Carthamus tinctorius Enhances the Antitumor Activity of Dendritic Cell Vaccines via Polarization toward Th1 Cytokines and Increase of Cytotoxic T Lymphocytes

Carthamus tinctorius (CT), also named safflower, is a traditional Chinese medicine widely used to improve blood circulation. CT also has been studied for its antitumor activity in certain cancers. To investigate the effects of CT on the dendritic cell (DC)-based vaccine in cancer treatment, cytokine secretion of mouse splenic T lymphocytes and the maturation of DCs in response to CT were analyzed. To assess the antitumor activity of CT extract on mouse CD117+ (c-kit)-derived DCs pulsed with JC mammal tumor antigens, the JC tumor was challenged by the CT-treated DC vaccine in vivo. CT stimulated IFN-γ and IL-10 secretion of splenic T lymphocytes and enhanced the maturation of DCs by enhancing immunological molecule expression. When DC vaccine was pulsed with tumor antigens along with CT extract, the levels of TNF-α and IL-1β were dramatically increased with a dose-dependent response and more immunologic and co-stimulatory molecules were expressed on the DC surface. In addition, CT-treated tumor lysate-pulsed DC vaccine reduced the tumor weight in tumor-bearing mice by 15.3% more than tumor lysate-pulsed DC vaccine without CT treatment. CT polarized cytokine secretion toward the Th1 pathway and also increased the population of cytotoxic T lymphocytes ex vivo. In conclusion, CT activates DCs might promote the recognition of antigens and facilitate antigen presentation to Th1 immune responses.


Introduction
Carthamus tinctorius (CT), also named safflower, is a traditional Chinese medicine widely used to improve blood circulation [1], extending the coagulation time in mice and exhibiting a significant antithrombotic effect [2]. However, CT is used not only for its traditional medicinal purposes but is also effective for treating breast cancer [3]. The oil extracted from the seed of CT is reported to contain alkane-6,8-diols, which have the activity to inhibit 12-Otetradecanoylphorbol-13-acetate-induced tumor promotion in two-stage carcinogenesis in mouse skin. In addition, Nferuloylserotonin and N-(p-coumaroyl) serotonin strongly inhibit the melanin production of Streptomyces bikiniensis and B16 melanoma cells [4,5]. These compounds are suggested to have potential antitumor effects. In addition, CT also is neuroprotective for cerebral ischemic injury in vivo and in vitro [2]. Recently, N-(p-coumaroyl)serotonin and N-(p-coumaroyl)tryptamine, active ingredients in CT, were shown to strongly inhibit the production of proinflammatory cytokines (IL-1α, IL-1β, IL-6, IL-8 and TNFα) from lipopolysaccharide-stimulated human monocytes [6]. Notably, extracts of CT exhibit a vast diversity of bioactivities, including immunomodulation, anti-infarction, antiallergic, anti-inflammatory and antiestrogenic effects, as well as functioning as a hemostatic agent to promote blood coagulation. Table 1 summarizes the diverse bioactivities of CT and delineates the therapeutic implications for this potent herbal medicine.
Dendritic cells (DCs) are professional antigen-presenting cells that stimulate immune responses by presenting endogenous or exogenous antigens to T-helper lymphocytes [31]. Immature DCs can be differentiated from monocytes and bone marrow progenitor cells by treatment with GM-CSF and IL-4 [32,33], after which the cells gain strong phagocytic activity but not antigen presentation activity. Immature DCs are stimulated with maturation signals to express more surface immunological molecules (such as CD86, CD80, Inhibitory effects on the production of antibody and delayed-type hypersensitivity reaction in vivo; and the responsiveness of mixed lymphocyte reaction and production of IL2 in vitro Lu et al. [7] Antilipid peroxidation effects Jin et al. [8] Triterpene Anti-inflammatory activity against 12-O-tetradecanoylphorbol-13-acetate-induced inflammation Akihisa et al. [9] Polysaccharides Antitumor activities through toll-like receptor 4/NF-κB pathway in the cell assay Ando et al. [10] Hydroxysafflor yellow A Anti-thrombotic and -infraction effects and as a neuroprotective agent against cerebral ischemic damage Wang et al. [1] Zhu et al. [2] Zhang et al. [11] Zhu et al. [12,13] Carthamin, safflower yellow Antiproliferative and pro-apoptotic activities in hepatic stellate cells Chor et al. [14] Reduction of cerebral infarction volume along with increase of bcl-2 and decrease of caspase-3 Luo et al. [15] Improving functions of cardiac contraction and dilation, increasing coronary blood flow and strengthening the bcl-2 protein expression Zhang and Jiang [16] Water soluble substances Cytotoxic effects on the rat nervous cells and may cause teratogenesis Nobakht et al. [17] Antimycotic properties especially against Aspergillus fumigatus Blaszczyk et al. [ Immunomodulatory effects mainly associated with anti-inflammatory activities Takii et al. [6] Serotonin derivatives Antioxidant effects mainly for prevention of atherosclerosis through inhibition of LDL oxidation and postischemic myocardial dysfunction Koyama et al. [19] Hotta et al. [20] Trechelosides Anabolic effects and antiestrogenic activities on bone through promotion of osteoblastic differentiation and inhibition of bone resorption Kim et al. [21] Jang et al. [22] Yoo et al. [23] Yuk et al. [24] Hong et al. [25] Linoleate High linoleate in dietary oil supplements increase serum concentrations of prostaglandin F2α metabolite Grant et al. [26] Leaf Flavonoids Antioxidative effects Lee et al. [27] Kinobeon A A preventive effect on singlet oxygen and acting as a tyrosinase inhibitor Kambayashi et al. [28] Kanehira et al. [29,30] MHC-I and MHC-II) for antigen presentation, leading to a strong immune response against foreign antigens [34]. TNF-α and CD40L can stimulate immature DCs to mature in vitro and may show potential for clinical use [35], suggesting that enhancement of DC maturation is required for improvement of DC immunotherapy. In the clinic, DC-based immunotherapies are being intensively studied for their application in the treatment of certain diseases, including cancer and infectious diseases [36][37][38]. Scientists are trying to improve the phagocytic activity or antigen presentation activity of DCs for use in immunotherapy [39,40]. Certain herbal medicines can also improve DC function [41]. To date, CT has been used as a folk medicine in cancer adjuvant therapy, but its function has not been proven. In this study, we studied the immunomodulatory effects of CT on cytokine secretion and surface immunologic molecules of DCs in vitro and antitumor activities of CT-treated DCs in an animal model of breast cancer.

Cytokine Secretion from Mouse Splenocytes Induced by CT
Extract. The splenocytes were homogenized from the spleen of healthy BALB/c mice, and the splenic T lymphocytes were isolated by passage through nylon wool columns [42]. The purity of T cells was confirmed by flow cytometry to be >90%. The mouse splenic T lymphocytes were treated with various concentrations of CT extract (5, 10 and 20 μg/mL of culture medium) for 24 h. The medium was collected to determine the concentrations of the cytokines IL-2, IFN-γ and IL-4. Cytokine levels were determined by the DuoSet ELISA kit as described in the manual.

Maturation of Mouse DCs by CT Extract.
Bone marrow cells were taken from BALB/c mice and then suspended in RPMI-1640 medium containing 10% FBS. The cell suspension was allowed to stand at room temperature for 10 min to remove the cell clots, and then the cell suspension was centrifuged at 800 g for 5 min at 4 • C and washed with PBS (pH 7.4) containing 0.5% FBS and 2 mM EDTA. The cells were allowed to attach to a 100-mm culture plate for 2 h and the floating cells were removed gently. The cells were then treated with GM-CSF (100 ng/mL) and IL-4 (100 ng/mL) at 37 • C, 5% CO 2 for 6 days, following the modifications of Zheng et al. [43]. After 6 days of differentiation, the DCs were treated with various concentrations of CT extract (5, 10 and 20 μg/mL of culture medium) for 72 h. After CT treatment, the DCs were collected for flow cytometric analysis of surface immunological molecules (CD80, CD86, MHC-I and MHC-II). In brief, the cells were stained with the specific FITCconjugated antibody.

Isolation of CD117 + Bone Marrow Cells.
Bone marrow cells were collected as above and suspended in PBS (pH 7.4) containing 0.5% FBS and 2 mM EDTA prior to the isolation of CD117 + cells. CD117 + cells were isolated using magneticactivated cell sorting (Miltenyi Biotec Inc, CA, USA) as described in the manual. Briefly, 1 × 10 7 cells were added to 20 μl anti-CD117 + provided by the kit for 15 min and then washed with PBS to remove excess unbound antibodies. The treated cells were loaded on a column, from which the CD117 − cells were eluted with PBS buffer in the presence of a magnetic field. The CD117 + cells were then eluted in the absence of a magnetic field and were then differentiated to DCs by adding GM-CSF and IL-4 as described above.

Assessment of Cytokines Released from JC Lysate-Pulsed
CT-Treated CD117 + -Derived DCs. After differentiation of CD117 + cells to DCs for 6 days, DCs were pulsed with JClysate and treated with or without CT extract (5, 10 and 20 μg/mL) for an additional 3 days. The conditioned medium was collected for determination of the concentrations of IL-2, IL-10, TNF-α and IL-1β [44].

Preparation of DC Vaccine and JC Tumor
Animal Experiment. CD117 + cells were cultured in differentiation medium (GM-CSF and IL-4) for 6 days followed by cultivation in fresh RPMI medium in the presence or absence of CT (20 μg/mL) for 3 days. JC cells (3-4 × 10 6 ) were lyzed in 1 mL RPMI-1640 medium containing 10% FBS by freezing and thawing four times, and the lysate was stored at -80 • C as a tumor antigen resource [45] to pulse the DCs.
BALB/c female mice (4-6 weeks) were purchased from the National Laboratory Animal Center (Taiwan, ROC). JC cells (3 × 10 6 per 150 μl PBS) were subcutaneously inoculated in the flank of BALB/c mice. Twenty-four tumorbearing mice were grouped (n = 8/group) as follows: untreated control, DC vaccine and CT-treated groups. In the DC vaccine group and CT-treated DC vaccine group, mice were injected intraperitoneally with the JC-pulsed DC cell suspension (1.7 × 10 6 per 0.2 mL PBS) and the CT-treated JC-pulsed DC cell suspension (1.7 × 10 6 per 0.2 mL PBS) on day 13, respectively. The tumor growth of JC tumor-bearing mice was measured by a caliper every 4-6 days and calculated using the equation, tumor weight (mg) = length (mm) × width (mm 2 )/2, until the tumor reached 2 × 10 3 mm 3 . On day 32, mice were euthanized by CO 2 , the tumors were excised and weighed and the spleens of each mouse were homogenized into a single cell suspension pulsed with or without CT-treated DCs to measure Th1-and Th2-related cytokines. The splenocytes were also re-stimulated with the previous corresponding JC-pulsed DC vaccine and CTtreated JC-pulsed DC vaccine for flow cytometric analysis of cytotoxic T lymphocytes. All mice received humane care, and the study protocol followed the guidelines of the Institutional Animal Care and Use Committees of the Development Center for Biotechnology (accredited by AAALAC).

Effects of CT-Treated DCs on the Cytokine Secretion and
Cytotoxic T Lymphocytes of Mouse Splenocytes. DC vaccine was prepared as described above and cultured in AIM-V medium (Gibco, USA), and splenic T lymphocytes were taken from the normal and the JC tumor-bearing mice and cultured in RPMI-1640 medium (Gibco, USA). In brief, splenic T lymphocytes were partially purified using a nylon wool column [46]. Different cytokines in response to CT (20 μg/mL)-treated DCs were determined when DCs (2 × 10 5 /mL) were pulsed with splenocytes from tumor-bearing mice at a cell : cell ratio of 1 : 10, 1 : 20 and 1 : 40 for 48 h at 37 • C, 5% CO 2 . After cocultivation of splenocytes and DCs, the medium was collected and stored at -20 • C prior to cytokine analysis. Cytokines were analyzed by ELISA as described above. The splenocyte culture was continued for an additional 72 h and then subjected to flow cytometric analysis, in which specific T lymphocytes were gated by staining with FITC-anti-CD3 and PE-anti-CD8.

Statistical Analysis.
Data were analyzed by one-way ANOVA using SPSS statistical software followed by the Dunnett's test for multiple comparisons between each group. A P-value < .05 was considered to indicate a significant difference.

IFN-γ and IL-10 Secretion from Mouse Splenic T Lymphocytes.
To determine the effect of CT extract on the stimulation of mouse resting T lymphocytes, the splenic T lymphocytes were partially purified and treated with various concentrations of CT extract for 24 h. The supernatants were collected and evaluated for the levels of IL-2, IFN-γ and IL-4 and IL-10. Figure 1 shows that CT extract stimulated the production of IL-10 > IFN-γ in a dose-dependent manner ( Figure 1). However, no such stimulation was observed for IL-2 and IL-4.

Expression of Immunological Molecules in the Immature
DCs. To determine whether the CT extract stimulated the maturation of DCs, mouse bone marrow cells were isolated and differentiated into immature DCs by adding GM-CSF and IL-4 for 6 days. The immature DCs were treated with various concentrations of CT extract (5, 10 and 20 μg/mL) for 48 h. Figure 2 shows that CT extract promoted the expression of DC surface markers (CD86 < MHC-I = MHC-II = CD80) in a dose-dependent manner (Figure 2).

Enhancement of Maturation of DCs by CT Extract.
To study whether the pattern of cytokine secretion was altered in DCs stimulated by CT extract in the presence of tumor  -20) in the absence of GM-CSF and IL-4 for additional 48 h. The medium was collected, and the concentrations of secreted cytokines were determined. Data from triplicate points are expressed as the mean ± SE. Statistical analysis was performed by oneway ANOVA followed by Dunnett's test. A P-value < .05 was considered to reflect a significant difference. * P < .05 , * * P < .01, * * * P < .001.
antigens, the immature DCs were pulsed with JC tumor lysate along with different concentrations of CT extract (5, 10 and 20 μg/mL) for 48 h. The conditioned medium was analyzed for IL-2, IL-10, TNF-α and IL-1β by ELISA and the DCs were subjected to analysis of surface immunological molecules (CD80, CD86, MHC-I and MHC-II). The results showed that CT extract increased TNF-α and IL-1β levels in a dose-dependent manner (Figure 3). However, the level of IL-2 was reduced when DCs were pulsed with tumor lysate. IL-10 decreased with an increase in the concentration of CT extract, suggesting that CT extract might increase the secretion of TNF-α and IL-1β to enhance the maturation of immature DCs. Treatment with CT extract maintained the high profile of cell surface markers, such as CD80, CD86, MHC-I and MHC-II on the CD117 + -derived CDs in the presence of tumor antigen (Figure 4).

Inhibition of Tumor Growth by CT Treated-DC Vaccine.
To study the antitumor activity of CT-treated DC vaccine, BALB/c mice were inoculated with JC tumor cells and challenged with a single dose of CT-treated DC vaccine. The JC tumor-bearing mice were challenged with DC vaccine and CT-treated DC vaccine on day 13, when tumor reached the size of 5 mm (length) × 5 mm (width). Results showed that CT-treated DC vaccine and DC vaccine decreased the tumor mass by 32.5% and 17.2%, respectively, compared to the untreated control ( Figure 5). This result suggested that CT might increase the antitumor activity of the DC vaccine.

Polarization of Cytokine Secretion to Th1
Response. To determine the Th1 and Th2 cytokine secretion in DC vaccine treatment, the tumor-bearing mice that had been challenged with DC vaccine were sacrificed and their splenocytes subjected to cytokine analysis, in which those cells were restimulated with the previous corresponding DC vaccine or CT-treated DC vaccine ex vivo. Splenocytes of the CT/DC vaccine compared with those of the CT-untreated DCvaccine showed increased or identical production of IL-2, IL-10 and IFN-γ. However, IL-4 production was notably decreased in splenocytes of mice treated with the CT/DC vaccine ( Figure 6). The secretion of cytokines was not increased in the sham splenocytes when those cells pulsed with DC vaccine or CT-treated DC vaccine with lack of JC antigen. This result suggested that either the DC vaccine or CT-treated DC vaccine elicited antitumor activity through a Th1 response, and that CT extract might also enhance the immune response of the DC vaccine.

Discussion
DCs are professional antigen-presenting cells that have been enlisted for use in vaccines against cancer. DCs can acquire tumor antigens by cocultivation with tumor cells or pulsation with tumor lysates to become activated. After maturation, the DCs present the tumor antigens to lymphocytes and trigger the immune response cascade. In this study, CT stimulated splenic T lymphocytes to secrete IFN-γ and IL-10, suggesting that CT can immunomodulate T lymphocyte function. Heightened expressions of CD80, CD86, MHC-I and MHC-II imply that CT stimulates maturation of antigen-presenting cells; moreover, an increase in the expression of MHC-I and II molecules, together with an increase in CD8-positive T cells, suggests that HLA-mediated presentation of tumor antigens accelerates after treatment with CT extract. CT also improved the antitumor activity of the DC vaccine as evidenced by a reduction in tumor weight.
The ex vivo analysis of cytokine secretion and lymphocyte population suggested that CT polarizes the immune response toward the Th1 pathway by increasing the secretions of IL-2 and IFN-γ, but not IL-4, and consequently produces more cytotoxic T lymphocytes to elicit antitumor activity than DC vaccine without CT treatment. It has been reported that stimulated DCs secrete TNF-α, IL-1β and IL-10 during the maturation process and then polarize the T lymphocytes toward the Th1 pathway [47], which is consistent with our observations shown in Figure 3.
DCs are widely distributed in the human body with different morphologies, such as Kupffer cells in liver [48] and Langerhans cells in the skin [49]. Unlike chemotherapy which produces severe side effects during treatment, the use of DCs provides an alternative strategy against tumors [50]. The functions of DCs are improved in such as the maturation, antigen presentation and regulatory cytokine secretion, which has survival benefits in cancer patients [51]. Although the CT-treated DC vaccine was intraperitoneally injected to activate immunity in this study, oral administration of CT extract might promote the recognition of antigens and facilitate antigen presentation via intestinal DCs, and thus this attractive approach is worthy of further investigation. However, many traditional medicinal herbals have being studied for immunomodulatory activities, such as Melilotus suaveolens Ledeb [52] and Tanacetum parthenium [53]. In literatures, both herbal plants were found to demonstrate their anti-inflammation activities in a monocytic cellbased assay system and exhibited effects on regulating the production of chemokines. Due to these recent studies, M. suaveolens Ledeb [52] and T. parthenium have been implicated to exhibit potential therapeutic benefits in treating many inflammation-related diseases, such as cancers, atherosclerosis and rheumatoid arthritis. Therefore, herbals in treating cancers might have activities via the modulation of cytokine profiles. In this article, our results suggest that CT could promote immunity through the activation of DCs per se that do not alter the cytokine secretion during immune responses of tumor lysate-pulsed DCs toward the Th1 pathway.