Cordycepin Enhances the Cytotoxicity of Human Natural Killer Cells against Cancerous Cells

Abstract

Cancer treatment with natural killer (NK) cell immunotherapy is promising. NK cells can recognize and kill cancer cells without sensitization, making them a potential cancer treatment alternative. To improve clinical efficacy and safety, more research is needed. Enhancing NK cell function improves therapeutic efficacy. Due to its potent apoptosis induction, Cordycepin, a bioactive compound from Cordyceps spp., inhibits cancer cell growth. Cordycepin has immunoregulatory properties, making it a promising candidate for combination therapy with NK cell-based immunotherapy. Cordycepin may enhance NK cell function and have clinical applications, but more research is needed. In this study, cordycepin treatment of NK-92 MI cells increased THP-1 and U-251 cell cytotoxicity. Cordycepin also significantly increased the mRNA expression of cytokine-encoding genes, including tumour necrosis factor (TNF), interferon gamma (IFNG), and interleukin 2 (IL2). NK-92 MI cells notably secreted more IFNG and granzyme B. Cordycepin also decreased CD27 and increased CD11b, CD16, and NKG2D in NK-92 MI cells, which improved its anti-cancer ability. In conclusion, cordycepin could enhance NK cell cytotoxicity against cancerous cells for the first time, supporting its use as an alternative immunoactivity agent against cancer cells. Further studies are needed to investigate its efficacy and safety in clinical settings.

INTRODUCTION

Natural killer (NK) cell is a type of cytotoxic lymphocyte that plays a crucial role in the innate immune system as a defensive effector cell against cancer/tumour and microbial infection without need for prior immunization required.¹⁾ NK cells are able to specifically target and diminish cancerous cells with both cytolytic and cytokine-producing activity.²⁾ Activated NK cells release cytolytic granules containing perforin, a pore forming glycoprotein and granzyme, a group of serine proteases that mediate cancer cell apoptosis.³⁾ NK cells can also secrete cytokines such as interferon gamma (IFNG) and tumour necrosis factor (TNF) alpha under the stimulation of immunoregulatory interleukin to further manipulate adaptive immunity.^2,4) With their anti-cancer potential, NK cells have attracted considerable interest in the realm of cancer immunotherapy. However, the use of enriched NK cells from blood, whether autologous or allogenic, necessitates additional processing to eliminate T lymphocytes and has some drawbacks, including unpredictable variability of yield, genetic alteration during expansion steps, and difficulty in manipulating or engineering.^5,6) Given the limitations of NK cells derived from blood, a continuously expanding NK cell line is a favourable substitute. Despite the establishment of numerous human NK cell lines, the NK-92 and its interleukin 2 (IL2)-independent derivatives, NK-92 MI and NK-92 CI, have consistently displayed excellent cytotoxicity against a variety of cancer cells and can be efficiently cultivated with the full spectrum of activating receptors such as CD28, NKG2D, NKp30, NKp44, and NKp46.^7–11) Particularly, NK-92 MI with its high IL2-producing capability,¹²⁾ makes the prominent candidates to be used in cancer immunotherapy. However, since NK-92 MI, along with its parental NK-92, is classified as an early mature NK cell with the lack of Fc gamma receptor IIIa/b (CD16) and several inhibitory receptors, its cytotoxic mechanism relies heavily on the perforin-granzyme cytolytic pathway and its cytotoxic effector molecules without antibody-dependent cell cytotoxicity (ADCC).¹³⁾ The cytotoxicity of NK-92 MI cells was also found to be varied and declined during long-term in vitro cultivation.¹⁴⁾ Therefore, NK-92 MI cell modulation and the search for potent agents that can maintain or boost its anti-cancer activity are also promising ways to make the therapy even more effective.

Cordycepin, 3′-deoxyadenosine, is the renowned natural compounds isolated from Cordyceps militaris.^15,16) This unique nucleoside derivative possesses a wide-range of biological activities, including immunoregulatory and apoptosis-based anticancer.^17,18) However, the mechanisms underlying such bioactivities is still elusive, and not yet fully elucidated. Since cordycepin can exhibit the remarkable influences in both immunity and anticancer aspects, it is possible to ameliorate NK cell functions, and could be one of alternative routes of its anticancer mechanisms.

In this study, we investigated the biological influences of cordycepin on NK-92 MI cells, including the physiological properties, focusing mainly on its surface markers/receptors and the cytokine and granzyme producing efficacy. Besides, the capacity of cordycepin in activating NK cell cytotoxicity against cancerous cells, THP-1 and U-251 cells, was also examined. This study, for the first time, elucidated the mechanism and verified the evidence that cordycepin could enhance the anti-cancer activity of NK cells.

MATERIALS AND METHODS

Cell Culture

A natural killer cell line, NK-92 MI (Catalogue No. CRL-2408), was purchased from the American Type Culture Collection (ATCC, Manassas, VA, U.S.A.), and cultured in the complete growth medium which was Eagle’s minimum essential medium (α-MEM) containing 1.5 mg/mL sodium bicarbonate (Sigma-Aldrich, St. Louis, MO, U.S.A.), 2 mM L-glutamine, 0.2 mM inositol (Sigma-Aldrich), 0.1 mM 2-mercaptoethanol (Gibco, CA, U.S.A.), 0.02 mM folic acid (Sigma-Aldrich), 12.5% horse serum (Sigma-Aldrich) and 12.5% fetal bovine serum (FBS) (HyClone, Logan, UT, U.S.A.). A human glioblastoma astrocytoma cell line, U-251 (Catalogue No. ECACC 09063001), were purchased from the European Collection of Authenticated Cell Cultures (ECACC, Salisbury, U.K.), and cultured in Dulbeccoʼs modified Eagleʼs medium (DMEM) media containing 10% FBS, and 1% (v/v) penicillin-streptomycin (HyClone). A human leukemia monocytic cell line, THP-1 (Catalogue No. TIB-202) were purchased from ATCC, and cultured in RPMI 1640 (HyClone) containing 10% FBS and 1% (v/v) penicillin–streptomycin. All cells were maintained in a humidified incubator with 5% CO₂ environment at 37 °C.

NK-92 MI Cell Viability against Cordycepin and NK-92 MI Cell Cytotoxic Activity on Cancerous Cells

Cordycepin stock solution was prepared at 25 mg/mL by dissolving 98.8% purified cordycepin powder (Sigma-Aldrich) in dimethyl sulfoxide (DMSO) solution (Sigma-Aldrich) to be used in the experiment. To assess NK-92 MI cell viability against cordycepin, NK-92 MI cells were seeded on 96-well microplates at a density of 2 × 10⁴ cells/mL to perform 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Sigma-Aldrich) and lactate dehydrogenase (LDH) assay using LDH cytotoxic assay kit (Abcam, Cambridge, U.K.; Catalogue No. ab65393). For MTT assays, cells were treated with cordycepin at varying concentrations (0, 1.25, 2.5, 3.75, 5, 15.6, 31.2, 62.5, 125, and 250 µg/mL) for 24 h. At a final concentration of 0.5 mg/mL, MTT solution was added to cell culture media and incubated for 3 h at 37 °C in the dark. The medium was removed, and DMSO was used to dissolve the formazan crystal. The absorbance was measured at 570 nm, using a SPECTROstar Nano microplate reader (BMG Labtech, Ortenberg, Germany). For LDH assay, cells were treated with cordycepin at varying concentrations (0, 1.25, 2.5, 5, 10, 20, 40, 80, 160, and 320 µg/mL) for 24 h. Then, microplates were centrifuged at 300 × g for 10 min at room temperature. The culture supernatants were then collected in new 96-well microplates, to perform lactate LDH assay following the manufacturer’s instruction and the absorbance at 450 nm was also recorded using a SPECTROstar Nano microplate reader.

To observe the effect of cordycepin on NK-92 MI cytotoxicity against cancerous cells, NK-92 MI cells were pre-treated with cordycepin at sub-toxic concentration for 24 h before performing LDH-based cytotoxicity assays. The target cells (Cancerous cell) 2500 cells were plated, and on the next day, the effector cells (NK-92 MI cells) were added at various ratios (1 : 1, 1 : 5, and 1 : 10, target cells: effector cells). After 24 h of co-culture, 50 µL of culture media was collected and used in LDH assay using. The value of experimental LDH release was calculated by subtracting the value of spontaneous LDH release from effector cells at corresponding dilutions. NK cytotoxicity of each condition was calculated using the following equation;

  

The experiments were performed in triplicates.

RNA Isolation and PCR Analysis

NK-92 MI cells were treated with cordycepin at varying concentrations (1, 5, 10, 25, 50, 100, 250, and 500 µg/mL) for 24 h. mRNA was extracted and isolated using the commercial NucleoSpin RNA kit (Macherey-Nagel, Dueren, Germany) and it was converted into a cDNA using cDNA synthesis kits (Toyobo, Osaka, Japan). RT-PCR was performed by using 2X Taq Master Mix (Vivantis, Selangor Darul Ehsan, Malaysia) in Biorad/C1000Touch Thermocycle (BioRad, Hercules, CA, U.S.A.) according to the manufacturer’s instructions. The amplified cDNA products were electrophoretically separated on 1.5% agarose gel and visualized by RedSafe™ Nucleic Acid staining (iNtRON Biotechnology, Gyeonggi-do, Korea). The relative expression level of a target gene was quantified by normalization with the internal control glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene. The primers used in the experiment were listed as follows: TNF 5′-GAGCACTGAAAGCATGATCC (forward) and 5′-CGAGAAGATGAT CTGACTGCC (reverse); IFNG 5′-CTCTTGGCTGTTACT GCCAGG (forward) and 5′-CTCCACACTCTTTTGGATGCT (reverse); IL2 5′-AGAACTCAAACCTCTGGAGGAAG (forward) and 5′-GCTGTCTCATCAGCATATTCACAC (reverse); GAPDH 5′-ACCTGACCTGCCGTCTAGAA (forward) and 5′-TCCACCACCCTGTTGCTGTA. The names of genes followed the HGNC nomenclature.

Release of IFNG and Granzyme B

NK-92 MI cells were cultured in α-MEM complete medium or α-MEM complete medium with 2.5 µg/mL cordycepin for 24 h prior to co-incubation with the target THP-1 or U-251 cells at target-effector ratios of 1 : 10 for 24 h. Supernatants of culture medium were collected by centrifugation at 2000 × g for 10 min and were then detected for the release of both IFNG and Granzyme B using enzyme-linked immunosorbent assay (ELISA) kits (Catalogue Nos. ab174443 for IFNG and ab235635 for Granzyme B, Abcam), according to the manufacturer’s instructions.

Flow Cytometry

NK-92 MI cells were cultured in α-MEM complete medium or 2.5 and α-MEM complete medium with 5 µg/mL cordycepin at 37 °C for 24 h. Cells were washed twice with phosphate buffered saline (PBS) and stained with antibodies mixed in 100 µL staining buffer (PBS with 1% fetal calf serum (FCS)) for 15–30 min at 25 °C in the dark. The following antibodies were used at concentrations of 1 : 100: phenylephrine (PE) anti-human CD27 (Biolegend, 356406), fluorescein isothiocyanate (FITC) anti-Human CD11b (BioLegend 301330), FITC anti-human CD16 (BioLegend Cat. No. 302006), PE/Cyanine5 anti-human CD56 (BioLegend 362516), PE anti-human CD314 (NKG2D) (BioLegend Cat. No. 320806). After two washes, cells were suspended in 50–100 µL PBS or staining buffer and analyzed on a Cytomics FC500 Flow Cytometer (Beckman Coulter, Indianapolis, IN, U.S.A.).

Statistical Analysis

IBM SPSS Statistics, version 16.0 (SPSS Inc., Chicago, U.S.A.), was used for statistical analysis. All tests were performed three times, and data were expressed as mean standard deviation (S.D.). ANOVA, followed by Tukey’s test at * p < 0.05 and ** p < 0.01 was used to evaluate whether there were significant changes between treatments.

RESULTS

Effects of Cordycepin on NK-92 MI Cell Viability and Cytotoxic Activity against Cancerous Cell

To assess the effect of cordycepin on cell viability, NK-92 MI cells were treated with different concentrations of cordycepin diluted in the complete growth medium for 24 h prior to determining the cell viability by using MTT assays and LDH assay. As shown in Fig. 1A, the treatment of 1.25–31.2 µg/mL and 1.25–20 µg/mL of cordycepin showed no significant effect on the cell viability of NK-92 MI cells according to MTT assays and LDH assays, respectively. However, the treatment with 40 µg/mL cordycepin or higher significantly declined cell viability of NK-92 MI cells in the dose-dependent manner compared to the control group without cordycepin (* p < 0.05, ** p < 0.01). Then, the augmentation effects of cordycepin on NK-92 MI cytotoxic activity against THP-1 acute myeloid leukemia cells and U-251 glioblastoma cells were observed. For these experiments, NK-92 MI cells were pre-treated with the sub-toxic dose of cordycepin at 2.5 µg/mL. Subsequently, both target cancerous cell types were separately co-cultured with the effector NK-92 cells at different target-effector ratios (1 : 1, 1 : 5, and 1 : 10) in an equally mixed medium consisting of the optimal media for both NK-92 MI and each cancerous cell type. Significantly, the pre-treatment of NK-92 cells with 2.5 µg/mL cordycepin led to the enhancement of NK-92 MI cytotoxic activity against both THP-1 cells and U-251 cells (Figs. 1B, C).

Fig. 1. Effects of Cordycepin on NK-92 MI Cell Viability and Cytotoxic Activity against Cancerous Cells

(A) Effects of cordycepin on NK-92 MI cell viability. The MTT assay (Left) and LDH assay (Right) were used to assess cell viability after treating NK-92 MI with different concentrations of cordycepin for 24 h. Significantly difference was relatively compared to the control (0 µM) at: * p < 0.05, and ** p < 0.01. Then, effector NK-92 MI cells were cultured with 2.5 µg/mL of cordycepin for 24 h prior to co-culturing with (B) target THP-1 acute myeloid leukemia cells, and (C) U-251 glioblastoma cells at various target-effector ratios (1 : 1, 1 : 5, and 1 : 10) and the co-culture setting was then incubated for 24 h. The LDH assay was also used to assess cancer cell death. Cell death percentages (%) are used to represent the data. Data were expressed as % NK cell cytotoxicity against the THP-1 and U-251 cells. Significantly difference was relatively compared to the control (Non-treated NK-92 MI) at: * p < 0.05, and ** p < 0.01. The experiments were performed in triplicate.

Effect of Cordycepin on mRNA Expression of Genes Encoding Cytokines in NK-92 Cells

To determine whether the cordycepin enhancement of the NK-92 MI cytotoxic activity against THP-1 cells and U-251 cells was due to cytokine gene regulation, the mRNA expression of genes encoding cytokines, including TNF, IFNG and IL2 was measured by RT-PCR. After the treatment of cordycepin at various concentrations (1.25, 2.5, 5, 10, and 20 µg/mL) for 24 h, the mRNA expression of TNF, IFNG, and IL2 were upregulated in the cordycepin-treated groups compared to the control group (Figs. 2A–D). The results revealed that the cytotoxicity enhancement of NK-92 MI cells by cordycepin treatment could relate to the induction of cytokine-encoding gene expression.

Fig. 2. The Effect of Different Cordycepin Concentrations on Cytokine mRNA Expression in NK-92 after 24 h of Treatment

(A) Densitometric analysis for mRNA expression of cytokine genes. (B–D) The relative expression TNF, IFNG, and IL2 were determined using GAPDH as a reference gene, respectively. The values were expressed as the mean standard deviation (n = 3). (* p < 0.05 and ** p < 0.01 versus control cells with no cordycepin treatment).

Effect of Cordycepin on IFNG and Granzyme B Production and Secretion by NK-92 Cells

According to the properties of mature NK cells, the secretion of IFNG is essential to the early phases of the immune response against cancer,¹⁹⁾ while granzyme B secretion is one of the crucial step to trigger cancer cell apoptosis.^20,21) The effect of cordycepin on IFNG and granzyme B secretion by NK-92 MI cells was determined. NK-92 MI cells were pre-treated with 2.5 µg/mL for 24 h and then co-cultured with each target cancerous cells at the target-effector ratio of 1 : 10 for 24 more hours. In both pre-treatment and co-culture steps, the supernatants of culture medium were collected to perform ELISA for such cytokine and granzyme detection. Pre-treatment with 2.5 µg/mL cordycepin caused statistically significant enhancement in IFNG and Granzyme B production and secretion (Fig. 3). The IFNG concentrations found in supernatants were 211.7 ± 6.9, 242.8 ± 27.6, 332.6 ± 31.1 pg/mL for the pre-treatment with cordycepin at 2.5 µg/mL and the co-incubation with target THP-1 and U-251 cells, respectively, which were 1.1–1.8 times higher than the IFNG production levels of untreated NK-92 MI cells (184.1 ± 10.4 pg/mL) (Fig. 3A). Moreover, the granzyme B concentration found in supernatants were 201.7 ± 14.1, 223.3 ± 7.1, 288.3 ± 51.8 pg/mL for the pre-treatment with cordycepin at 2.5 µg/mL and the co-incubation with target THP-1 and U-251 cells, respectively. These values were 1.2–1.8 times higher than the granzyme B production levels of untreated NK-92 MI cells (Fig. 3B). The results demonstrated that cordycepin enhanced the production and secretion of both IFNG and granzyme B in NK-92 MI cells after the treatment, which could lead to its enhanced cytotoxicity against cancerous cells.

Fig. 3. Effect of Cordycepin on IFNG and Granzyme B Production and Secretion by NK-92 MI

NK-92 MI were incubated with 2.5 µg/mL cordycepin for 24 h prior to co-culture with target THP-1 acute myeloid leukemia cells and U-251 glioblastoma cells at target-effector ratios (1 : 10) and the co-culture setting was then incubated for 24 h. (A) IFNG and (B) Granzyme B in culture supernatant were then determined by ELISA. The experiments shown in this study were performed in triplicate (* p < 0.05; ** p < 0.01).

Effect of Cordycepin on the Expression of Surface Markers and Receptors of NK-92 MI Cells

Since the development stage and function of NK cells could be observed from the expression of their surface makers and receptors, the effects of cordycepin on such marker expression was monitored using flow cytometry including T-cell activation antigen S152 (CD27), integrin subunit alpha M (CD11b), neural cell adhesion molecule 1 (CD56), Fc gamma receptor IIIa/b (CD16) and killer cell lectin like receptor K1 (NKG2D). The treatment of NK-92 MI cells with 2.5 and 5 µg/mL cordycepin upregulated the expression of CD11b, CD16 and NKG2D (Figs. 4B, C, E) but conversely downregulated CD27 (Fig. 4A). Additionally, the expression of CD56 were maintained at the same level despite of the cordycepin treatment (Fig. 4D). These results indicate that cordycepin could affect the functional properties and even the developmental stage of NK-92 MI cells.

Fig. 4. Effect of Cordycepin on the Expression of Surface Markers/Receptors of NK-92 MI

Representative flow cytometry analyses for the expression of (A) CD27, (B) CD11b, (C) CD16, (D) CD56, and (E) NKG2D were obtained from NK cells treated with cordycepin at 0, 2.5, and, 5 µg/mL, respectively.

DISCUSSION

The natural killer cell line, NK-92, originally derived from a 50-year-old male patient with rapidly progressing non-Hodgkin’s lymphoma,²²⁾ is a highly cytotoxic and continuously growing cell line and has been reported to counteract a broad spectrum of cancer cells in vitro and currently used in numerous clinical studies.^6,23) Even as the closest resembling NK cell line, its IL2-dependent property proved to be the major limitation for long-term cultivation and prolonged treatment. Therefore, efforts have been made to engineer an IL2-independent variants of NK-92 cell line including NK-92 MI which was engineered to produce autologous IL2 by particle-mediated gene transfer of the hIL-2 cDNA (MFG-hIL2 vector)¹²⁾ to overcome such limitation and become more favourable for both research-based and clinical applications. There were several clinical trials of NK-92-based cancer immunotherapy against different types of cancer such as leukemia, Merkel cell carcinoma and pancreatic cancer²³⁾ either independent or with the combination of radiation or chemotherapy. Although none of the patients reported severe adverse effects, no substantial clinical responses were observed. Moreover, long-term in vitro cultivation of NK-92 MI cells was found to cause the reduction of their cytotoxicity against four leukemia cell lines¹⁴⁾ which could be the burden for the clinical application. Therefore, the efficacy and durability of the injected NK-92 in the clinical therapy approach might be improved by modifying or boosting its cytotoxic activity utilizing particular synthetic or phytochemical stimulators.²⁴⁾ Since, water extract (WE) of C. militaris containing cordycepin was previously discovered to act as an immune stimulator and increase phenotypic and functional maturation of dendritic cells (DC) by upregulating CD40, CD54, CD80, CD86, and MHC class II expression,²⁵⁾ in this study, we observed the biological activities of cordycepin, the unique 3′ deoxyadenosine from Cordyceps militaris, as NK cell stimulator.

To primarily determine the optimal sub-toxic concentration cordycepin to be used for NK cell stimulation, the viability of NK-92 MI cells was assessed following incubation with various concentrations of cordycepin (Fig. 1A). 1.25–20 µg/mL of cordycepin was found to show no significantly negative influence on NK-92 cell viability compared to the control group without cordycepin. Considering the most significant mRNA upregulation of TNF, IFNG and IL2 by 2.5 µg/mL of cordycepin treatment (Fig. 2), this was the optimal sub-toxic cordycepin concentration to be carried out in further experiments. Previously, researchers reported that treatment of cholangiocarcinoma cells (CCA) with cordycepin prior to and during co-culturing with NK-92 cells significantly increased the death of such cancerous cells as compared to solitary cordycepin or NK treatment. The relying mechanism was elucidated to be due to the sensitizing effect of cordycepin on cancerous cells via the increase in tumor-necrosis factor related apoptosis-inducing ligand (TRAIL) receptor (DR4 and DR5) expression.²⁶⁾ However, the effect of cordycepin on NK-92 during such a co-culture situation was neglected and remained unclear. Therefore, this study aimed to investigate the effect of cordycepin mainly in NK-92 MI and tested whether cordycepin could stimulate its cytotoxic activity against cancerous cell. Twenty-four hours of incubation of NK-92 MI cells with 2.5 g/mL cordycepin significantly (p < 0.01) increased their cytotoxicity against both THP-1 cells, representing liquid cancer cell type, and U-251 cells, representing solid malignant cancer cell type (Figs. 1B, C). Further experiment suggested that the augmentation of NK-92 MI cytotoxicity may relate to the upregulation of TNF, IFNG and IL2 (Fig. 2), the enhancement of IFNG and granzyme B secretion (Fig. 3), the activation of the CD16, CD11b and NKG2D and the suppression of CD 27 (Fig. 4).

Several studies have shown that cordycepin has potent immunomodulatory properties regarding the cytokine regulation. Agreeable with the results, C. militaris extract containing cordycepin was reported to increase IFNG and IL12 level in blood of participants after oral administration.²⁷⁾ In methotrexate-induced splenocytes, cordycepin has been shown to drastically improve IL2, interferon, and tumour necrosis factor production.²⁸⁾ Oppositely, studies reported the suppression of T lymphocyte function with the reduction of IL2 and IFNG level²⁹⁾ and the downregulation of TNF in LPS-induced RAW 264.7 macrophage.³⁰⁾ The effect of cordycepin on cytokine regulation may be varied from cell type to cell type. IL2, first identified as a T cell growth factor, is a 15.5–16 kDa globular glycoprotein comprised of four antiparallel α helices.³¹⁾ Despite the fact that IL2 is exclusively produced by T cells and not NK cells,³²⁾ it is required for their proliferation and cytotoxicity via the synthesis of lytic molecules such as perforin and Granzyme B.³³⁾ IL2 stimulation was reported to restore impaired NK cell cytotoxicity caused hypoxic settings in in multiple myeloma.³⁴⁾ With the genetically modification, NK-92 MI could independently produce IL2 sufficient their own proliferation. Study reported that IL2 up-regulated the expression of interleukin 12 (IL12) receptors, IL12RB1 and IL12RB2, and played an important role in maintaining the expression of signal transducer and activator of transcription 4 (STAT4), a critical STAT protein involved in IL12 signaling in NK cells which subsequently enhance IFNG expression and production.³⁵⁾ Therefore, cordycepin-induced IL2 upregulation could be one of the plausible causes of NK-92 MI cytotoxicity augmentation. As for IFNG, it is a homodimeric cytokine that regulates various aspects of immune system responses, including NK cell actions, by forming a positive feedback loop.³⁶⁾ The upregulation of IFNG expression and secretion in this study could potentially take part in the cytotoxicity enhancement via the activation of death receptor expression on target cancerous cell and initiate pro-apoptotic signaling pathways.³⁷⁾ IFNG also increases NK cell cytotoxicity and promotes the expression of activating receptors on the surface of NK cells, such as NKp46 and NKG2D, promoting the recognition and killing of cancerous cells.³⁸⁾ Additionally, the quantification of CD56 expression on the surface of activated NK cells was found to be correlated to the evaluation of cytotoxicity and IFNG production,³⁹⁾ which is applicable with the maintaining of surface CD56 expression in this study. TNF, the homodimer proinflammatory cytokine, was also upregulated by cordycepin to augment NK 92 MI cytotoxicity. TNF could assist the effects of IL2 on NK cell proliferation, differentiation, and activation, as well as on NK cell-mediated cytotoxicity against target cells in vitro.⁴⁰⁾ Besides, as a death receptor ligand, TNF binds to death receptors on the surface of target cancerous cells, which the expression was activated by IFNG, to activate cancer apoptotic cell death.⁴¹⁾ Other than the cytokine, the secretion of granzyme B, the secreted serine protease that is found in the lytic granules of NK, along with perforin, a pore-forming glycoprotein,⁴²⁾ was also enhanced by cordycepin treatment. The improved level of granzyme B may entered the cytoplasm of the target cancer cell after being secreted and causes apoptotic cell death³⁾ hence causing the cytotoxicity amplification.

Considering the alteration of surface markers/receptors of NK 92 MI caused by cordycepin treatment, this could shed some light on the mechanism of the functional augmentation effect of cordycepin on NK cells. Generally, NK-92 cell line and its derivative, NK-92 MI, express of CD56^bright with the absence of CD16^6,11) indicating its immature 4a/4b development stage.¹⁾ Its cytotoxic mechanism highly depends on the perforin-granzyme cytolytic pathway and its cytotoxic effector molecules including TNF-superfamily members but not ADCC.¹³⁾ To overcome such limitation, researchers modified NK-92 to establish various CD16-positive NK cell lines using a variety of strategies. Zhao et al. engineered the NK-92 cell line using lentivirus-based transfection of CD16/CAR to obtain the NK92-41BB cell line with high granzyme-mediated cytotoxicity and antibody-dependent cellular cytotoxicity.⁴³⁾ Cheng et al. utilized the adaption medium X–VIVO10 with human platelet lysates to establish a CD16-expressing population designated as oNK-1.⁴⁴⁾ Similarly to the activation of CD16 expression by chemicals in adaption medium, cordycepin could activate the CD16 expression in NK-92 MI cells. This finding suggests that cordycepin may have potential as an alternative method for generating CD16-expressing NK cells for use in immunotherapy. Further studies are needed to investigate the efficacy and safety of this approach. Additionally, resembling in murine NK cells, the density of CD27 and CD11b were reported to further functionally distinguish the NK subsets and used to indicate NK cell deficiency in patients.⁴⁵⁾ The downregulation of CD27 and the upregulation of CD11b could somehow affected NK-92 MI cytotoxicity. The downregulation of CD27 expression in combination with the expression of CD11b was reported to be an indicator of cytolytic effector cells within human NK cell subsets. Compared to CD27^- NK cells, CD27⁺ NK cells in the blood were found to produce lower levels of perforin and granzyme B with less cytotoxicity.⁴⁶⁾ The transmembrane activating receptor, NKG2D was also found to be drastically upregulated by cordycepin. NKG2D binds to specific ligands such as MICA on the surface of target cancerous cells and activates NK cells to produce cytotoxic molecules such as granzyme B.⁴⁷⁾ Target cell recognition by NKG2D also leads to the production of IFNG and TNF to facilitate tumour clearance.^48,49) NKG2D downregulation could be found in cancer patients due to the effect of transforming growth factor beta (TGF) secretion by cancer cells and caused cancer immune evasion.^50,51) Therefore. the NKG2D upregulation was another crucial mechanism behind the enhancement of cytotoxicity by cordycepin.

CONCLUSION

This study investigated the potency of cordycepin to enhance the cytotoxicity of NK cells against cancerous cells (THP-1 and U-251 cancer cells) (Fig. 5). It was found that cordycepin exhibited potent NK cell augmentation potential through the activation of TNF, IFNG and IL2 production, and the enhancement of exocytosis granzyme B secretion. Importantly, cordycepin could induce the maturation of NK-92 MI cells by decreasing the expression of CD27 and increasing the expression of CD11b, CD16, and NKG2D. This evidence supported the further development of the clinical applications of cordycepin as an anti-cancer and immune-enhancing agent and by elevating NK cell activity.

Fig. 5. Schematic Illustration of the Biological Activity of Cordycepin on the Cytotoxicity Enhancement of NK-92 MI against Cancerous Cells

Acknowledgments

This work was supported by Suranaree University of Technology (SUT) Research and Development Fund.

Author Contributions

NC: conceptualization, investigation, methodology, and data analysis.

PK: investigation, methodology, data analysis, and contribution to manuscript writing.

PN: conceptualization, methodology, data analysis, contribution to manuscript writing, and funding acquisition.

All authors have read and approved the final version of the manuscript.

Conflict of Interest

The authors declare no conflict of interest

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