Lycopene Prolongs the Lifespan and Enhances the Cytotoxicity of NK Cells after Ex Vivo Expansion

Lycopene is a nonprovitamin a carotenoid mainly found in fruits and vegetables, which has been reported to possess a variety of biological effects. The properties of lycopene on human natural killer (NK) cells after ex vivo expansion were assessed in the present study. Results showed that lycopene has a positive effect on NK cells viability and cytotoxicity. Aging and apoptosis started from the fourth week onwards in the cultured NK cells which were obtained from the peripheral blood mononuclear cells (PBMC). Supplemented with lycopene (5μM) can restore the decreased viability and cytotoxicity of NK cells and reduce NK cells apoptosis caused by aging during fourth-sixth week culture. Its anti-apoptosis effect on NK cells may be related to lycopene can down regulated the expression of caspase 3 and 9 genes. Furthermore, lycopene can enhance the IFN-γ expression in gene and protein level after 7d treatment. However, lycopene did not affect the functional receptor’s (NKG2A, NKG2D, NKp30 and NKp44) expression on NK cells. These results indicated that lycopene has a positive effect on NK cells. As a health product, it may help to prolong the lifespan and enhance the cytotoxicity of NK cells after ex vivo expansion.


Introduction
As a well-known red carotenoid component of tomato, lycopene has drawn a lot of attention because of its potential biological functions. Lycopene is recognized has most antioxidant effect among carotenoids (1), its antioxidant effect is two times higher than carotene and ten times higher than tocopherol (2). Lycopene has more potent proliferation inhibition of human cancer cells than either α-carotene or β-carotene (3). Studies reported lycopene has ability to induce cell-cycle arrest and apoptosis in many human cancer cell lines, such as breast cancer cells (4,5), prostate cancer cells (6), Hep3B human hepatoma cells (7), T-84, HT-29, MCF-7 and DU145 (8). The anti-tumor effects of lycopene may be related to modulate intracellular reactive oxygen (ROS)-mediated cell growth (9); modulate cell cycle proteins such as β-tubulin, CK8/18, CK19 and heat shock proteins (10) or affect the PI3K/Akt signaling pathway (11).
Lycopene also shows multi-immunomodulatory activity. Kobayashi (12) has reported that lycopene can recover the intrathymic T cell differentiation in tumorous mice than control. Watzl B (13) assessed the effect of low-carotenoid diet supplemented with tomato (providing high amounts of lycopene) on immune functions in healthy men. Their study revealed that juice intervention has the positive influence on lymphocyte proliferation, NK cell cytotoxicity and IL-2 secretion by a time-delayed modulation in healthy men.
Natural killer (NK) cells are large granular lymphocytes which are defined by presence of CD56 and absence of CD3 on the cell surface (CD56 + CD3 -) (14). NK cells comprise 5%-10% of all peripheral blood lymphocytes (15), can lyse virally infected cells or oncogenically transformed cells and secrete many kinds of cytokines without requiring prior immunization. NK cells exert cytotoxicity on oncogenically transformed cells specifically and efficiently, which make them play an important role in tumor identification and surveillance.
β-carotene is a cognate material of lycopene; many studies have confirmed the positive effect of β-carotene on NK cells. Watson (16) reported the number of NK cells increased substantially in peripheral blood mononuclear cells (PBMC) from individuals supplemented with β-carotene. Enhanced NK cell cytotoxicity was also observed in human subjects who given oral β-carotene reported by Prabhala (17). Chew and Park (18) reported that non-provitamin A carotenoids such as lycopene, were more active than β-carotene in enhancing cell-mediated and humoral immune response in animals and humans. However, a few studies have been down about lycopene effect on human NK cells.
In the present study, the viability, apoptosis, cytotoxicity, receptor expression and cytokines secretion of primary human NK cells treated by lycopene after ex vivo expansion were delineated to assess if there were positive effect of this 24 Lycopene Prolongs the Lifespan and Enhances the Cytotoxicity of NK Cells after Ex Vivo Expansion antioxidant on NK cells lifespan, activity and function.

Cells
Primary human NK cells were prepared according the methods described in Huang et al (19). Briefly, peripheral venous blood (10mL) from healthy donors (n=10) was collected in heparinized tubes. Blood sample collecting procedure was conforming to the informed consent guidelines of the Ethic Committees of Northwestern Polytechnical University. Blood samples were diluted 2-fold by PBS (sterile, 1×). PBMCs were collected by using Lymphocyte Separation Liquid (Haoyang TBD, Tianjin, China) according the instructions and washed twice with PBS (sterile, 1×), then re-suspended in RPMI-1640 media (Gibco, Rockville, MD, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco, Rockville, MD, USA), 100U/mL IL-2 (Peprotech, Rocky Hill, NJ, USA), 100μg/mL of penicillin and streptomycin (Genview, Carlsbad, CA, USA). After counted carefully, PBMCs were co-cultured with equal numbers of stimulating cells (genetically modified K562 cells, prepared according to (20) and irradiated).
K562 cells (purchased from American Type Culture Collection, ATCC, Manassas, VA) and stimulating cells were cultured in RPMI-1640 cell media supplemented with 10% FBS, 100μg/mL of penicillin and streptomycin and routinely cultured at 37 o C in 5%CO2 incubator.

Lycopene Treatment
Lycopene was prepared to storage solution (0.5mM, 100×working concentration) in DMSO. NK cells (5×10 5 each) were suspended in 990μL of RPMI-1640 and cultured in 24-well plates (Costar, New York, USA) at 37 o C in a 5%CO2 incubator with 10μL of the lycopene storage solutions for 1-3 weeks. In this assays, the working concentrations of lycopene was 5μΜ. Control group contain 10μL DMSO to exclude its effect on cells.

Cell Viability and Apoptosis
After lycopene treatment in different time interval, NK cells in each group were centrifuged, washed with PBS, and re-suspended in 600μL of RPMI-1640 media. 20μL of CCK-8 (Cell Counting Kit-8, Dojindo, Japan) was added to a 200μL cell suspension per well in a 96-well plate in triplicate for each group. The cells were then incubated for 2h at 37 o C in a 5% CO2 incubator. The absorbance (A) values of each well was recorded at 450nm in microplate reader (BioTek Synergy4, USA).
The apoptosis of NK cells was assessed by AnnexinV-FITC/PI method (Apoptosis Detection Kit, Beyotime Institute of Biotechnology, Haimen, China). After treated with lycopene in different time interval, NK cells in each group were centrifuged and washed 3 times with PBS, stained with AnnexinV-FITC and PI following the instructions and analyzed by flow cytometry.

Cytotoxicity
The cytotoxicity of NK cells were carried out according the procedure described in (21). Briefly, after lycopene treatment, NK cells (8×10 5 ) were centrifuged and washed 3 times with PBS, then re-suspended in 400μL of RPMI-1640. 100μL of NK cells (2×10 5 ) were plated into each well of a 96-well plate with 100μL of K562 cells (4×10 4 ) in triplicate. The effector control wells contained 100μL of NK cells (2×10 5 cells) and 100μL of medium. The target control wells contained 100μL of K562 cells (4×10 4 cells) and 100μL of medium, to make the effector-to-target ratio (E:T) 5:1. Plate was incubated at 37°C in a 5%CO2 incubator. After 4h coculture, 20μL of CCK-8 was added to each well and the plate was then incubated for another 2h. Recorded the A values at 450nm in a Microplate Research Reader (Bio-Tek Synergy HT). Cytotoxicity was determined by evaluating the rate of NK cells killed K562 cells, the killing rate was calculated as following equation (22): (Ae, average A450 of triplicate wells for NK cell control; At, average A450 of triplicate wells for K562 cells control; Ae+t, average A450 of triplicate wells for NK cells plus K562 cells).

mRNA Expression Analysis
After lycopene treatment, TRIzol reagent (Invitrogen, Carlsbad, CA, USA) was used to isolate total RNA from NK cells (1×10 7 ). The mRNA levels of apoptosis related gene (FasL, caspase3, 6, 7, 8, 9, and 10), cytokines (IFN-γ, perforin, and Granzyme-B), and important functional receptors of NK cells (NKG2D, NKG2A, NKp30 and NKp44) were evaluated by relative quantification PCR (RT-qPCR) with SYBR Green random mixing method. Glyceraldehyde-3-phosphate dehydrogenase (GADPH) was used as the internal control. The 2 -△△ Ct method was used to calculate relative changes of gene expression (23). All primers used here were shown in Table 1. RT-qPCR was performed according to the instructions of the TransStart Top Green qPCR SuperMix kit (TransGenBiotech, Beijing, China). The reaction mixtures were incubated for 30 min at 48°C, followed by 40 cycles of PCR at 94°C for 5s, 55°C for 15s, and 72°C for 10s. At the end of 40 cycles (CFX96, BIO RAD, USA), a melting curve analysis was performed to confirm the presence of only a single amplified product of the expected size.

Receptors Expression
After lycopene treatment for 7d, NK cells were centrifuged, washed with PBS, and divided into 4 groups (1×10 5 cells each) and stained with PE-conjugated mouse anti-human NKG2A (R&D Systems, Minnesota, USA), NKG2D, NKp30 and NKp44 (BD Bioscience, California, USA). All cells were analyzed by using flow cytometry.

ELISA Assay of IFN-γ, Perforin and Granzyme-B
ELISA Kits (BD Bioscience, CA, USA) were used to assess the secretion of IFN-γ, perforin and Granzyme-B. NK cells (8×10 5 ) in each lycopene treated group were centrifuged, washed with PBS and re-suspended in 400μL RPMI-1640 (IL-2 free), mixed with 1.6×10 5 K562 cells in 100μL RPMI-1640, Ex vivo expanded NK cells were harvested and stained with CD56-PE and CD3-FITC mAbs after 3 weeks culture. The purity of NK cells (CD56 + CD3 -) was shown in Fig. 1. From the one donor, the percentage of NK cells in PBMC after expansion was 91.17%, and the mean percentage of NK cells form 10 donors was 90.82%±0.39% (n=10).

Data Analysis
SPSS 16.0 statistical software (IBM, New, York, USA) were used here to perform the statistical analysis. The data were presented as the mean±SD. All results were analyzed using the analysis of variance (ANOVA). Multiple comparisons used LSD test to evaluate the significance of differences between groups. Statistical significance was defined as p<0.05. lycopene treated group respectively. The variation tendency of NK cells cytotoxicity was similar to its viability. As show in Fig. 2B, the cytotoxicity of NK cells decreased significantly since the 5 th week culture, which decreased to 31.4%±2.3% compared with the data (89.6%±1.1%) obtained from 2d (3 th week) (p<0.05). And in lycopene treated group, the cytotoxicity were 42.1%±2.8% in 5 th week. It was suggested that lycopene increase the NK cells viability and cytotoxicity in the 5 th -6 th weeks ex vivo culture.

NK cells purity
Flow cytometry assay of primary human NK cells from one donor (for instance); the percentage of NK cells (CD56 + CD3 -) in PBMCs was 91.17% after 3 weeks ex vivo expansion. Figure 1. Flow cytometry assay of primary human NK cells.

NK Cells Viability and Cytotoxicity
There was no obvious effect of lycopene on NK cells viability in short term treatment. It was shown in Fig.2A, the A values (reported the viable NK cells) were 0.864±0.028, 0.858±0.013 and 0.845±0.012 in 2, 4 and 7days (4 th week) respectively in lycopene treated group compared with 0.856±0.019, 0.857±0.014, and 0.834±0.017 in control. With time, the A values of NK cells in control were decreased rapidly, but which decreased marginally in lycopene treated group. The A values in these group were 0.524±0.018 (14d/5 th week), 0.329±0.1 (21d/6 th week) in control and then incubated for 4h at 37°C in 5% CO2 incubator (K562 0.683±0.016 (14d/5 th week) and 0.47±0.041 (21d/6 th week) in cells were used to stimulate the cytokine secretion of NK cells according (24). Culture supernatant from lycopene treated group was assessed by corresponding ELISA Kits. Each sample were performed twice, A values were recorded at 450nm. Curve Expert 13.0 was employed to create the standard curve according to the A values of standards. The quantities of these cytokine secreted by NK were calculated from these standard curves.

NK Cells Apoptosis
There was no obvious apoptosis of NK cells was observed under inverted phase contrast microscope in 3 weeks ex vivo culture, therefore the NK cells apoptosis was assessed after 7d (4 th week of ex vivo culture) in present lycopene. Over 7d lycopene treatment and stained with Annexin-V and PI, NK cells apoptosis were tested by flow cytometry and showed in Fig. 3. After 3 weeks ex vivo expansion, NK cells were routinely cultured in 1640 medium. It was noticed that, since the 4 th weeks, NK cells were seen to be aging and apoptosis gradually. However, lycopene show the positive effect to slow down this process. In routinely cultured (control) group, the early apoptosis (AnnexinV + /PI -) of NK cells were17.1±3.46%, 20.5±2.7%, and 18.8±4% in 4 th , 5 th and 6 th week. And the early apoptosis (AnnexinV + /PI -) of NK cells in present of lycopene were 5.6%±2.1%, 17.8%±2.6%, and 15.8%±3.1% respectively in 4 th , 5 th and 6 th week. Meanwhile, the late apoptosis/necrosis NK cells (AnnexinV + /PI + ) were 15.9%±3.1%, 20.1%±2.3%, and 56%±3.5% in control and 8.32%±2.3%, 10.6%±3.9%, and 35.4%±4.3% in lycopene treated groups in these weeks. These results suggested that lycopene could relieve the aging caused NK cells apoptosis and necrosis to a certain extent during long time ex vivo culture.
Part A: A values of NK cells treated with lycopene for 2-21d; Part B: Killing rates of NK cells treated with lycopene for 2-21d. Data represents the mean±SD of six independent experiments. One-way ANOVA and LSD tests were performed. *: p<0.05 compared with the control (n=6).

RT-qPCR
The levels of IFN-γ and perforin proteins in the NK cells supernatants were performed in Fig.4. It was noticed that, IFN-γ level was significantly increased after 7d lycopene treatment, which was increased to 466±19pg/mL compared with the 389±17pg/mL in the control. The concentration of perforin was not changed obviously. There were about 610ng/mL perforin in lycopene treated and control groups. The ELISA result of Granzyme-B was not showed because which was hardly detected in the supernatants of NK cells.
The expression profiles of genes involved in apoptosis and NK cell functions after lycopene treatment were depicted in Table 2 and 3. The mRNA level of caspase 3 and caspase 9 genes increased gradually since 4 th week culture compared with the 3 weeks cultured cells (data not show). And lycopene treatment can decrease this gene expression (Table 3). After 7d lycopene treatment, the IFN-γ mRNA increased significantly (8.81 fold) compared with the control ( Table 2, p<0.05). There was no significant change on NK cell functional receptors mRNA expression after lycopene treatment.

ELISA
Lycopene (5μM) treated NK cells were stimulated with K562 cells, supernatants were collected in different time intervals and then the concentrations of IFN-γ and perforin in them were detected. Each column represents mean±SD of four independent experiments. One-way ANOVA and LSD tests were performed, *: p<0.05 compared with control (n=4).

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Lycopene Prolongs the Lifespan and Enhances the Cytotoxicity of NK Cells after Ex Vivo Expansion Part A: NK cells were stained with PE-conjugated mAbs and analyzed by flow cytometry (for instance). Part B: The bar graph represents the mean±SD from four independent experiments. One-way ANOVA and LSD test (n=4). Figure 5. Protein expression profiles of the NK cell receptors NKG2A, NKG2D, NKp30, and NKp44 after lycopene treatment.

Receptor expression
Flow cytometry assays indicated that lycopene display no significantly regulation of NK cells functional receptor. The positive percentages of NKG2A, NKG2D, NKp30 and NKp44 were not changed obviously.

Discussion
As a kind of common natural pigment in many vegetables and fruits, lycopene is a carotenoid without provitamin-A activity and also a major component found in human serum and other tissues among the carotenoids (25). Antioxidant p r o p er ti e s of lycopene are recognized to be primarily responsible for its beneficial properties. Recently, study suggested that there were other bioactivities of lycopene including modulation of intercellular gap junction communication, immune system and metabolic pathways (25). Immune cells, especially lymphocytes are particularly sensitive to oxidative stress because of these highly active cells constantly generate reactive oxidative products (ROS) in their normal cellular activity (18,26). Accumulated ROS are highly reactive and can destroy cellular membranes, cellular proteins and nucleic acids. For this reason, antioxidants such as lycopene may help to protect lymphocyte activity and functions.
Literature shows that lycopene inhibited 7-KC-induced macrophage apoptosis by reduced the increase in ROS production, and NOX-4, Hsp 70 and Hsp 90 expression. It also limited caspase-3 activation and modulate the effects of 7-KC on AKT, Bcl-2, Bcl-xL and Bax (27). Li (28) reported that lycopene could protect marrow mesenchymal stems from ischemia-induced apoptosis in vitro by activate phosphoinositide-3 kinase (PI3K)/Akt. Mills (29) reported that lycopene influences lymphocyte proliferation significantly through its effects on processes involved in early cellular activation.
In the present study, the effect of lycopene on human primary NK cells was assessed. The results show that, the viability and cytotoxicity of NK cells decreased gradually since the 4 th week ex vivo culture, and accompanied by increased early apoptotic and late apoptotic/necrotic cells, which may cause by aging and accumulated ROS. The RT-qPCR result shows that, after lycopene treatment, the gene of caspase-3 and caspase-9 down-regulated significantly. It was known that, caspase-8 and caspase-10 are the upstream enzymes of caspase-3 and 9. However, transcription of caspase 8 and 10 did not changed significantly. Analogically, the downstream enzymes, caspase 6 and 7 also did not change obviously after lycopene treatment. It may explain that, when lycopene down-regulated the transcription of caspase-3 and 9 gene, the decreased caspase-3 and 9 proteins interfere the signal transmission of the apoptosis signal pathway. Therefore, the aging induced apoptosis of NK cells were relieved by supplement lycopene, and it enhanced the viability and cytotoxicity of NK cells Lutein is another carotenoid with no provitamin A activity. Studies show that, dietary lutein increased IFN-γ mRNA expression in splenocytes and decreased apoptosis in blood leukocytes of tumor bearing mice compared to unsupplemented one (30,31). Our results show that, 7d lycopene treated also significantly increases the NK cells IFN-γ expression in gene and protein levels. The increased IFN-γ secretion could helps to increase the cytotoxicity of NK cells.

Conclusions
Lycopene has the positive effect on NK cells by prolonging its lifespan, increasing its cytotoxicity and decreasing its apoptosis, which can be used as a health product to increase immunity.