Ethyl acetate fraction of Tiwai onion (Eleutherine palmifolia (L), Merr) increases the cytotoxicity of NK-92 cells against HeLa CCL-2 cells

 

Nurul Hasanah¹*, Karyono Mintaroem², Loeki EnggarFitri³, Noorhamdani⁴

¹Doctoral Programme in Medical Science, Faculty of Medicine, Universitas Brawijaya, Laboratory of Histology, Faculty of Medicine, Universitas Mulawarman

²Laboratory of Pathology Anatomy, Faculty of Medicine, Universitas Brawijaya.

³Laboratory of Parasitology, Faculty of Medicine, Universitas Brawijaya.

⁴Laboratory of Microbiology, Faculty of Medicine, Universitas Brawijaya.

 

ABSTRACT:

 

 

INTRODUCTION:

The immune system has an immune surveillance function that recognizes and destroys abnormal cells before developing into a tumor or killing of a tumor^1-3. The immune effectors such as B cells, T cells, Natural Killer (NK), Natural Killer T (NKT), interferon (IFN) types I and II, and perforin (pfn) have been known to be related to immune surveillance⁴.

 

 

Cancer cells express immunogenic antigens that stimulate the response of the immune effector⁵ and ultimately lead to the destruction of cells undergoing a transformation and malignant cells^4,6.

 

Studies conducted on invasive cervical cancer prove that tumor cells can escape NK cells lysis through the expression of human leucocyte antigen-G (HLA-G) molecules. Human leucocyte antigen-G can be expressed as membrane-bound and soluble isoforms, by immune cells that infiltrate tumors and circulating in the peripheral blood of cancer patients. Human leucocyte antigen-G inhibit immune response and induce regulatory and suppressor cell production, which facilitates tumor cells to escape from immune surveillance^7,8.

 

Natural Killer (NK) cells kill their target cells through two mechanisms that require direct contact between the effector and the target cell. In the first pathway, cytoplasmic granule toxin as the main membrane-destroying protein known as perforin and its family serin protease or granzyme are secreted by exocytosis and together induce apoptosis in the target cell⁹. The granular exocytosis pathway will strongly activate cell-death pathways through the activation of apoptotic cysteine protease (caspase) or independent of caspase¹⁰.

 

Herbal is available for the treatment of cancer, many herbal formulations are available for cancer treatment but the side effects can be minimized¹¹. Tiwai onion (Eleutherine palmifolia (L), Merr) is a medicinal plant that has been believed as an anti-cancer treatment by the people of East Kalimantan. Scientific research that supports the claim of the anti-cancer potential of this plant has not been able to completely explain the underlying mechanisms, both in vivo and in vitro.

 

MATERIAL AND METHOD:

Sample fractionation:

Tiwai onion plant was collected from Samarinda, East Kalimantan Indonesia in 2018. The crude methanol extract of Tiwai onion was obtained from extraction by 60% methanol in water and then fractionated with n-hexane separatory funnel. Then-hexane fraction was fractionated further with ethyl acetate¹². Before every treatment, ethyl acetate fraction of tiwai (EAFT) was dissolved in DMSO 1% and diluted with complete medium to obtain the dosages of 125, 250, 500, and 1000μg/ml.

 

Human cell line co-culture:

The HeLa CCL-2 cell lines (ATCC) were grown in α-MEM supplemented with Glutamax-I 4500mg/L glucose (Invitrogen), 10% FCS (BioWhittaker), 1000U of Penicillin, and 10 ǔg/ml Streptomycin. The cells were cultured in flask 25cm² and maintained at 37°C and 5% CO₂ until 80% confluent¹². The 10⁴ cells/well was seeded into the 2 plates 24 well which one plate was covered with a round coverslip at the bottom of the well for analysis of HLA-G secretion in membrane and supernatant and the other plate was not given a coverslip for analysis of apoptosis and granzyme secretion. Each well was added 500ng/ml IL-10 (Elabscience). The plates were incubated 48 hours in a 37⁰C at 5% CO₂. NK cells were co-cultured into these wells with a ratio of 1:1¹³. Plates were put in an incubator for 20 hours then treated with EAFT at 0 (KI), 125 (KII), 250 (KIII), 500 (KIV), and 1000 (KV) μg/ml and incubated for 24 hours.

Measurement of membrane-bound HLA-G secretion with immunofluorescence:

Cells attached to the coverslip were fixed for 10 minutes in absolute methanol at 4°C. Samples were then rehydrated in PBS Tween 0,05% 3 times, and add PBS Triton X 1000 0, 1% for 5 min. Cells were incubated for 60 min in Blocking buffer 1 % (BSA 0,1g in PBS 10 ml). The HLA-G (4H84) mAbs (Santa Cruze; Cat#sc-21799) 1:400 were applied and incubated overnight following by incubations with biotinylated goat anti-mouse mAb and FITC-labeled streptavidin 1:1000 in the dark. Samples were then mounted in object-glass and observed under a fluorescence microscope, (Olympus).

 

Measurement of soluble-HLA-G with Elisa (HLA-G Elisa kit: Elabscience (Cat#E-EL-H1663):

The standard working solution added to the first two columns: Each concentration of the solution is added in duplicate, to one well each, side by side (100uL for each well). The samples were added to the other wells (100 uL for each well). The plate covered with the sealer and incubated for 90 min at 37℃. The liquid was removed out of each well, do not wash. Immediately 100μL of Biotinylated Detection Ab working solution added to each well. The plate covered with the sealer and gently mix up and incubated for 1 hour at 37°C. The solution was aspirate from each well, and 350uL of wash buffer was added to each well. For 1~2 minutes was soaked and the solution was aspirated from each well and pat it dry against clean absorbent paper. This wash step repeated 3 times. 100μL of HRP Conjugate working solution was added to each well, was covered with the plate sealer and incubated for 30 min at 37°C. The solution was aspirate from each well, repeat the wash process for five times. 90μL of substrate reagent was added to each well. Cover with a new plate sealer. Incubate for about 15 min at 37°C. The plate was protected from light. 50μL of stop solution was added to each well. The optical density (OD value) of each well was determined at once with a micro-plate reader at 450 nm.

 

Measurement of Granzyme B with Elisa (Human Granzyme B Elisa Kit: Biolegend (Cat#439207, 439208):

The plate was washing 4 times with at least 300μL of 1X Wash Buffer per well and blot any residual buffer by firmly tapping the plate upside down on absor­bent paper. 50μL of Assay Buffer added to each well that will contain either standard dilutions or samples. 50μL of standard dilutions or samples added to the appropriate wells. The plate was sealed with a Plate Sealer and incubate the plate at room temperature for 2 hours while shaking at 200rpm. The contents of the plate discard into a sink, then washed the plate 4 times with 1X Wash Buffer. 100μL of Human Granzyme B Detection Antibody solution added to each well, the plate was sealed and incubated at room temperature for 1 hour while shaking. The contents of the plate were discarded into a sink, then the plate washed 4 times with 1X Wash Buffer. 100μL of Avidin-HRP E solution added to each well, the plate was a seal and incu­bated at room temperature for 30 minutes while shaking. The contents of the plate discarded into a sink, then the plate washed 5 times with 1X Wash Buffer. For this final wash, wells were soaked in 1X Wash Buffer for 1 minute for each wash. This will help minimize the background. 100μL of Substrate Solution F added to each well and incubated for 30 minutes in the dark. Wells containing human Granzyme B should turn blue in color with an intensity proportional to its concentration. The reaction was stopped by adding 100μL of Stop Solution to each well. The solution color should change from blue to yellow. The absorbance was read at 450 nm immediately.

 

Cell apoptosis assay with Flow cytometry¹⁴:

Hela and NK cells were co-cultured on a 24-well plate and then treated with EAFT. After incubation, the suspended cells (NK cells) are washed three times with PBS to remove all effector cells. HeLa cells are washed with PBS, then collected and colored using Apoptosis FITC Annexin V I detection devices (BD Biosciences). The percentage of apoptotic cells was the sum of the percentage of cells stained with only annexin V and cells stained with both annexin V and PI.

 

Statistical analysis:

From the data obtained, a normality test was carried out using the Kolmogorov-Smirnov test. The data obtained will be tested by ANOVA (p <0.05), and differences between groups were further tested by Least Significant Difference (LSD)¹⁵.

 

RESULT:

Induction of apoptosis in Hela CCL-2 cells after co-cultured with NK cells:

Flow cytometry analysis of Annexin V-FITC/PI dual staining was used to examining changes of phosphatidylserine exposure, the apoptotic marker, due to inducing capacity of EAFT in Hela CCL-2 cell. EAFT induced apoptosis in Hela CCL-2 cells in a dose-dependent manner. Administration of EAFT in culture was able to significantly increase apoptosis (p<0,05) among doses and when compared with the same dose but without NK cells. This shows that NK cells have a role in increasing apoptosis and EAFT has a role in increasing NK cells cytotoxicity. These results indicated that cell death caused by EAFT treatment occurred primarily through apoptosis (Fig.1).

 

Figure 1. Ethyl acetate fraction of Tiwai onion -induced apoptosis in Co-culture of Hela CCL-2 + NK cells (a) assay with Annexin V/PI through flow cytometry. (b) Mean of apoptosis in the histogram form (*P<0.05 vs KI)

 

Inhibition of membrane-bound HLA-G and soluble-HLA-G in Hela CCL-2 cells after co-cultured with NK cells:

The expression of membrane-bound HLA-G Hela CCL-2 cells after co-culture with NK cells are shown in Figure 2. This study demonstrated that HLA-G was secreted on Hela cell membranes that cultured with NK cells after IL-10 induction with a mean of 26.27 arbitrary units (AU) field of view and HLA-G secretion then decreased after exposure to EAFT 125μg/mL : 19.23 AU, 250μg/mL : 14.27 AU, 500μg/mL : 13.15 AU and the lowest secretion after exposure to EAFT 1000 μg/mL : 5.74 AU. Microscopic images of Hela cells expressing HLA-G on their membranes were obtained by examination using a fluorescence microscope as shown in Figure 2 which shows the reduction in green fluorescence according to the increase in treatment dosage.

 

 

Figure 2. Ethyl acetate fraction of Tiwai onion inhibits membrane-bound HLA-G secretion in the co-culture of Hela CCL-2 + NK cells (a) assay with Immunofluorescence, the cell with green fluorescence is HeLa cell, the cell with orange color is NK cell: magnification 400x. (b) Mean of the membrane-bound HLA-G secretion and (c) soluble HLA-G production in co-culture of Hela CCL-2 + NK cells data in the histogram form (*P<0.05 vs KI)

Stimulation of granzyme secretion after co-culture Hela-NK cells:

From this study, the highest mean granzyme secretion was found in the KII group (187.448pg/mL) and the lowest in the KV group (12.332pg/mL) as shown in Figure 3.

 

 

Figure 3. Histogram of the mean of the granzyme production co-culture of Hela CCL-2 + NK cell (*P<0.05 vs KI)

 

DISCUSSION:

Tiwai onion (Eleutherine palmifolia (L)., Merr) is a medicinal plant that has been believed as an anti-cancer treatment by the people of East Kalimantan. The immune effectors such as Natural Killer (NK) has been known to be related to immune surveillance through its cytotoxicity ability. Cervical cancer which exposed by IL-10 originates from innate or adaptive immune cells in the microenvironment. Although IL-10 acts as an effector and regulator of the immune system, in cancer cells this cytokine increases the expression of HLA-G which is a non-classical MHC class 1 molecule. Increased HLA-G expression in cancer cells is related to immune-evasion, through NK cells¹⁶.

 

HLA-G has comprehensive suppressive functions exerted in multiple steps to impair anti-tumor immune responses by interacting with receptors expressed on immune cells such as CD85j/immunoglobulin-like transcript 2 (ILT2) and CD56dim NK cells expressed by some NK cells¹⁷.

 

In this study, EAFT administration in co-culture significantly increasing apoptosis among doses and when compared with the other group at a similar dose without the presence of NK. This result shows that NK cells have a role in increasing apoptosis and ethyl acetate fraction has a role in increasing NK cell activity (a comparison chart of apoptosis before and after co-culture).

 

Increased apoptosis is thought to be related to the effect of EAFT in suppressing membrane-bound and soluble HLA-G. This is evidenced in this study, increases of dose EAFT can decrease in membrane-bound and soluble HLA-G. In this study, EAFT can reduce membrane-bound HLA-G when compared to controls. EAFT effect on soluble HLA-G shows interesting results because on exposure EAFT at the dose 125 μg/mL increases HLA-G soluble (7-fold) (Fig.2c) Further, the increase in soluble HLA-G is not in line with the decrease in apoptosis. In controls, although soluble HLA-G was lower than exposure to ethyl acetate fraction dose 125μg/mL apoptosis was lower. This raises the possibility that 1) membrane-bound HLA-G play a greater role in influencing NK cell activation, 2) according to the research method, NK cells, and HeLa cells have communicated before being exposed to EAFT, so because of the proximity of the two cells, the NK receptor responds more quickly HLA-G membranes compared to those released to the medium (soluble).

 

Increased apoptosis and NK cell activity in EAFT exposure are thought to be related to the suppression of membrane-bound and/or soluble HLA-G. If this hypothesis is correct, the cytokine (or chemokine) secretion of NK cells will increase with the same trend. If there is no suppression it will happen HLA-G/ILT2 interaction impairs the expression and functions of chemokine receptors in NK cells¹⁸. Trogocytosis is a rapid process of transferring cell membrane fragments containing molecules from one cell to another during cell-to-cell contact¹⁹. Immune cells such as activated NK cells, T cells, and monocytes can rapidly acquire membrane fragments containing functional HLA-G from other cells (i.e., HLA-G+ immune or tumor cells) in their vicinity by the process of trogocytosis. The acquired HLA-G molecule can immediately reverse the functional phenotype of the recipient from effector to regulatory cells²⁰. When activated, NK cells acquired HLA-G1 from M8-HLA-G1 tumor cells, NK-HLA-G1acq+ cells lost their cytolytic functions and ceased cell proliferation. Moreover, NK-HLA-G1acq+ cells behaved as suppressor cells capable of suppressing the cytolytic functions of other NK cells²¹.

 

But in this study, granzyme secretion increased significantly compared to controls at ethyl acetate fraction doses (125 and 250μg/mL) and decreased with increasing ethyl acetate fraction doses( Fig.3). The mechanisms that occur when high HLA-G expression is involved in HLA-G/receptor (particularly ILT2 mediated immune suppression have been documented in previous studies and include impairment of immune cell proliferation, differentiation, cytotoxicity, cytokine secretion, and chemotaxis^18,22,23. This HLA-G/ILTs interaction causes phosphorylation of ITIMs and recruits protein tyrosine phosphatase Src homology 2 (SH2) domain-containing proteins such as SHP-1 and SHP-2, which initiates the inhibitory signaling cascade^24,25. HLA-G1 and HLA-G5 isoforms could suppress NK cell cytolysis in a manner dependent on the HLA-G expression, and HLA-G1 and HLA-G5 isoforms had an additive effect on NK cytolysis suppression^26,27. In this study, HLA-G expression was inhibited resulting in increased cytotoxicity with the secretion of granzyme at low doses and decreased with increasing EAFT doses. The granzyme was also secreted by NK cells before being exposed to EAFT. This can be explained that there has been an interaction between Hela target cells and NK cells in immune synapse through their receptors before EAFT exposure for 20 hours incubation period, so that after being given EAFT exposure for 24 hours to then measure the levels in supernatant (extracellular) granzyme levels are already decreased. From the explanation of the above process, it is known that the process of granzyme secretion by NK cells takes place immediately after the NK cell is in contact with the HeLa target cell. After that granzyme undergoes exocytosis out of the NK cells into the gap and in short time endocytosis into the HeLa cells. This assumption is reinforced by the results of research on the existence of electrostatic interactions between granzyme and the target cell membrane. Granzyme binds the target cell membrane with electrostatic interactions because granzyme is very positively charged with pI ~ 9-11, and the surface of the cell is negatively charged)^28,29. Granzyme which is positively charged enters the target cell through electrostatic interaction with the negative charge on the surface structure of the target cell. This is evidenced by previous studies on mice with reduced granzyme B uptake and cytotoxicity in poor cation conditions²⁹. Therefore ethyl acetate fraction is also thought to have a direct effect on NK cells activity in transduction signals that regulate cytokine expression and secretion (interferon, perforin, etc.).

 

CONCLUSION:

EAFT Onion (Eleutherinepalmifolia(L)., Merr) can increase the cytotoxicity of NK-92 cells against HeLa CCL-2 cells

 

ACKNOWLEDGMENT:

We are grateful to Biomedical Laboratory Faculty of Medicine Universitas Brawijaya Malang, Indonesia for excellent technical assistance and the collecting of Hela CCL-2 and NK -92 cells in the research.

 

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

 

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Accepted on 10.02.2020           © RJPT All right reserved

Research J. Pharm. and Tech 2020; 13(6): 2854-2858.