Anticancer effects of thymoquinone on prostate cancer cells and natural killer cell activity

Introduction

Globally, the burden of cancer incidence and death is increasing rapidly (1). Cancer is a complex disease and there is no single treatment that is effective for all types (2,3). With an estimated 1,414,259 new cases worldwide each year, prostate cancer (PC) is the second most frequent malignancy and the top cause of death for men worldwide (4). The average age at diagnosis is 66 years old, and PC incidence and mortality rise with age globally (3). There are 158.3 new cases diagnosed per 100,000 males, with 293,818 additional cases projected by 2040 (5). Moreover, African-American men have been reported to experience higher incidence rates and roughly double the mortality rate from PC than Caucasian men. This discrepancy has been explained by social, environmental and genetic variations (2).

PC that is detected early may not show any symptoms, often advances slowly and may be treated with little-to-no intervention. The most frequent symptoms, however, are nocturia, increased frequency of urine and difficulty urinating, which are indicative of prostatic enlargement. In more advanced stages of the disease, patients may experience back discomfort and urine retention due to metastasis to the axis skeleton, the most common site of bone metastatic disease (6).

The risk of PC is notably influenced by diet and exercise. For example, dietary factors are associated with the observed variations in PC incidence rates between countries and ethnic groups (7,8). However, the majority of studies concentrate on the genes implicated in the inherited form of PC as well as the mutations that take place in the acquired form (6,7). Therefore, a thorough analysis of PC epidemiology and risk factors may aid in understanding the relationship between genetic abnormalities and the function of the environment in causing these alterations and/or encouraging tumor progression. These differences in PC incidence imply that the etiology of PC is markedly influenced by environmental variables. However, underdiagnosis, disparities in screening techniques and access to healthcare may also contribute to variations in incidence (6).

Due to several biological and tumor microenvironmental factors, PC presents substantial treatment challenges. For example, prostate tumors often have a low tumor mutational burden, meaning that the immune system targets fewer neoantigens (9). Furthermore, immunosuppressive cells, such regulatory T cells, tumor-associated macrophages and myeloid-derived suppressor cells, are present in the PC immunosuppressive tumor microenvironment. The normal immune response against cancer cells is hampered by these cells, which produce a ‘cold’ tumor environment with limited immune cell penetration (10). Furthermore, although androgen deprivation therapy is initially successful, certain patients experience resistance to the treatment, which can result in metastatic castration-resistant PC. Changes in the tumor microenvironment and immune evasion strategies are associated with this development (11).

Using immune checkpoint inhibitors, cancer vaccines, chimeric antigen receptor T-cell treatment and combination medicines, immunotherapy offers potential treatments by using the immune system to target and kill cancer cells. By reducing antitumor immune responses or modifying tumor immunogenicity, several immune system components cooperate to either protect the host against necessary tumor progression, enhance tumor escape, or both (12). This process is known as cancer immuno-altering (12,13). Immune spots, which are resistant cell surface receptors that regulate the suppression or activation of immune reactions, serve an important role in different phases of the immune responses. For example, both PD-1 (Programmed Death-1) and cytotoxic T-Lymphocyte-Associated Protein 4 CTLA4, prevent overactivation of the immune system once binding to their ligands (14). The optimum outcome for controlling tumors is immune system activation (13). In addition to surgery, chemotherapy, radiation and targeted medicines, cancer immunotherapy has become a crucial therapeutic approach. Furthermore, cancer immunotherapy has demonstrated encouraging outcomes with several cancer types and can be used in conjunction with other treatments. Consequently, immunotherapy has been reported to have notable benefits: In one study (15) immunotherapy improved lung cancer treatment PD-1 inhibitor to standard chemotherapy, increased objective response rate and progression free survival. In addition, another study showed that combining immunotherapy with radiation enhance anti-tumor immune response through increasing the release of tumor antigen following radiation (16).

A subset of lymphocytes, known as natural killer (NK) cells, is essential to the innate immune response against malignancies and infections (17,18). NK cells are able to recognize and target cancerous and contaminated cells (19,20), and ~90% of the circulating NK cell population is made up of CD56 and CD16 cells, which cause cell lysis by releasing cytolytic granules that include granzyme and perforin. Together, NK cells and interferon-γ (IFN-γ) cause target cells to undergo apoptosis (21,22). For aberrant cells, NK cells increase antibody-mediated cytotoxicity and cytotoxic T lymphocytes act in a similar manner (23).

NK cells help to defend the body against foreign (pathogens) and endogenous (cancer) threats. Extensive research has been performed with the goal of identifying an efficient method for targeting impacted cells by utilizing NK cell pathways (24) (Fig. 1). However, notwithstanding the promising results of this research, tumor cells develop a defense mechanism that thwarts the cytotoxicity caused by NK cells (25,26).

Figure 1. Cytotoxicity mechanism of NK cells. By evaluating the net input of activating and inhibitory signals from target cells through NK cell surface receptors, NK cells discriminate healthy cells from target cells. However, several tumors develop mechanisms to escape NK cell immune responses by modifying their cell surface molecules, such as low immunogenicity. Following the identification of the target cell by NK cells, the stimulation domain is activated. This is initiated by the secretion of a cytolytic enzyme, known as perforin, stored in NK cell vesicles. Perforin creates pores in the cell membrane of the target cell, allowing the passage of granzymes into the target cell and starting protein digestion (cytolysis). NK cells express TRAIL and FasL, which interact with TRAIL receptors and FAS ligands in cancer cells, respectively. This interaction results in cell death, signaling complex stimulating apoptosis. NK, natural killer; TRAIL, TNF-related apoptosis inducing ligand; TRAILR, TRAIL receptor; MHC, major histocompatibility complex; FasL, FAS ligand. Created in BioRender.com.

Research is investigating the use of NK cells to treat (27–29) PC. NK cells can directly target and eliminate PC cells, resulting in direct tumor cytotoxicity. Moreover, research has reported that, both in vitro and in vivo, genetically modified NK cells, such as prostate-specific membrane antigen (PSMA)-targeted chimeric antigen receptor-NK cells, have strong cytotoxic effects on PC cells. They can specifically identify and eliminate PSMA-expressing PC cells, providing a targeted therapy strategy. Therefore, these altered NK cells may be employed as an ‘off-the-shelf’ therapeutic option, enabling a sustainable and economical treatment technique (27).

Additionally, PC cells usually develop defenses against immune detection, such as by producing soluble NK cell receptor D (NKG2D) ligands to obstruct the recognition of NK cells. However, NK cells can adapt to these challenges, and strategies are being developed to make them more effective against these tumor escape tactics (28). Investigating NK cell function against PC is challenging as they produce TGF-β, IL-10 and regulatory T cells, inhibiting NK activation, and have low infiltration of NK cells making it hard to study their direct interaction with PC. PC is a useful model for researching NK cell-mediated therapy, for example due to the immune microenvironment dynamics, in which PC tumors are distinguished by a distinct immune milieu that consists of both immuno-suppressive and immune-activating components (29–32). As studies have reported that NK cells in patients with PC frequently have compromised cytotoxic function, this environment offers a realistic setting for researching how NK cells interact with tumor cells and the factors determining their efficacy (29,33). This malfunction provides a clear target for therapeutic intervention trials targeted at restoring NK cell activity and is associated with the course of the disease. Furthermore, research employing preclinical models has reported that NK cells are capable of efficiently inhibiting the proliferation of PC cells resistant to castration. These results highlight how effective NK cell-based treatments can be in treating advanced PC (30). Nevertheless, PC uses several strategies to avoid immune monitoring, especially from NK cells. These tactics include modulating the expression of NK cell receptors, secreting immunosuppressive substances and changing the expression of ligands (28). PC cells can decrease the expression of ligands such NKG2D and DNAX accessory molecule 1 that are recognized by NK cell receptors. Moreover, immunosuppressive cytokines such as TGF-β are abundant in the tumor microenvironment. The release of TGF-β causes the activating receptors of NK cells, including NK cell P30-related protein and NKG2D, to be downregulated, which reduces the cytotoxic effects (31). Furthermore, PC can cause NK cells to exhibit a fatigued phenotype, which is typified by decreased activating receptor expression and decreased cytotoxic activity. This fatigue is associated with NK cells expressing more inhibitory receptors, such as T cell immunoglobulin mucin 3 and programmed cell death protein 1 (PD-1), which results in weakened antitumor responses (32). Additionally, programmed death-ligand 1 (PD-L1) expression on cancer cells may be upregulated in PC with hypoxic circumstances. Immune evasion is facilitated by the inhibition of the cytotoxic function of NK cells caused by the interaction between PD-L1 and its receptor PD-1 (34).

Finally, due to their ability to treat numerous diseases, several plants are regarded as useful therapeutic instruments in a wide range of medical disorders. Moreover, different medicinal plants have been used worldwide to alleviate the symptoms of several illnesses for millennia (35). One well-known example with a wide range of applications is Nigella sativa, also known as dark cumin. For two millennia, populations around the world have utilized this plant, a dicotyledon of the Ranunculaceae family, as a snack, spice and nutritional supplement (36). Thymoquinone (TQ), the main active ingredient in the black seeds, has garnered interest in both traditional medicine and contemporary therapeutic research (37,38). Therefore, the present study aimed to assess how TQ, which comprises ~40% of Nigella sativa (38), affects the cytotoxic activity of NK cells against the PC3-RFP cell line. The results could aid in comprehending the potential therapeutic impact of TQ on NK cells for cell-based immunotherapy.

Materials and methods

Blood collection

Blood samples were collected at King Abdulaziz University Hospital (Jeddah, Saudi Arabia) between August and November 2021 from 10 healthy volunteers six male, four female and aged 25–45 years in EDTA tubes (BD Vacutainer) and used for NK cell isolation. All experiments were performed at King Faisal Specialist Hospital and Research Centre (Jeddah, Saudi Arabia). Inclusion criteria for enrolled participants include general good health with no chronic diseases, recent infections and immunosuppressive medication or vaccination. Exclusion criteria were smoking, pregnancy or breastfeeding, autoimmune disease and malignancy.

MACS positive isolation

NK cells were purified by positive selection of CD56 cells (CD56 MicroBeads, human, 130-050-401, Miltenyi biotec) and MACS column (130-042-201, Miltenyi biotec, USA) was used according to the manufacturer's instruction.

Cell lines and culture

Transfected PC3 cells with red fluorescence (PC3-RFP cells) with the pDSRed-monomer-Hyg-C1 plasmid to produce hygromycin-resistant cells that express RFP were provided by AlShaibi et al (39). The cells were stored at King Fahd Medical Research Center (Jeddah, Saudi Arabia) and used in the present study.

PC3-RFP cells were cultured in high-glucose DMEM (cat. no. 11965118; Thermo Fisher Scientific, Inc.), with sodium pyruvate L-glutamine and Phenol Red, and supplemented with 10% penicillin-streptomycin (10,000 U/ml; cat. no. 15140122; Thermo Fisher Scientific, Inc.). NK cells were cultured in NK MACS Medium (cat. no. 130-092-657; Miltneyi Biotec B.V. & Co. KG), supplemented with 20% FBS (cat. no. 12103C; Sigma-Aldrich; Merck KGaA) and 0.1 mM β-mercaptoethanol (Sigma-Aldrich; Merck KGaA), IL-2 (human animal-component free, recombinant, expressed in E. coli, ≥98%; cat. no. SRP3085; Sigma-Aldrich; Merck KGaA). The cells were routinely maintained in humidified incubator at 37°C and 5% CO2.

TQ preparation

TQ (cat. no. 274666-1G; Sigma-Aldrich; Merck KGaA) was prepared by dissolving 4.926 mg TQ crystal in 10 µl anhydrous dimethyl sulfoxide (≥99.9%; cat. no. 276855; Sigma-Aldrich; Merck KGaA) and 9,990 µl NK medium to yield a stock solution. The final DMSO concentration in all TQ working solutions (50 and 25 µM) was 0.1%. Vehicle controls containing 0.1% DMSO in NK medium without TQ were included in all experiments.

Cell cytotoxicity of TQ on tumor (PC3-RFP) and NK cells

Cell cytotoxicity was analyzed using the CytoTox® 96 Non-Radioactive Cytotoxicity Assay kit (Promega Corporation). Briefly, PC3-RFP was plated at a density of 15×103 cells per well in a 96-well flat-bottom plate and incubated overnight at room temperature. Subsequently, NK cells were co-cultured at an effector cell/target cell ratio of 1:2 in the presence of different concentrations (25 and 50 µM) of TQ for 5 h. CytoTox 96 lysis buffer was added and incubated for 45 min at room temperature. CytoTox 96 reagent was then added to each well and incubated for 30 min at room temperature in the dark, and then the stop solution was added. The absorbance was measured at 680 and 490 nm.

Flow cytometry

Following positive selection of NK cells were stained with CD56-PE (DAKO; Agilent Technologies, Inc.) at 25°C for 30 min in the dark. The cells were then analyzed using flow cytometry (Novocyte Flow Cytometer; Agilent Technologies, Inc.). Phycoerythrin (PE) detection channel was the analyte detector, CD56-PE was the analyte reporter, and NovoExpress software (version 1.6.3; Agilent Technologies, Inc.)used for data acquisition and analysis.

ELISA

PC-3-RFP and NK cells (1:2; 15–30×103) were cocultured in the presence of TQ for 5 h at 37°C. Cell-free supernatants were harvested and ELISA was used for detection of IFN-α (Human IFN-α ELISA Kit; cat. no. BMS216 Invitrogen™; Thermo Fisher Scientific, Inc.), granzyme B [Human granzymes B (Gzms-B) ELISA kit; cat. no. E0899Hu; Shanghai Korain Biotech Co., Ltd.] and perforin [Human Perforin/Pore-forming protein (PF/PFP) ELISA Kit; cat. no. E0070Hu; Shanghai Korain Biotech Co., Ltd.] were used. Absorbance was measured at 450 nm using a microplate reader.

Statistical analysis

The data were analyzed using GraphPad Prism 9 (IBM Corp.) and the results are presented as the mean ± standard deviation of three independent experiments. Statistical significance was assessed using one-way analysis of variance and Tukey's multiple comparison test. Spearman correlation coefficient was used for correlation tests. P<0.05 was considered to indicate a statistically significant difference.

Results

Flow cytometry analysis

Using flow cytometry, the positivity of CD56 was determined to assess the purity of the isolated NK cells by comparison with the defined cutoff values obtained with unstained control cells. The NK cell purity was 95.7% (Fig. 2).

Figure 2. NK cells dot plot before treatment. CD56 positivity was determined by comparison with defined cut-off values obtained from unstained control cells. The NK cell purity was 95.7%. NK, natural killer.

Cytotoxic effect of TQ on NK cells with PC3-RFP

The cytotoxicity of NK cells was evaluated in co-culture with PC3-RFP cells. The effector (NK cells) and target (PC3-RFP) ratio was 1:2 and different concentrations of TQ (25 and 50 µM) were added in the culture. In the presence of TQ, there was a significant increase in NK cell cytotoxicity: Both concentrations of TQ (25 and 50 µM) significantly upregulated NK cell cytotoxicity in PC3-RFP cells, with cytotoxicity at 85.35% compared with 55.1% in the control group of NK cells co-cultured with tumor cells. Cell cytotoxicity was also significantly increased in tumor cells treated with both 25 and 50 µM TQ in the presence of NK cells, compared with tumor cells treated with the same TQ concentrations in the absence of NK cells (Fig. 3).

Figure 3. NK cell cytotoxicity against PC3-RFP cells in the presence or absence of TQ. The effector (NK cells) and target (PC3-RFP) ratio was 1:2. *P<0.05; **P<0.01; ***P<0.001. NK, natural killer; TQ, thymoquinone; TC, tumor cells.

Effect of TQ on the activity of NK cells

Co-culture of NK cells with PC3-RFP cells significantly increased the production of perforin, granzyme B and IFN-α from NK cells compared with NK cells alone using both concentrations of TQ (25 and 50 µM). NK cells co-cultured with PC3-RFP cells and treated with 50 µM TQ had significantly increased production of perforin and granzyme B production compared with the control NK cells co-cultured with tumor cells. The higher 50 µM TQ dose increased the production of perforin and granzyme B (0.80 and 1.24 ng/ml, respectively) by the NK cells more than the lower 25 µM dose (0.50 and 0.76 ng/ml, respectively). Furthermore, IFN-α production by the NK cells was significantly increased in the presence of 25 and 50 µM TQ (0.5 and 0.7 ng/ml, respectively) compared with the control NK cells co-cultured with tumor cells (0.3 ng/ml), with a greater increase observed in the presence of 50 µM TQ (Fig. 4).

Figure 4. Production of major cytokines against PC3-RFP cells by NK cells in the presence or absence of TQ. TQ enhances the release of (A) perforin, (B) granzyme B and (C) IFN-α, the major secreted cytokines of NK cells. *P<0.05; **P<0.01; ***P<0.001. NK, natural killer; TQ, thymoquinone; TC, tumor cells.

The correlation between individual cytokines and NK cytotoxicity was analyzed using the Spearman correlation coefficient. Although there was no significant correlation between the cytokine concentrations and NK cytotoxicity; NK cells released higher levels of IFN-α in response to PC3-RFP cells compared to the other cytokines (Fig. 5).

Figure 5. Spearman correlation coefficients (2-tailed) of perforin, IFN-α and granzyme B cytokines, and natural killer cytotoxicity with PC3-RFP cells.

Discussion

Several studies have reported the immunomodulatory effect of TQ on immune cells including NK cells (40–43). The current study focused on investigating the effect of TQ on the cytotoxicity of NK cells and its anticancer activity against PC3-RFP cells. The results demonstrated that treating cancer cells with a high dose of TQ in the presence of NK cells enhanced NK cell cytotoxicity more than NK cells co-cultured with cancer cells alone or in cells with a lower dose of TQ Furthermore, NK cells cultured with cancer cells treated with both TQ concentrations (25 and 50 µM) exhibited considerably increased NK cell cytotoxicity compared with cancer cells treated with TQ alone. This indicates that the antitumor effect of TQ is associated with its stimulation of NK cell function.

Majdalawieh et al (44) assessed how Nigella sativa extract affected the immune systems of mice and reported that the extract activated macrophages, increased NK cell antitumor activity, changed the Th1/Th2 cytokine balance toward a Th1-dominant response, and markedly increased splenocyte proliferation. Despite the fact that TQ, a crucial component of N. sativa, was not isolated, the results indicate that the extract enhances antitumor immunity by inducing both innate and adaptive immunological responses, potentially through TQ (44). Furthermore, a study by Sjs et al (45) produced a nanomedicine based on TQ and reported that administration of the nanomedicine to triple negative breast cancer cell line MDA-MB-231 was associated with apoptosis, DNA damage, slowing down of the cell cycle and prevention of cell division (45). According to these findings, TQ may be a potential strategy for both preventing and treating cancer (46,47).

Nigella sativa may affect cancer pathophysiology via several signaling pathways, such as inducible nitric oxide synthase, p53, ROS, TNF and caspases. These mechanisms have been demonstrated by numerous in vitro and in vivo studies, which suggest that N. sativa concentrates can be used to treat several malignant tumor types at different phases of carcinogenesis (40–42). Additionally, the medicinal plant is well-known for its potent protective benefits against the development and spread of tumors, as well as its anti-inflammatory and immune-stimulating properties, all of which make it a useful supplement to cancer treatment plans. Its impact on the NK cells may be responsible for these effects.

It has been reported that TQ combined with ionizing radiation, such as γ-radiation, has a synergistic lethal effect on breast cancer cells in vitro (42,43,48–50). Moreover, according to the present study, TQ administration directly increased the capacity of NK cells to lyse human PC (PC3-RFP) cells. TQ functions as an antimetabolic medication and may inhibit the development of colorectal cancer carcinogenesis by controlling the glycolytic metabolic pathway and the PI3/AKT axis (51). Additionally, pretreatment with TQ-pH-sensitive liposomes (PSL) decreased cancer marker enzymes, restored the relative weight of the lung and enhanced the activity of antioxidant enzymes in serum, according to a lung cancer study that prepared TQ in a particular formula as TQ-PSL to increase its solubility. Histopathological analysis revealed that TQ-PSL protected lung tissues by decreasing oxidative stress, suppressing inflammatory mediators and inducing apoptosis in pre-cancerous cells (52). TQ itself may be also useful as a treatment for lung adenocarcinoma as it has been reported to inhibit tumor cell proliferation, induce lung cancer cells to undergo apoptosis, markedly reduce TNF and NF-kB activity, and arrest cells in the S phase of the cell cycle (53). TQ has also been widely used in biomaterial treatments (54). Furthermore, TQ has demonstrated notable anti-hepatocellular carcinoma potential, modulating the ERK and p38 signaling pathways, the. Cytotoxic effect of TQ is enhanced by low ERK phosphorylation or ERK inhibition as elevated p-ERK activity protect hepatocellular carcinoma from TQ toxic effect (55,56). TQ and TNF-related apoptosis inducing ligand may cooperate to trigger apoptosis in hepatocellular carcinoma by mediating DNA damage, according to Zhang et al (57) TQ also has an effect on other cancers. For example, TQ markedly increased the amount of reactive oxygen species (ROS) produced by human pancreatic cancer cells whilst simultaneously suppressing the migration and proliferation of cancer cells. Moreover, natural quinones such as TQ may be effective antimetastatic treatments for pancreatic cancer based on these findings (58). As TQ successfully inhibits the fusion of autophagosomes and lysosomes, cancer cells incur apoptosis. Furthermore, TQ causes apoptosis by increasing ROS levels (59). TQ may also function as an immunomodulatory drug that boosts anticancer immune activity, as reported in other in vitro and in vivo research (48,59). It has been also reported that TQ may work in conjunction with chemotherapy and radiation to improve treatment results and reduce treatment side effects (59,60).

Research by Shimasaki et al (61) indicates that NK cells are essential for the regulation of tumor development and metastasis. NK cells contribute to the induction of adaptive anticancer T cell and B cell responses in addition to their function in the early defenses against infection and cancer (61,62). Additionally, NK cells can swiftly eliminate nearby cells that have surface markers associated with neoplastic transformation (53). The direct immune-stimulating impact of TQ on NK is well supported by the current data. In the present study, TQ increased the NK cell production of IFN, which is consistent with other research (4,47) which have reported that the cytotoxic effect of NK cells on tumor cells was increased in the presence of TQ, with increased secretion of perforin, granzyme B, and IFN-α, and that TQ promoted the cytotoxic activity of NK cells against breast cancer MCF-7 cells (63). The results of the present study also supported earlier research by demonstrating the inhibitory action of TQ on human PC PC3-RFP cells. Notably, TQ therapy directly increased the capacity of NK cells to kill PC3-RFP cancer cells. In the NK + TQ + PC3-RFP group in the present study, IFN activity increased, indicating higher concentration. Subsequently, the rise in granzyme B was comparable with earlier research (60,61,64) which showed granzyme B trigger apoptosis by cleaving (BID) BH3 Interacting-Domain Death Agonist pro apoptotic protein of Bcl2 family, causing mitochondrial distribution and activating caspases. This process is crucial for NK cells to induce death in cancer cells (65). Although BID activity was not measured in the present study, previous studies demonstrated the importance of mitochondrial disruption by BID to reach the lethal effect supporting the importance of granzyme B mediated pathways in NK cells cytotoxicity on caspases and causing cell death (64,65). Therefore, granzyme activity measurements are important in studies on NK cytotoxicity.

Furthermore, according to the findings of the present study, TQ affects NK cells, which then directly stimulates the immune system. The TQ-induced immunoreactivity of NK cells against PC3-RFP cells increased when 50 µM TQ was applied, compared with 25 µM. This indicated that the stimulation of NK cells by TQ was associated with its cytotoxic effect on PC3-RFP cells. However, although certain signaling molecules implicated in producing the TQ immunostimulatory action in NK cells have been identified, the specific signaling pathways and molecular targets in these cells are still unknown. It has been suggested that TQ increases NK cell-mediated cytotoxicity by upregulating pro-apoptotic markers such as BAX and BID and downregulating anti-apoptotic proteins such as BCL-2 and myeloid cell leukemia 1 (66). TQ prevents NF-κB, a transcription factor implicated in inflammatory reactions, from activating. TQ also improves NK cell function and lowers pro-inflammatory cytokine expression by inhibiting NF-κB activation, which helps to strengthen antitumor immunity (67). Moreover, by upregulating PTEN, TQ disrupts the PI3K/AKT signaling pathway and reduces AKT activity. This downregulation enhances NK cell-mediated cytotoxicity and promotes apoptosis in cancer cells by reducing cell survival and proliferation signals. Furthermore, antioxidant enzymes are upregulated when TQ stimulates the Nrf2 pathway. By shielding NK cells from oxidative stress, this modulation preserves their functioning and increases their antitumor activity (68). Therefore, more in vitro and in vivo studies are required to identify the target receptors and intracellular and extracellular components that have unique activities in the signal transduction pathways associated with TQ changes in NK cells.

Despite, the promising results of the capacity of TQ to increase NK cell cytotoxicity against PC3-RFP PC cells in vitro, a number of limitations of the present study should be noted. The in vitro design of an experiment cannot fully replicate the intricate interactions in the tumor microenvironment, including tumor-induced immunosuppression, cytokine gradients and immune cell trafficking. In addition, the study did not explore the precise molecular mechanisms behind TQ-induced increased NK cell activity, necessitating further investigation into signaling pathways, receptor expression changes and gene regulatory networks. Moreover, the study used PC3-RFP, a single PC cell line; however, its potential application to other subtypes or primary tumor cells is uncertain due to its heterogeneity. Finally, the observed effects may be influenced by the NK cell source and activation state, with variability in cytotoxic responses potentially introduced by incomplete donor characterization, isolation techniques and baseline NK cell activity. Future therapeutic development depends on determining the ideal concentration range that maximizes tumor cell killing without compromising NK cell viability or function.

In conclusion, the present study demonstrated that TQ, a bioactive substance obtained from Nigella sativa, significantly increases the cytotoxic activity of NK cells against PC3-RFP cells. As a potential supplement to immunotherapeutic approaches in the treatment of PC, the findings imply that TQ may enhance the innate immune response, specifically by increasing NK cell-mediated tumor cell lysis. Furthermore, the potential of TQ as an immunomodulatory agent with anticancer effects is supported by the observed upregulation of NK cell cytotoxicity in its presence. Future research should clarify the precise molecular mechanisms by which TQ improves NK cell function, including its effects on activation receptors, cytokine production and intracellular signaling pathways, in order to build on the present findings. Clinical trials and in vivo research are also necessary to confirm the therapeutic potential of TQ in a physiological setting and evaluate its safety and effectiveness when used in conjunction with currently available immunotherapies. Furthermore, investigating the impact of TQ on different subtypes of PC and its association with the tumor microenvironment may elucidate its suitability for personalized medicine.

Acknowledgements

The authors would like to thank Ms Samar A. Zailaie (King Faisal Specialist Hospital and Research Centre, Jeddah) for technical assistance and scientific advice.

Funding

Funding: No funding was received.

Availability of data and materials

The data generated in the present study may be requested from the corresponding author.

Authors' contributions

NIT and NuAA contributed to the study design. NoAA performed the study. NAK and HFA performed the data analysis. NIT, NuAA and HFA performed the data interpretation. HFA and NoAA drafted the manuscript. NIT, NoAA, HFA and NuAA revised the manuscript. All authors critically reviewed the manuscript. NIT and NuAA confirm the authenticity of all the raw data. All authors have read and approved the final manuscript.

Ethics approval and consent to participate

The present study was approved by the Biomedical Ethics Research Committee of King Abdulaziz University Faculty of Medicine (approval no. No640-20). Written informed consent was obtained from all subjects involved in the study.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References