin and granulysin. Innate Immun 2013; 20: 301–311.

27.

Lopez

Brennan

Whisstock

, et al. Protecting a serial killer: pathways for perforin trafficking and self-defence ensure sequential target cell death. Trends Immunol 2012; 33: 406–412.

28.

Davis

Gallone

Cytolytic granules supply Ca²⁺ for their own exocytosis via NAADP and resident two-pore channels. Commun Integr Biol 2013; 6: e24175.

29.

Gates

Lopes

NP.

Characterisation of flavonoid aglycones by negative ion chip-based nanospray tandem mass spectrometry. Int J Anal Chem 2012; 2012: 259217.