Studying the Anticancer Effects of Thymoquinone on Breast Cancer Cells through Natural Killer Cell Activity.

Cancer immunotherapy is quickly growing and can now be viewed as the "fifth column" of cancer treatment. In addition, cancer immunotherapy has shown promising results with different kinds of cancers and may be used as a complementary therapy with various types of treatments. Thus, "immuno-oncology" is showing astounding advantages. However, one of the main challenges that face this type of therapy is that cancer cells can evade immune system elimination through different mechanisms. Many studies were done to overcome this issue including adding immune stimulants to generate synergistic effects or by genetically modifying NK cells themselves to be stronger and more resistant. Nigella sativa, also known as black cumin, is a well-known example of a widely applicable herbal medicine. It can effectively treat a variety of diseases, such as hypertension, diabetes, bronchitis, gastrointestinal upset, and cancer. The anticancer qualities of Nigella sativa appear to be mediated by an immune-modulatory effect that stimulates human natural killer (NK) cells. These are a type of lymphocyte and first line of defense against pathogens. Objectives. In this study, we investigated the therapeutic effect of thymoquinone, a major component of Nigella sativa, on the cytotoxic pathways of NK cells. Methods. NK cells were cultured with breast cancer cell line Michigan Cancer Foundation-7 (MCF-7); and were treated with Thymoquinone. The cytotoxicity of NK cells on cancer cells was measured. The cultured media were then collected and measured via enzyme-linked immunosorbent assay (ELISA) for concentrations of perforin, granzyme B and interferon-α (IFN-α). Results. The cytotoxic effect of NK cells on tumor cells was increased in the presence of thymoquinone, with an increased release of perforin, granzyme B, and IFN-α. Conclusion. Thymoquinone promotes the cytotoxic activity of NK cells against breast cancer MCF-7 cells.

Shenkang injection protects against renal fibrosis by reducing perforin expression through the STING/TBK1/IRF3 signaling pathways in natural killer cells.

BACKGROUND: Immune activation, chronic inflammation, and renal interstitial fibrosis (RIF) are associated with chronic kidney disease (CKD). The herbal formula, Shenkang injection (SKI), has been reported to attenuate RIF. However, the mechanisms by which SKI alleviates renal fibrosis, especially the role of natural killer (NK) cells, are unknown and require exploration. PURPOSE: This study aimed to determine the mechanisms by which SKI alleviates RIF. METHODS: Differential gene expression between CKD mice and control groups was explored using bioinformatics analysis. To reveal how SKI reduces RIF in CKD, a CKD mouse model was established using folic acid for in vivo studies, and human kidney-2 cells were used for in vitro experiments. The effects of various SKI doses were then determined. Immunohistochemical staining, Enzyme-linked immunosorbent assay, western blotting, and quantitative real-time PCR were used for pathological and molecular expression detection. RESULTS: We first investigated the potential immune dysfunction in CKD using bioinformatics analysis. Some differentially expressed genes were enriched in immune-related functions. The expressions of perforin and interferon (IFN)-γ, which are mainly released by NK cells, were significantly higher in patients with CKD (p< 0.05). In vivo experiments showed that SKI alleviated renal fibrosis in a folic acid-induced renal fibrosis model. Serum creatinine and blood urea nitrogen levels were reduced in the high-dose SKI-treated group. Additionally, the mRNA and protein expression levels of type IV collagen and alpha-spinal muscular atrophy were reduced. Biochemical detection showed that SKI could also downregulate the activity of NK cells (by decreasing the expressions of perforin and IFN-γ). Increased levels of stimulator of interferon genes (STING)/TANK-binding kinase 1 (TBK1)/IFN regulatory factor 3 (IRF3), phosphorylation of TBK1, and IRF3 in FA-induced RIF mice were alleviated by SKI treatment, which was consistent with the results of in vitro experiments. CONCLUSION: These results demonstrated that SKI could decrease the activation of NK cells via the STING/TBK1/IRF3 signaling pathway, thereby alleviating RIF and protecting renal function in CKD. This may provide valuable evidence supporting the clinical use of SKI in the treatment of patients with CKD.

Allergen immunotherapy modulates sensitivity of Treg cells to apoptosis in a rat model of allergic asthma.

AIM: To study the apoptosis of Foxp3+ Treg cells following Alstonia scholaris pollen sensitization-challenge and following allergen immunotherapy. MATERIALS & METHODS: Wistar rats were sensitized-challenged with Alstonia scholaris pollen and were further given intranasal immunotherapy. For the analysis of the apoptotic proteins on Treg cells by flow cytometry, multiple gating procedures were followed. RESULTS: Allergen sensitization-challenge increases Annexin-V, Fas, FasL, caspases-8, 9, 3 cytochrome-C, APAF-1, Bax, perforin-1 and granzyme-B on Treg cells which is decreased following intranasal immunotherapy. On the other hand, Bcl-2 expression is decreased in allergy and increased by immunotherapy. CONCLUSION: Apoptosis of Treg cells is increased following allergen sensitization-challenge via extrinsic, intrinsic and perforin/granzyme pathways and allergen immunotherapy decreased the sensitivity to apoptosis of Treg cells.

Immunomodulatory properties of medicinal mushrooms: differential effects of water and ethanol extracts on NK cell-mediated cytotoxicity.

Medicinal mushrooms have been used for centuries in Asian countries owing to their beneficial effects on health and longevity. Previous studies have reported that a single medicinal mushroom may produce both stimulatory and inhibitory effects on immune cells, depending on conditions, but the factors responsible for this apparent dichotomy remain obscure. We show here that water and ethanol extracts of cultured mycelium from various species (Agaricus blazei Murrill, Antrodia cinnamomea, Ganoderma lucidum and Hirsutella sinensis) produce opposite effects on NK cells. Water extracts enhance NK cell cytotoxic activity against cancer cells, whereas ethanol extracts inhibit cytotoxicity. Water extracts stimulate the expression and production of cytolytic proteins (perforin and granulysin) and NKG2D/NCR cell surface receptors, and activate intracellular signaling kinases (ERK, JNK and p38). In contrast, ethanol extracts inhibit expression of cytolytic and cell surface receptors. Our results suggest that the mode of extraction of medicinal mushrooms may determine the nature of the immunomodulatory effects produced on immune cells, presumably owing to the differential solubility of stimulatory and inhibitory mediators. These findings have important implications for the preparation of medicinal mushrooms to prevent and treat human diseases.

The preclinical pharmacokinetic disposition of a series of perforin-inhibitors as potential immunosuppressive agents.

The cytolytic protein perforin is a key component of the immune response and is implicated in a number of human pathologies and therapy-induced conditions. A novel series of small molecule inhibitors of perforin function have been developed as potential immunosuppressive agents. The pharmacokinetics and metabolic stability of a series of 16 inhibitors of perforin was evaluated in male CD1 mice following intravenous administration. The compounds were well tolerated 6 h after dosing. After intravenous administration at 5 mg/kg, maximum plasma concentrations ranged from 532 ± 200 to 10,061 ± 12 ng/mL across the series. Plasma concentrations were greater than the concentrations required for in vitro inhibitory activity for 11 of the compounds. Following an initial rapid distribution phase, the elimination half-life values for the series ranged from 0.82 ± 0.25 to 4.38 ± 4.48 h. All compounds in the series were susceptible to oxidative biotransformation. Following incubations with microsomal preparations, a tenfold range in in vitro half-life was observed across the series. The data suggests that oxidative biotransformation was not singularly responsible for clearance of the compounds and no direct relationship between microsomal clearance and plasma clearance was observed. Structural modifications however, do provide some information as to the relative microsomal stability of the compounds, which may be useful for further drug development.

Cryptotanshinone induces inhibition of breast tumor growth by cytotoxic CD4+ T cells through the JAK2/STAT4/ perforin pathway.

Cryptotanshinone (CPT), is a quinoid diterpene isolated from the root of the Asian medicinal plant, Salvia miotiorrhiza bunge. Numerous researchers have found that it could work as a potent antitumor agent to inhibit tumor growth in vitro, buith there has been much less emphasis on its in vivo role against breast tumors. Using a mouse tumor model of MCF7 cells, we showed that CPT strongly inhibited MCF7 cell growth in vivo with polarization of immune reactions toward Th1-type responses, stimulation of naive CD4+ T cell proliferation, and also increased IFN-γ and perforin production of CD4+ T cells in response to tumor-activated splenocytes. Furthermore, data revealed that the cytotoxic activity of CD4+ T cells induced by CPT was markedly abrogated by concanamycin A(CMA), a perforin inhibitor, but not IFN-γ Ab. On the other hand, after depletion of CD4+ T cells or blocked perforin with CMA in a tumor-bearing model, CPT could not effectively suppress tumor growth, but this phenomenon could be reversed by injecting naive CD4+ T cells. Thus, our results suggested that CPT mainly inhibited breast tumor growth through inducing cytotoxic CD4+ T cells to secrete perforin. We further found that CPT enhanced perforin production of CD4+ T cells by up-regulating JAK2 and STAT4 phosphorylation. These findings suggest a novel potential therapeutic role for CPT in tumor therapy, and demonstrate that CPT performs its antitumor functions through cytotoxic CD4+ T cells.

Ganoderma lucidum stimulates NK cell cytotoxicity by inducing NKG2D/NCR activation and secretion of perforin and granulysin.

Ganoderma lucidum (G. lucidum) is a medicinal mushroom long used in Asia as a folk remedy to promote health and longevity. Recent studies indicate that G. lucidum activates NK cells, but the molecular mechanism underlying this effect has not been studied so far. To address this question, we prepared a water extract of G. lucidum and examined its effect on NK cells. We observed that G. lucidum treatment increases NK cell cytotoxicity by stimulating secretion of perforin and granulysin. The mechanism of activation involves an increased expression of NKG2D and natural cytotoxicity receptors (NCRs), as well as increased phosphorylation of intracellular MAPKs. Our results indicate that G. lucidum induces NK cell cytotoxicity against various cancer cell lines by activating NKG2D/NCR receptors and MAPK signaling pathways, which together culminate in exocytosis of perforin and granulysin. These observations provide a cellular and molecular mechanism to account for the reported anticancer effects of G. lucidum extracts in humans.

Natural killer cell populations and cytotoxic activity in pigs fed mother's milk, formula, or formula supplemented with bovine lactoferrin.

BACKGROUND: Natural killer (NK) cells are components of the innate immune defense system, and their levels differ between breast and formula-fed (FF) infants. Lactoferrin (Lf) modulates NK cell cytotoxicity ex vivo. We hypothesized that dietary bovine Lf (bLf) would increase NK cell populations and cytotoxicity. METHODS: Piglets were sow-reared (SR), FF, or 1 g/l bLf-fed (LF) for 21 d. NK cells (CD3(-)CD4(-)CD8(+)) in blood (peripheral blood mononuclear cells (PBMCs)), spleen, and mesenteric lymph node (MLN) were determined by flow cytometry. PBMC NK cells were tested for cytotoxic activity against target K562 cells ex vivo in the presence of media (unstimulated), interleukin-2, or bLf. NK cell mRNA expression was determined by reverse transcription-quantitative PCR. RESULTS: SR and LF piglets had more NK cells in MLN (P = 0.0097) and spleen (P = 0.0980) than FF piglets. In PBMCs, SR piglets had more NK cells than FF piglets (P = 0.0072); LF piglets were intermediate and not different from FF or SR piglets. NK cell intelectin-2 mRNA expression was 2.5-fold higher (P = 0.0095) in LF than SR or FF piglets. NK cells in SR piglets exhibited greater (P < 0.0001) cytotoxic activity than those in LF or FF piglets, which was supported by greater perforin mRNA expression. CONCLUSION: Dietary bLf increased blood NK cell populations and NK Lf receptor expression but not NK cell cytotoxicity.

Depletion of ascorbic acid impairs NK cell activity against ovarian cancer in a mouse model.

Ascorbic acid (Vitamin C) administration has been used to prevent infectious diseases in public or as a therapeutic agent by the physicians in treatment of several diseases. Ascorbic acid is also involved in immune cell functions and immune responses, although the mechanisms by which it exerts effects on immune cells against cancer cells are not fully understood at the normal plasma level. In this study, we used the mice lacking l-gulono-γ-lactone oxidase (Gulo), the enzyme required for the biosynthesis of ascorbic acid, to characterize the effects of ascorbic acid on NK cell cytotoxicity against ovarian cancer cells, MOSECs (murine ovarian surface epithelial cells). Gulo(-/-) mice depleted of ascorbic acid survived for a shorter time than the normal control or Gulo(-/-) mice supplemented with ascorbic acid after tumor challenge regardless of treatment with IL-2. CD69 and NKG2D expression was clearly reduced in NK cells isolated from mice depleted of ascorbic acid as compared to that in the normal control and the mice supplemented with ascorbic acid. We also observed that IFN-γ secretion by NK cells isolated from Gulo(-/-) mice depleted of ascorbic acid was decreased after NK cells were co-cultured with MOSECs. Furthermore, the mRNA expression of perforin and granzyme B genes was also significantly decreased in NK cells isolated from mice depleted of ascorbic acid. Taken together, our results suggest that ascorbic acid at the normal plasma concentration has an essential role in maintaining the NK cytotoxicity against cancer cells.

The proposed mechanism of action during different pain management techniques on expression of cytolytic molecule perforin in patients after colorectal cancer surgery.

The postoperative period is accompanied with neuroendocrine, metabolic and immune alteration which is caused by tissue damage, anesthesia, postoperative pain and psychological stress. Postoperative pain contributes to dysfunction of immune response as a result of interaction between central nervous and immune system. The postoperatively activated hypotalamo-pituitary-adrenocortical axis, sympathic and parasympathic nerve systems are important modulators of immune response. According to bidirectional communication of immune and nervous system, appropriate postoperative pain management could affect immune response in postoperative period. Although the postoperative suppression of immune response has been reported, a very little are known about the influences of different pain management techniques on cytotoxic function of immune cells in patients with colorectal cancer in early postoperative period. Perforin is a cytotoxic molecule expressed by activated lymphocytes which has a crucial role in elimination of tumor cells and virus-infected cells, mostly during the effector's phase of immune response. Immune compromise during the postoperative period could affect the healing processes, incidence of postoperative infections and rate and size of tumor metastases disseminated during operation. The pharmacological management of postoperative pain in patients with malignancies uses very different analgesic techniques whose possible influence on cytotoxic functions of immune cells are still understood poor. For decades the most common way of treating postoperative pain after colorectal cancer surgery was intravenous analgesia with opiods. In the last decade many investigations pointed out that opiods can also contribute to postoperative suppression of immune response. Epidural analgesia is a regional anesthesia technique that acts directly on the origin of pain impulses and pain relief can be achieved with small doses of opiods combined with local anesthetics. Local anesthetics potentate analgesic properties of opiods but per se are also acting as antiinflammatory drugs. Afferent neural blockade by epidural analgesia attenuates neuroendocrine stress response. We propose that epidural analgesia could be more convenient that intravenous analgesia in maintenance of immunological homeostasis that is altered by surgical stress, tumor growth and pain.

Enhancement of natural killer cell activity in healthy subjects by Immulina®, a Spirulina extract enriched for Braun-type lipoproteins.

Immulina®, a commercial extract of Arthrospira (Spirulina) platensis is a potent activator of THP-1 monocytes and CD4+ T cells IN VITRO and enhances several immunological functions in mice. We further characterized Immulina® by determining that Braun-type lipoproteins are responsible for a major portion of the IN VITRO monocyte activation exhibited by this material. In order to understand the effect of Immulina® on NK cell activity, a pilot study was conducted on ten healthy North American individuals who supplemented their diet with Immulina® (400 mg/day) for seven days. We observed a 40% average increase in the killing of K562 tumor cells by NK cells (p < 0.01) after Immulina® supplementation. In a separate placebo-controlled, crossover study involving 11 healthy Danish subjects, we observed increased mRNA expression of the NK cell marker NKG2D by 37% (p = 0.02) and by 55% (p = 0.0003) after administration of Immulina® (200 mg and 400 mg per day, respectively) for seven days. The mRNA expression of the NK- and T-cell marker perforin increased by 75% (p = 0.008) after administration of 400 mg Immulina® per day. Both markers displayed significant dose-dependent effects (p = 0.0003 and p = 0.02, respectively). The ratio between CD56 (bright) and CD56 (dim) NK cells was not affected by Immulina® administration. In summary, two independent studies showed enhancement of NK cell activity following administration of Immulina® for seven days.

Zinc transporter ZIP8 (SLC39A8) and zinc influence IFN-gamma expression in activated human T cells.

The zinc transporter ZIP8 is highly expressed in T cells derived from human subjects. T cell ZIP8 expression was markedly up-regulated upon in vitro activation. T cells collected from human subjects who had received oral zinc supplementation (15 mg/day) had higher expression of the activation marker IFN-gamma upon in vitro activation, indicating a potentiating effect of zinc on T cell activation. Similarly, in vitro zinc treatment of T cells along with activation resulted in increased IFN-gamma expression with a maximum effect at 3.1 microM. Knockdown of ZIP8 in T cells by siRNA decreased ZIP8 levels in nonactivated and activated cells and concomitantly reduced secretion of IFN-gamma and perforin, both signatures of activation. Overexpression of ZIP8 by transient transfection caused T cells to exhibit enhanced activation. Confocal microscopy established that ZIP8 is localized to the lysosome where ZIP8 abundance is increased upon activation. Loss of lysosomal labile zinc in response to activation was measured by flow cytometry using a zinc fluorophore. Zinc between 0.8 and 3.1 microM reduced CN phosphatase activity. CN was also inhibited by the CN inhibitor FK506 and ZIP8 overexpression. The results suggest that zinc at low concentrations, through inhibition of CN, sustains phosphorylation of the transcription factor CREB, yielding greater IFN-gamma expression in T cells. ZIP8, through control of zinc transport from the lysosome, may provide a secondary level of IFN-gamma regulation in T cells.

Neem leaf glycoprotein induces perforin-mediated tumor cell killing by T and NK cells through differential regulation of IFNgamma signaling.

We have demonstrated augmentation of the CD3-CD56+ natural killer (NK) and CD8+CD56_ T-cell-mediated tumor cell cytotoxicity by neem leaf glycoprotein (NLGP). These NK and T cells were isolated from the peripheral blood of head and neck squamous cell carcinoma patients with a state of immunosuppression. NLGP induces TCRalphabeta-associated cytotoxic T lymphocyte (CTL) reaction to kill oral cancer (KB) cells. This CTL reaction is assisted by NLGP-mediated up-regulation of CD28 on T cells and HLA-ABC, CD80/86 on monocytes. CTL-mediated killing of KB cells and NK-cell-mediated killing of K562 (erythroleukemic) cells are associated with activation of these cells by NLGP. This activation is evidenced by increased expression of early activation marker CD69 with altered expression of CD45RO/CD45RA. NLGP is a strong inducer of IFNgamma from both T and NK cells; however, IFNgamma regulates the T-cell-mediated cytotoxicity only without affecting NK-cell-mediated one. Reason of this differential regulation may lie within up-regulated expression of IFNgamma-receptor on T-cell surface, not on NK cells. This NLGP-induced cytotoxicity is dependent on up-regulated perforin/granzyme B expression in killer cells, which is again IFNgamma dependent in T cells and independent in NK cells. Although, FasL expression is increased by NLGP, it may not be truly linked with the cytotoxic functions, as brefeldin A could not block such NLGP-mediated cytotoxicity, like, concanamycin A, a perforin inhibitor. On the basis of these results, we conclude that NLGP might be effective to recover the suppressed cytotoxic functions of NK and T cells from head and neck squamous cell carcinoma patients.

Dietary omega-3 polyunsaturated fatty acids can inhibit expression of granzyme B, perforin, and cation-independent mannose 6-phosphate/insulin-like growth factor receptor in rat model of small bowel transplant chronic rejection.

BACKGROUND: The aim of our work was to investigate the effects of omega-3 polyunsaturated fatty acids on apoptosis and granzyme B, perforin, and cation-independent mannose 6-phosphate/insulin-like growth factor receptor (CI-MPR) expression of intestinal epithelial cells of chronic rejection after small intestinal transplantation. METHODS: Small bowel transplantation was performed in a rat combination of 3 groups: group 1, Lewis-to-Lewis; group 2, F344-to-Lewis, dietary corn oil; group 3, F344-to-Lewis, dietary fish oil. All recipients were killed at 16 weeks posttransplantation. The apoptosis rate of mucosal cells was evaluated by flow cytometry. The expression of granzyme B, perforin, and CI-MPR was analyzed by reverse transcriptase PCR. RESULTS: A high apoptotic rate was observed when the allografts demonstrated 1 or more histologic features of chronic rejection. omega-3 Polyunsaturated fatty acids decreased in rate of the apoptosis, and it can inhibit the expression of granzyme B, perforin, and CI-MPR. CONCLUSIONS: omega-3 Polyunsaturated fatty acids can suppress the rejection to mucosal cells of allograft at the time of chronic rejection in small intestinal transplantation, which may be significant in increasing the surviving rate of allograft, delaying the chronic dysfunction, and prolonging the lifetime of both allograft and acceptor.

Heat shock suppresses human NK cell cytotoxicity via regulation of perforin.

Human natural killer (NK) cell, which is an important lymphocyte for immune surveillance, is highly sensitive to heat, but the nature of its response to and its mechanistic regulation by heat remain unclear. Here we determined the effect of in vitro heat shock and in vivo hyperthermia on human NK cell cytotoxicity. Human peripheral blood mononuclear cells (PBMC) obtained from healthy volunteers were subjected to heat shock in vitro (42 degrees C, 1 h). PBMC from cancer patients receiving intentional hyperthermia (42 degrees C, 1 h) for cancer therapy were also obtained. NK cytolytic activity was determined in these samples. NK cell cytotoxicity was down-regulated by heat shock in vitro at 5 h, but at 24 h after heat shock, the NK cytotoxicity was comparable to that with its respective control. Furthermore, we observed that the mRNA and protein expression levels of perforin, which is the cytolytic granule of NK cells, were regulated by heat shock in a similar manner as NK cytotoxicity at 5 h and at 24 h after heat shock. Heat regulation involved the perforin protein in CD56(dim) but not in CD56(bright) NK cell subset. Heat shock neither induced cell death nor altered the expression of some NK activating receptors and adhesion molecules. Moreover, whole-body hyperthermia at 42 degrees C for 1 h of cancer patients also suppressed the cytotoxicity of NK cells but recovered to basal level 1 week after hyperthermia. Heat shock in vitro and in vivo temporarily represses the cytotoxicity of human NK cells.