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Introduction

In China, herbs and their products are widely used in clinical treatment, with increasing popularity. Matrine (MT) and oxymatrine (OMT), two types of alkaloid components in the roots of Sophora species, have various pharmacological activities and anti-inflammatory, anti-allergic, anti-virus, anti-fibrotic and cardiovascular protective effects (1). At present, OMT has been widely used in the treatment of hepatitis B and liver fibrosis in China (2). Furthermore, OMT may exert its anticancer activities through various channels, primarily by inhibiting cancer cell proliferation (3), inducing cell cycle arrest (4) and differentiation (5), accelerating apoptosis (6), restraining agiogenesis (7), inhibiting metastasis and invasion (8), and preventing or reducing chemotherapy- and radiotherapy-induced toxicities (9). However, these previous studies are mostly limited to observations of superficial phenomenon and lack systematic investigation using modern molecular biology techniques. The precise mechanism underlying the anticancer activity of OMT remains largely unknown.

Dendritic cells (DCs) serve a critical role in antigen capturing, processing and presentation (10). In the event of infection or inflammation of the body, microbial infection and other factors may promote the maturation of DCs, and thus initiate a T cell-mediated immune response (11,12). There are a range of effector T cells, including immunogenic cluster of differentiation (CD)4+ T helper (Th) cells, cytotoxic CD8+ T cells and in particular, tolerogenic regulatory T cells (Tregs), termed the DC-Treg system. In principle, DCs are associated with the two principal types of immunity, innate and adaptive. Therefore, DCs may be an ideal target for the development of immunotherapies and an adjuvant to convert their function between tolerogenic and immunogenic may be desirable. It is important to identify and develop strategies that may improve the efficacy of DC-mediated antitumor immunotherapy.

The immune status of the systemic or local microenvironment in tumor hosts may determine the responsiveness to chemotherapy (13). The immunomodulatory activity of OMT has been demonstrated in rheumatoid arthritis (13), chronic hepatitis B (14) and colitis models by shifting the Th subsets (15). However, to the best of the authors' knowledge, the potential effect of OMT on the DC-mediated antitumor immune response has not yet been studied. In the present study, the effects of OMT on DC maturation, and the subsequent simulation of CD4+ T cell polarization and cytokine secretion in vitro were examined. Furthermore, whether the immunomodulatory ability of OMT may reverse drug-resistance in A549 lung cancer cells was investigated.

Materials and methods

Subjects

Male NSCLC patients and healthy controls between the age of 40–55 were enrolled in this study. The median age was 46.8 years (range: 40–54) in NSCLC patients (n=13), and 45.3 years (range: 41–52) in healthy controls (n=15). Inclusion criteria for the present study were patients with histological proven NSCLC staging II–IV, who were primarily diagnosed in The Third Affiliated Hospital of Sun Yat-sen University between January 2017 and December 2017. All enrolled patients had no previous treatment with molecular target therapy, chemotherapy or radiotherapy. Exclusion criteria were chronic systemic diseases (including hypertension, diabetes and coronary heart disease) or immune systemic diseases (including HIV, organ transplantation and tumors). All subjects joined voluntarily with informed consents. This present study received ethical approval from the Institutional Review Board of Sun Yat-sen University.

Isolation and culture of DCs

Blood samples (50 ml) were obtained from each subjects. Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll (GE Healthcare Life Sciences, Little Chalfont, UK) density gradient centrifugation at 1,200 × g and 4°C for 20 min. PBMCs were cultured for 2 h and non-adherent cells were removed. The DC cell culture medium was prepared by adding recombinant human (rh) granulocyte-macrophage colony-stimulating factor (GM-CSF; 100 ng/ml) and rh interleukin (IL)-4 (100 ng/ml) to RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing 10% fetal bovine serum (FBS; Hyclone; GE Healthcare Life Sciences, Logan, UT, USA). PBMCs were incubated for 8 days at 37°C and prepared for subsequent experiments.

Detection of DC surface markers

Cells were treated with lipopolysaccharide (LPS; 100 ng/ml), OMT (1 mg/ml) and OMT (1 mg/ml) + LPS (100 ng/ml) for 48 h. According to preliminary experiments in the present study, oxymatrine inhibited the proliferation of dendritic cells, and the effect of inhibiting proliferation was observed at OMT concentration of 0.8 mg/ml. However, the inhibitory effect was not significantly increased with the increase of concentration, so the concentration of OMT in this experiment was chosen to be 1.0 mg/ml (data not shown). The cultured DCs were collected and centrifuged at 1,000 × g for 5 min at 4°C. The supernatant was discarded and the cells were washed three times with PBS. In total, 5 µl CD83 antigen (CD83)-phycoerythrin (PE; 556855; BD Biosciences, San Jose, CA, USA), T-lymphocyte activation antigen CD86 (CD86)-PE (560957; BD Biosciences), CD11 antigen-like family member C (CD11c)-PE (555392; BD Biosciences) and major histocompatibility complex II (MHC II)-PE(555812, BD Biosciences) were added to each tube. Subsequent to gentle mixing, the cells were incubated at room temperature in the dark for 30 min. The cells were washed twice with PBS and resuspended in 100 µl PBS, and the DC surface markers were detected using a flow cytometer (BD Accuri C6; BD Biosciences).

Isolation and culture of CD4+ T cells from patients with non-small cell lung cancer (NSCLC)

A total of 10 patients with newly diagnosed NSCLC were enrolled to collect 20 ml blood prior to any treatment. Subsequently, PBMCs were isolated using the Ficoll density gradient method at 1,200 × g, 4°C for 20 min. In total, 1×106 cells were used to isolate CD4+ T cells using a magnetic cell separation system. The selected cells were cultured in RPMI-1640 medium containing 10% FBS for subsequent experiments.

Co-culture of DCs and T cells

The DCs were treated with control, LPS (100 ng/ml), OMT (1 mg/ml) and OMT (1 mg/ml)+LPS (100 ng/ml) for 48 h. Cells were subsequently centrifuged at 1,000 × g for 5 min at 4°C and the supernatant was discarded. Subsequent to washing three times with PBS, the DCs and peripheral blood-derived CD4+ T cells from patients with NSCLC were implanted into Matrigel-coated Transwell chambers; DCs were seeded into the lower chambers and CD4+ T cells into the upper chambers at 1:5, 1:10 and 1:20 ratios. Cells were cultured in RPMI-1640 medium containing 10% FBS for 48 h; a non-contact cell co-culture system was established.

qPCR for forkhead box protein P3

DC cells and T cells were co-cultured at a ratio of 1:5, 1:10 and 1:20, respectively, as described above. CD4+ T cells were collected and total RNA was extracted using TRIzol (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocols. cDNA were synthesized using PrimeScript™ RT reagent kit with gDNA Eraser (Takara Bio, Inc., Otsu, Japan), strictly following the manufacturer's protocols. The target genes and controls were analyzed by RT-qPCR using SYBR-Green PCR Master Mix (Toyobo Life Science, Osaka, Japan) and the reactions were performed on ABI 7500 system (Applied Biosystems; Thermo Fisher Scientific, Inc.). Primers used in the study were as follows, U6 (housekeeping) primers: 5′-CGCTTCACGAATTTGCGTGTCAT-3′ (forward) and 5′-GCTTCGGCAGCACATATACTAAAAT-3′ (reverse); FOXP3 primers: 5′-CTGACCAAGGCTTCATCTGTG-3′ (forward) and 5′-ACTCTGGGAATGTGCTGTTTC−3′ (reverse). Reagents in the reaction included 0.5 µl of cDNA (1:20), 0.5 µl of forward primer and 0.5 µl of reverse primer, 10 µl of PCR Master Mix and 4.0 µl dH2O. Thermocycling conditions for PCR were 95°C for 5 min, and 40 cycles of 95°C for 15 sec, 65°C for 15 sec and 72°C for 32 sec. The average value was calculated by the relative quantitative method, and the gene expressions in the treatment group relative to the control group were calculated by the formula 2−ΔΔCq following Livak's method (16).

Detection of forkhead box protein P3 (FOXP3)+/CD4+ Tregs

The co-cultured CD4+ T cells were collected and centrifuged at 1,000 × g for 5 min at 4°C; the supernatant was discarded. Anti-Human FoxP3 Staining kit with CD4-Fluorescein Isothiocyanate and FOXP3-PE (560132; BD Biosciences) was used in the following experiments. The cells were washed three times with PBS, and CD4-Fluorescein Isothiocyanate (1:20) was added to each tube and incubated for 30 min at room temperature in the dark. For antibodies to enter the intracellular structure, cells were resuspended in fixation/permeabilization buffer (00–5223/00-5123; eBioscience; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocols. Following two washes with PBS, the cells were incubated for 30 min at room temperature in the dark. FOXP3-PE (1:20) was added and incubated for another 30 min in the same condition. Cells were subsequently washed twice and resuspended with the fixation/permeabilization buffer. The ratio of FOXP3+/CD4+ cells was detected using a flow cytometer (BD Accuri C6; BD Biosciences).

Detection of cytokines in supernatant of the co-culture system

The DCs were treated with control, LPS (100 ng/ml), OMT (1 mg/ml) and OMT (1 mg/ml) + LPS (100 ng/ml), and mixed with peripheral blood-derived CD4+ T cells from patients with NSCLC at ratios of 1:5, 1:10 and 1:20. After 48 h, the supernatant of the co-culture system was collected. The concentrations of IL-10, transforming growth factor (TGF)-β, IL-35, interferon (IFN)-γ, IL-2 and IL-12 in the supernatant were detected using ELISA kits: IL-10 (E-EL-H0103c), TGF-β (E-EL-0110c), IL-35 (E-EL-H2443c), IFN-γ (E-EL-0108c), IL-2 (E-EL-H0099c) and IL-12 (E-EL-H0150c; all from R&D Systems, Inc., Minneapolis, MN, USA).

Detection of A549 apoptosis following co-culture

The induction of A549 resistant cells was performed by gradually increasing the concentration of cisplatin from a starting concentration of 1.0 µM, and after 4 weeks, it was increased to 2.0 µM, and serially subcultured for ~100 passages. Then, 6 months later, the A549/DDP resistant cell line was established. The human lung adenocarcinoma cells A549 were purchased from Procell Life Science & Technology Co., Ltd. (Wuhan, China; cat. no. CL-0016). The cells were maintained in RPMI-1640 (Gibco; Thermo Fisher Scientific, Inc.) medium with 10% fetal bovine serum (FBS; Hyclone; GE Healthcare Life Sciences) and 1% penicillin-streptomycin solution (Beyotime Institute of Biotechnology, Haimen, China), and cells were cultured in a humidified atmosphere at 37°C with 5% CO2. Co-culture systems of A549/DDP plus DC or DC-T were established. OMT (1 mg/ml)-treated DCs and the FOXP3+/CD4+ T cells induced by DC-T co-culture (cell ratio, 1:5) were incubated with A549/DDP cells. The cells were seeded for 24 h in the upper and lower chambers of Transwell plates at ratios of 1:1, 1:5, 1:10 and 1:20. At the same time, 22 µM DDP was added to the A549/DDP medium for 24 h. The collected cells were stained using an Annexin V/Propidium Iodide kit (BD Biosciences) according to the manufacturer's protocol, and the apoptotic ratio was analyzed using a flow cytometer (BD Accuri C6; BD Biosciences).

Western blotting

A549/DDP cells were lysed using radioimmunoprecipitation assay buffer (50 mM Tris, 150 mM NaCl, 1% NPNP-40, 1% sodium deoxycholate and 0.05% SDS; pH 7.4) following co-culture with OMT-treated DCs/DC-T and treatment with DDP. Total protein was extracted and subsequently quantified using a bicinchoninic acid kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA). For western blotting, protein loading was set at 40 µg and an 12% SDS-PAGE gel was used for protein separation. The proteins were transferred to a nitrocellulose membrane and incubated with primary antibodies against FOXP3 (1:3,000; cat. no. ab191416), apoptosis regulator BAX, (Bax; 1:1,000; cat. no. ab32503), B cell lymphoma-2 (Bcl-2; 1:1,000; cat. no. ab32124) and β-actin (1:1,000; cat. no. ab8224) overnight at 4°C, and subsequently incubated with Goat Anti-Rabbit (1:1,000; cat. no. ab6702) or Anti-Mouse (1:1,000; cat. no. ab6708) (all from Abcam, Cambridge, UK) secondary antibodies for 1 h at room temperature. Chemiluminescence was used to develop the color of the membrane using an ECL kit according to the manufacturer's instructions (cat. no. FD8030; Guangzhou Fude Biological Technology Co., Ltd., Guangzhou, China). ImageJ 8.0 software (National Institutes of Health, Bethesda, MD, USA) was used to perform densitometric analysis.

Statistical analysis

Each experiment was repeated at least 3 times. Data were presented as the mean ± standard deviation. Statistical significance was determined by one-way analysis of variance with Bonferroni Correction using SPSS software version 19.0 (IBM Corp., Armonk, NY, USA). P<0.05 was considered to indicate a statistically significant difference.

Results

OMT promotes DC maturation

The monocytes were prepared from the peripheral blood of healthy adult volunteers. To investigate the effect of OMT, the immature DCs were cultured for 8 days containing 1 mg/ml OMT in medium supplemented with rhGM-CSF (100 ng/ml) and rhIL-4 (100 ng/ml). Bacterial LPS is the most common ‘maturation’ signal for DCs (17). With the LPS-treated (100 ng/ml) group (LPS-DCs) as a positive control and mononuclear cells as a negative control, the expression of DC surface markers was detected by flow cytometry analysis, as presented in Fig. 1. Compared with the negative control group, treatment with OMT (1 mg/ml) significantly increased the expression of CD83, CD86, CD11c and MHC II (P<0.001), and the expression level of these markers was higher when LPS and OMT were combined. These results demonstrated that OMT may promote DC maturation.

Figure 1. OMT promotes dendritic cell maturation. The expression levels of CD86, CD83, CD11c and MHC II were detected by flow cytometry. Compared with the control group and LPS group, treatment with OMT (1 mg/ml) increased the expression level of those markers (CD86-26.8%, CD83-33.6%, CD11c-23.4% and MHC II-22.5%), and when LPS and OMT were combined, the expression levels were higher compared with the OMT (1 mg/ml) group (CD86-38.8%, CD83-45.8%, CD11c-33.7% and MHC II-45.9%). ***P<0.001 vs. respective Ctrl. group OMT, oxymatrine; LPS, lipopolysaccharide; CD86, T-lymphocyte activation antigen CD86; CD83, CD83 antigen; CD11c, CD11 antigen-like family member C; MHC II, major histocompatibility complex II.

Differentiation of FOXP3+/CD4+ Tregs induced by OMT-DCs

To examine the response of allogeneic primary CD4+ T cells to DC stimulation pretreated with OMT (1 mg/ml; OMT-DC), the following experiments were performed. CD4+ T cells were isolated from the peripheral blood of patients with NSCLC. DCs were co-cultured with the primary CD4+ T cells for 48 h in Transwell plates at ratios of 1:5, 1:10 and 1:20. The expression levels of FOXP3 in CD4+ T cells were analyzed by flow cytometry, western blotting and qPCR.

As presented in Fig. 2, OMT-DCs stimulated the primary CD4+ T cells to express FOXP3, and the mRNA expression level of FOXP3 decreased as the DCs were more diluted. The FOXP3 protein assay showed that the total amount of FOXP3 was increased when LPS and OMT were used separately or in combination (especially when used in combination). However, in the case of the same treatment, the dilution of DC cells had no apparent effect on FOXP3 protein amount when groups of different DC cell dilution ratios were compared among each other. Whether there are translation level and turnover level factors regulating the expression of FOXP3 protein in this case deserves further investigation. Altogether, these results demonstrated that OMT-pretreated DCs may enhance FOXP3+/CD4+ Treg differentiation.

Figure 2. Differentiation of FOXP3+/CD4+ regulatory T cells induced by OMT-DCs. (A) Compared with the control group and LPS group, OMT-DCs stimulated the primary CD4+ T cells to express FOXP3, and the expression level of FOXP3 decreased with the decrease of co-culture cell ratio (1:5-3.8, 1:10-2.6 and 1:20-1.6%). When LPS and OMT were combined, the expression of FOXP3 was higher compared with the OMT-DCs group (1:5-5.3, 1:10-3.3 and 1:20-2.2%). (B) Expression of FOXP3 detected by western blotting and quantitative polymerase chain reaction presented similar results to the flow cytometry assay. ***P<0.001 vs. respective Ctrl. group. FOXP3, forkhead box protein P3; OMT, oxymatrine; DCs, dendritic cells; LPS, lipopolysaccharide; Ctrl., control; CD, cluster of differentiation.

Cytokine secretion stimulated by OMT-pretreated DCs

Inflammatory factors serve an important role in the immune response. ELISA was used to quantitatively analyze inflammatory factors in the co-culture supernatants. When OMT-DCs were co-cultured with T cells, a large number of anti-inflammatory factors, including IL-10, TGF-β and IL-35, and pro-inflammatory cytokines, including IFN-γ, IL-12 and IL-2, were secreted. Furthermore, the expression levels of those inflammatory factors decreased as the DCs were more diluted with T cells (Fig. 3). When LPS-DCs were co-cultured with T cells, it additionally promoted the secretion of inflammatory factors, in a similar manner to OMT-DCs. When LPS and OMT were applied simultaneously, the secretion of inflammatory factors reached the highest concentration. These results suggested that OMT-pretreated DCs may promote the primary T cells to polarize into FOXP3+/CD4+ Tregs, and enhance the anti-inflammatory and pro-inflammatory cytokines secretion. Based on these results, the co-culture ratio of DCs to CD4+ T cells was set at 1:5 for subsequent experiments.

Figure 3. Cytokine secretion stimulated by OMT-pretreated DCs. At different co-culture cell ratios, compared with the control group and LPS group, OMT-DCs stimulated the primary CD4+ T cell to secrete cytokines, including IL-10, TGF-β and IL-35, and pro-inflammatory cytokines, including IFN-γ, IL-12 and IL-2. When LPS and OMT were combined, the cytokine secretion was higher compared with the OMT-DC group. Furthermore, the expression levels of the inflammatory factors decreased as the DCs were more dilute. ***P<0.001 vs. respective Ctrl. group. OMT, oxymatrine; DCs, dendritic cells; CD, cluster of differentiation; LPS, lipopolysaccharide; IL, interleukin; TGF-β, transforming growth factor-β; IFN-γ, interferon-γ; Ctrl., control; Conc., concentration.

Reversal of DDP-resistance in A549/DDP by OMT-DC/OMT-DC-T

A DDP-resistant cell line, A549/DDP, was established. As presented in Fig. 4A, A549/DDP has a half maximal inhibitory concentration of 22 µM for DDP. In order to investigate the effect of OMT-pretreated DCs and OMT-DCs induced CD4+ T cells on the sensitivity of A549/DDP to antitumor drugs, OMT-stimulated DCs (OMT-DC) and OMT-DC-stimulated primary CD4+ T cells (OMT-DCs-T) were co-cultured with the A549/DDP cells at ratios of 1:1, 1:5, 1:10 or 1:20 for 24 h. At the same time, the A549/DDP cells were treated with 22 µM DDP for 24 h. Annexin V/PI staining was used to analyze the apoptosis of the A549/DDP cells.

Figure 4. Reversal of DDP-resistance in A549/DDP by OMT-DCs/OMT-DCs-T. (A) A549/DDP has a half maximal inhibitory concentration of 22 µM for DDP. (B) At the same cell co-culture ratio, OMT-DCs and OMT-DCs-T may increase the percentage of apoptotic cells in A549/DDP, and the apoptotic rate decreased accompanied by a gradual decrease in the co-culture ratio of OMT-DCs/OMT-DCs-T and A549/DDP, detected by flow cytometry. DDP, cisplatin; A549/DDP, DDP-resistant A549 cells; OMT, oxymatrine; DCs, dendritic cells; Inh, inhibition; PI, propidium iodide; FITC, fluorescein isothiocyanate; Q, quadrant; UL, upper left; UR, upper right; LL, lower left; LR, lower right; T, cluster of differentiation 4+ T cells.

As presented in Fig. 4B, OMT-DC and OMT-DC-T may increase the percentage of apoptotic cells in A549/DDP, and the apoptotic rate decreased accompanied by the gradual dilution of the OMT-DCs/OMT-DCs-T with A549/DDP.

The western blotting results (Fig. 5) demonstrated that the expression of apoptosis-associated protein Bcl-2 increased as the A549/DDP cell apoptosis decreased; however, the protein expression level of Bax decreased. These results suggested that OMT-pretreated DCs and DC-induced T cells may reverse DDP-resistance in a non-contactable co-culture cell model.

Figure 5. Reversal of cell apoptosis in A549/DDP by OMT-DCs/OMT-DCs-T. The expression of apoptosis-associated protein Bcl-2 was negatively associated with the A549/DDP apoptosis; however, the expression level of Bax protein was positively associated. A549/DDP, DDP-resistant A549 cells; OMT, oxymatrine; DCs, dendritic cells; Bcl-2, B cell lymphoma-2; Bax, apoptosis regulator BAX; T, cluster of differentiation 4+ T cells.

Discussion

Carcinogenesis is a multistep process; agents that are able to target one or more of these processes maybe ideal cancer chemopreventive agents. According to previous studies (18,19), OMT may exert anticancer activities through various channels. Notably, it was observed that all of these functions were a result of direct contact of OMT with tumor cells. The present study is, to the best of the authors' knowledge, the first study to demonstrate that the therapeutic efficacy of OMT may be indirectly achieved by activating the DC-mediated antitumor immune response in circulation and/or in the tumor microenvironment.

Monocyte-derived mature DCs serve a key role in the immune response. Activated DCs may release various cytokines that mediate T cell activation and efficiently present a subset of antigens to T cells (20). At present, the potential impact of OMT on the maturation and function of DCs has not yet been fully investigated. In the present study, it was identified that OMT may enhance DC maturation in the process of differentiation of mononuclear cells derived from human peripheral blood. The positive effect of OMT on proliferation was significant at 1 mg/ml, and the surface markers of DCs, including CD83, CD86, CD11c and MHC II, were increased, suggesting that OMT may promote DC maturation. Immature DCs facilitate tolerance toward cancer cells, whereas, fully mature DCs may strongly promote anticancer immunity (21). The immune inhibitory microenvironment of the tumor inhibits DC differentiation and maturation (22,23). Efficiency and the success of such an interaction is dependent on the maturation status of the DCs (24). These data provide insight to support the use of OMT-mediated anticancer immunotherapy in human cancer.

DCs are key regulators of adaptive immunity with the potential to induce T cell activation/immunity or T cell suppression/tolerance. DCs are capable of differentiating naïve T cells into a range of effector cells, including immunogenic CD4+ Th cells, cytotoxic CD8+ T cells and tolerogenic Tregs (25). In the present study, similar to LPS, OMT-activated DCs were able to subsequently promote the differentiation of T cells into FOXP3+/CD4+ Treg cells in vitro. In agreement with the present results, Ma et al (26) demonstrated that OMT significantly upregulated FOXP3 and downregulated nuclear receptor ROR-γ expression, thereby inducing Treg/Th17 imbalance and inhibiting inflammation of rheumatoid arthritis. In contrast, it was identified that treatment with OMT triggers an increase in Th1 cytokines and a decrease in Th2 cytokines, which enhances the immune response and improves inhibitory activities to hepatitis B virus (14). Accordingly, OMT may promote the immune response to remove viruses and relieve hyperinflammation to protect organ function in autoimmune disorders (26). Therefore, it was hypothesized that OMT may have bilateral modulation on DC-T interaction, which may be switched mutually, according to the specific pathophysiological setting.

It is generally accepted that FOXP3+/CD4+ Treg infiltration in tumor tissue serves a role in inhibiting antitumor immunity and contributing to immune tolerance (27–29), thus promoting the occurrence and progression of tumors. However, a number of previous studies demonstrated that FOXP3+/CD4+ Treg infiltration was positively associated with good prognosis in colorectal, head and neck cancer, NSCLC and breast cancer (30–33). Immunosuppression or immune escape has been implicated as a primary mechanism in the initial establishment of tumors. Along with the secondary infection and cytotoxic drug usage in advanced stages, the resultant chronic systemic inflammation may alter the antigenic landscape of tumor cells, rewire oncogenic signaling networks, protect against cell death and reprogramme immune cell functions (34). It represents a shared resistance mechanism to different therapy (35,36). At this stage, a higher proportion of FOXP3+/CD4+ Tregs may relieve hyperinflammation and therapy-induced injury, thereby contributing to improve the overall prognosis of patients. Therefore, the DC-T response to treatment with OMT may vary at each stage of tumor progression.

The supernatant of the DC-T co-culture medium contained anti-inflammatory factors (including IL-10, TGF-β and IL-35) and pro-inflammatory cytokines (IFN-γ, IL-12 and IL-2). Furthermore, pro-inflammatory and anti-inflammatory factor secretion decreased when the co-cultured DCs were more diluted with the primitive T cells. This suggested that the DC-T co-culture system may affect the final anti-/pro-inflammatory concentration in the tumor microenvironment. The therapeutic effect of OMT is partly achieved by immune modulation. The protection of OMT is associated with inhibiting pro-inflammatory cytokines in acute lung injury (37), pulmonary fibrosis (38), rheumatoid arthritis (13) and organic ischemia/reperfusion injury (39,40). However, Ning et al (41) demonstrated that OMT was able to directly induce antiviral cytokine secretions in the peripheral lymphocytes isolated from patients with chronic hepatitis B. Therefore, further study is required to clarify the specific pattern of OMT regulating DC-T interaction, and determining the ultimate immune balance seems to be particularly critical.

In the past 5 years, MT and OMT have been extensively studied for their cancer chemopreventive potential against various cancer types. However, only a number of previous studies have investigated the underlying mechanisms of drug-resistance reversion. It was demonstrated that OMT and its derivatives may reverse DDP resistance by inhibiting multidrug resistance-associated protein 1 expression and the downregulation of Bax/Bcl-2 in the human nasopharyngeal carcinoma cell line HONE1 (42). Wang et al (43) observed that MT induced mitochondrial apoptosis in DDP-resistant NSCLC cells via suppression of β-catenin/survivin signaling (43). In addition, OMT may additionally reverse taxol resistance in NCI-H520/TAX25 human lung cancer cells (44), whereas, MT was able to induce apoptotic effects in doxorubicin resistant K562 cells (45). Therefore, OMT and its derivatives maybe used as ideal adjuncts for cancer chemotherapy.

In contrast to a previous study (46), it was demonstrated that OMT-pretreated DCs and DCs-T coculture may enhance apoptosis of DDP/A549 cells in vitro, without direct contact of OMT with tumor cells. Therefore, it was hypothesized that OMT-induced DC maturation and activation may be key in promoting DDP/A549 apoptosis. At present, it is well documented that mature DCs are able to induce strong antitumor responses mediated by effective candidates, including cytotoxic CD8+ T cells and large numbers of cytokines, to enhance sensitivity of tumor cells to various chemotherapeutic drugs (47). Immunogenic cell death of tumor cells starts from the interaction between dendritic cells and cancer cells. Cytokines released during apoptosis of tumor cells promote the maturation of dendritic cells. Mature dendritic cells activate tumor-specific cytotoxic T cells, which shows the antitumor effect (48). Further investigations on the molecular mechanisms of DC-mediated antitumor capacity following OMT administration are required.

As DCs are largely responsible for the ability of the immune response to recognize and eliminate cancer cells, DCs have attracted interest for antitumor immunotherapy in recent decades (49). In order for DC-based immunotherapy to elicit potent antitumor immune responses, an immune stimulatory response, as opposed to a tolerogenic immune response, is required following the administration of DCs. At present, research has focused on identifying the key factors responsible for therapeutic success/failure to determine the full clinical potential use of DCs in cancer immunotherapy. Due to the low toxicity and wide range of biological activities, OMT and specific natural products, which have therapeutic selectivity or may preferentially kill cancer cells without significant toxicity to normal cells, are being considered for future cancer therapy.

In conclusion, the multi-targeted chemopreventive efficacy of OMT was demonstrated in the present study, focusing on its immunoregulatory capacity. OMT may increase the apoptosis of drug-resistant tumor cells by activating DC differentiation and function, thereby enhancing the antitumor immune response. The present data suggested that OMT is a promising reagent for the development of more effective DC-based immunotherapy or cancer vaccines. The results of the present study are not only applicable to patients with tumors; however, additionally have reference value for infectious and immune diseases, and provide a theoretical basis for the use of OMT in the clinical setting.

Acknowledgements

Not applicable.

Funding

The present study was supported by funds from the China National Natural Science Funds (grant no. 81472760) and the Science and Technology Planning Project of Guangdong Province, China (grant no. 2014A020212078).

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Authors' contributions

HL designed all the experiments, applied for project funding and wrote the final version of the paper. MZ performed the in vitro cell culture, including isolation and culture of DCs and CD4+ T cells, as well as co-culture of DCs and T cells. PL performed flow cytometeric detection, including detection of surface markers of DCs and the ratio of FOXP3+ Tregs/CD4+. HW enrolled and collected the baseline data for the NSCLC patients and healthy controls. HW completed the detection of cytokines in supernatants using ELISA kits. XL completed the qPCR and western blotting in overall experiments, and completed the detection of A549 apoptosis. JY performed the statistical analysis and wrote the paper in cooperation with HL. JY and HL dealt with editorial queries and the overall submission. All authors read and approved the final manuscript.

Ethics approval and consent to participate

All participants provided written consent, and the study was approved by the Institutional Review Board of Sun Yat-sen University.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References