- Primary research
- Open Access
Lycopene improves the efficiency of anti-PD-1 therapy via activating IFN signaling of lung cancer cells
© The Author(s) 2019
- Received: 22 August 2018
- Accepted: 18 March 2019
- Published: 21 March 2019
Monoclonal antibodies targeting programmed death-1 receptor (PD-1) and its ligand (PD-L1) have been developed to treat cancers including lung cancer. In this study, we aimed to investigate whether lycopene could promote the effect of anti-PD-1 treatment on lung cancer.
Tumor formation assay was conducted. Immune reactions were assessed by detecting several cytokine levels using enzyme-like immunosorbent assay. T cell activity was analyzed using cytometry. The mechanism of lycopene action was investigated using Western blot, quantitative real-time polymerase chain reaction and bisulfite sequencing analysis.
After the mice injected with Lewis lung carcinoma (LLC) cells were sacrificed, we found that combined lycopene and anti-PD-1 reduced the tumor volume and weight compared to control treatment. Cell apoptosis in the tumor tissues was significantly enhanced in mice with combined lycopene and anti-PD-1 treatment in comparison with those of either lycopene or anti-PD-1 alone. Furthermore, lycopene could assist anti-PD-1 to elevate the levels of interleukin (IL)-1 and interferon (IFN) γ while reduce the levels of IL-4 and IL-10 in the spleen of mice injected with LLC cells. Lycopene treatment increased the CD4+/CD8+ ratio in the spleen and promoted IFNγ-expressing CD8+ T cells in tumor tissues. Upon IFNγ stimulation, lycopene diminished PD-L1 expression via activating JAK and repressing phosphorylation of AKT.
Our results have demonstrated that lycopene could be used as a potential adjuvant drug to synergistically improve the efficiency of anti-PD-1 therapy.
- Lung cancer
- Programmed death-1 receptor (PD-1)
- Interferon (IFN) γ
- Interferon-regulatory factor (IRF) proteins
Lung cancer is one of the most malignant tumors and the leading cause of cancer death worldwide [1, 2]. Only 18% of patients survive from lung cancer . However, most patients are diagnosed in advanced stages with less than 5% patients survive for 5 years . The first-line treatment depends on genetic aberration of patients. For example, drugs targeting epidermal growth factor receptor (EGFR) and translocation of anaplastic lymphoma kinase (ALK) have been applied in patients with EGFR mutation or ALK translocation [4, 5]. However, for patients without those oncogenic drivers, cytotoxic chemotherapy is commonly applied. Nevertheless, poor prognosis and drug resistance are still obstructions for the efficiency of current treatments. Therefore, recent attempts have begun to focus on treating lung cancer by modulating the immune reactions.
Recently developed drugs that regulate specific immune checkpoints, and monoclonal antibodies targeting programmed death-1 receptor (PD-1) and its ligand (PD-L1), have all showed impressive anti-tumor effects [6–9]. PD-1 is a cell surface protein containing 288 amino acid. It has two ligands, PD-L1 and PD-L2. Studies have evidenced that high expression of PD-L1 has been observed in numerous cancers and is associated with poor outcomes . PD-L1 expression in tumor cells facilitates their escape from immune system surveillance. Among all patients with advanced non-small cell lung cancer (NSCLC), more than 20% showed PD-L1 expression in at least half of tumor cells [8, 11]. Therefore, improving the efficiency of anti-PD-1 therapy could yield promising outcomes.
Previous results have indicated that lycopene exerts anti-tumor effects by altering the methylation of genes through inhibiting DNA methyltransferase (DNMT) enzyme activity [12, 13]. DNMT inhibitors have been shown to enhance the efficacy of anti-PD-1 therapy in the treatment of lung cancer by activating interferon (IFN) signaling . Here, we sought to investigate whether lycopene could promote the effects of anti-PD-1 treatment on lung cancer. A series of results confirmed that lycopene promoted anti-PD-1 therapeutic efficiency of lung cancer by promoting IFNγ-expressing CD8+ cells infiltrated in tumor tissues and increasing IFNγ expression in tumor cells.
Tumor formation assay
The effect of combined therapy was detected in vivo. Lewis lung carcinoma (LLC) cells (1 × 106) were injected in the rear flak of C57BL/6 mice. Anti-mouse PD-1 antibodies (6 mg/kg) were administrated by intraperitoneal injection 3 days apart for 4 times. Lycopene (40 mg/kg) was intraperitoneally injected daily. Tumor volume and weight were measured. Tumor volume (mm3) was calculated using (length × width2)/2. The animal study was approved by the Ethical Committee of the Fifth People’s Hospital of Wuxi City. Mice were sacrificed to obtain tumor tissues.
Enzyme-like immunosorbent assay (ELISA)
The cytokine levels in the spleen were measured using ELISA. Briefly, the spleen of mice was obtained and minced in phosphate buffered saline. The levels of interleukin (IL)-2, interferon (IFN) γ, IL-4 and IL-10 were detected in triplicates by ELISA kits (Groundwork Biotechnology Diagnosticate, USA) according to the manufacturers’ instructions. The color absorbance was measured at 450 nm with an ELISA plate reader (SoftMax Pro 5.3).
Flow cytometry assay
The spleen or tumor tissues were mechanically disrupted using a gentleMACS™ dissociator (Miltenyi Biotec, Bergisch Gladbach, Germany) in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer to isolate cells for flow cytometry. Cells were then stained with fluorescently-labeled antibodies including anti-CD8 PE (Biolegend, San Diego, CA, USA), anti-CD4 (L3T4) FITC (Biolegend, San Diego, CA, USA) and anti-IFN-γ (BD Biosciences, Franklin Lakes, NJ, USA). For apoptosis analysis, perforin and granzyme B were employed.
Quantitative real-time polymerase chain reaction (qRT-PCR)
Primer sequences used for real-time PCR
Forward primer (5′–3′)
Reverse primer (5′–3′)
Bisulfite sequencing analysis
Primers for methylation-specific PCR
Forward primer (5′–3′)
Reverse primer (5′–3′)
Total proteins were extracted by cell lysis buffer (20 mM NaCl, 10% glycerol, 1% Triton X-100). To separate proteins, 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis was employed. Proteins were then transferred to Immobilon-P membranes (Millipore, Bedford, MA, USA) and incubated with primary antibodies overnight at 4 °C. Anti-DNMT1, anti-DNMT-3a, anti-DNMT3b, anti-PD-L1, anti-pJAK2, anti-JAK2, anti-p-STAT3, anti-STAT3, anti-p-AKT, anti-AKT and anti-β-actin were purchased from Sigma (St. Louis, MO, USA). All primary antibodies were diluted at 1:1000. After incubating with secondary antibody, the membranes were developed with Enhanced Chemiluminescence Plus Western Detection kit (Amersham Pharmacia Biotech, Piscataway, NJ, USA). For all quantization results of Western blotting, densitometry analysis was used. The values were obtained by dividing the optical density values of interested proteins by it of β-actin. The results were counted and plotted.
All data were presented as mean ± standard deviation (SD). Statistical analysis was performed using SPSS 19.0 (IBM., Chicago, IL, US) and Graphpad Prism 7 (Graphpad Inc., La Jolla, CA, US). Statistical significance was assessed using analysis of variance (ANOVA), unpaired t-tests or Mann–Whitney U-tests. p value of less than 0.05 was regarded as statistically significant.
Lycopene improves the efficiency of anti-PD-1 therapy in NSCLC
The combined lycopene and anti-PD-1 therapy alters the immune responses of mice and promoted death of tumor cells
Lycopene reduces the methylation levels of IRF1 and IRF7 promoters to promote IFNγ expression
The effect of lycopene on the expression of IFNγ-related genes in LLC cells
The effect and mechanism of lycopene on the expression of PDL-1 downstream of IFNγ signaling
About 20% of patients with advanced NSCLC showed responses to an antibody targeting PD-1, pembrolizumab [6, 15]. Investigations have demonstrated several factors that impact the sensitivity to pembrolizumab, including EGFR mutations, ALK rearrangements and mutational landscape . Additionally, few patients show strong reactions to anti-PD-1. Therefore, it is urgent to improve the efficiency of anti-PD-1 therapy.
In the present study, we demonstrated that both anti-PD-1 and lycopene alone had the ability to repress tumorigenesis. When combining anti-PD-1 therapy with lycopene, their anti-tumor effect was significantly increased. Although there were a series of studies demonstrating that lycopene intake was associated with decreased risks of prostate, gastric, breast, and lung cancers [17–21], whether it could synergistically function with anti-PD-1 therapy has not been reported. Here, we found that lycopene synergistically exerts anti-tumor function with anti-PD-1. In addition, cell apoptosis and cell death were promoted in the tumors of mice treated with combined lycopene and anti-PD-1. These results suggest that lycopene promotes anti-PD-1 effects on cytotoxic CD8 cells, which on the other hand increases cell death.
Since lycopene has been reported to play a role in the immune system, it is possible that the increased efficiency of anti-PD-1 therapy by lycopene is mediated through its functions in regulating immune response. Previous evidence indicated that lycopene inhibited inflammatory responses through blocking IκB kinase, reducing Toll-like receptor 4 expressions and the productions of several NF-κB-related cytokines such as Tumor necrosis factor-α, IL-1β and IL-6β [21, 22]. It is known that blocking PD-1 unleashes the T cells to target tumor cells. Therefore, based on the surprising effects of lycopene on anti-PD-1 treated lung cancer, we further investigated whether lycopene affected T cell activities. As expected, the productions of IL-2 and IFNγ were significantly increased in the spleen of mice treated with lycopene, whereas IL-4 and IL-10 were inhibited. The ratio of CD4+/CD8+ cells in the spleen was enhanced in lycopene-treated mice compared to untreated mice, indicating that lycopene is able to promote immune response by enhancing the activation, growth and differentiation of T cells, as well as impact T helper (Th)1/Th2 drift. In addition, increased IFNγ-expressing CD8+ cells in tumor tissues suggests that lycopene may disrupt the functions of PD-1 and PD-L1 to reduce tumor escaping from the immune system.
Interestingly, we found that lycopene promoted the mRNA expression levels of IFNγ and IFNβ and their downstream gene expression including IRF1, IRF7, CXCL9 and CXCL10. It is known that IRF1 is an important factor involved in apoptosis, which can be stimulated upon stimuli such as IFNγ and activated IFNβ gene . Loss of IRF1 expression or function has been reported to be associated with several human cancers such as breast cancer and gastric cancer [24–26]. Another IRF family protein, IRF7, also plays critical roles in tumors. A previous study reported that silencing IRF7 could assist tumor cell to escape from the immune system, leading to increased tumor metastasis . Essentially, IRF7 is also well-known by its regulatory role in the induction of IFNα/β genes . Therefore, the elevated expression of IFNβ may be caused by the impact of lycopene on IRF7. Although IRF3 is also a master regulator for Type I IFN expression, we did not observe a significant change of IRF3 expression upon lycopene treatment. Additionally, no difference in IRF8 expression was exhibited upon lycopene treatment. It is known that IFNγ induces IRF8 expression , and IRF8 expression could be repressed by DNA methylation . Here, we found that lycopene treatment diminished the methylation levels of IRF1/7 promoters but not IRF8. It may indicate that lycopene may not directly function in IRF1/7 promoter methylation. However, we observed that lycopene significantly repressed the expression level of DNMT3a but did not affect DNMT1 and DNMT3b. Previous studies have demonstrated that DNMT3a is a negative regulator of IRF8 . Therefore, this result further indicates that lycopene treatment affecting the mRNA levels of IRF1 and IRF7 may only be caused by its role in IFNγ. Further study is needed to explain how lycopene regulates DNMT3a but showed no effect on IRF8.
The role of IFNγ in anti-tumor immunity could be highlighted by resistance of tumors to interferons that leads to their evasion from the immune system . Recently, immune checkpoint blockade (ICB) has been implicated in cancer treatments, which blocks inhibitory receptors on the surface of intra-tumoral effector T cells [33, 34]. Results that ICB could induce production of intra-tumoral IFNγ in mouse models and human patients, as well as the dependence of tumor infiltration on IFNγ receptor by immune cells [35, 36], indicated a key role of IFNγ in rejecting tumor. Studies from patients resistant to anti-PD1 therapy have also provided strong support for ICB-induced IFNγ action directly on tumor cells [37, 38]. It would be interesting to further test these possibilities in the context of NSCLC.
T cell-secreted IFNγ was reported to mediate the induction of PD-L1 expression in melanoma microenvironment . In this study, we found that lycopene was able to decrease both mRNA and protein levels of PD-L1 upon IFNγ stimulation in LLC cells. This process may be mediated by the regulatory effects of lycopene on AKT signaling. However, no change in PD-L1 was observed in tumors, suggesting that lycopene could disrupt the induction of PD-L1 by T cell-secreted IFNγ.
In conclusion, this is the first study discovering the role of lycopene in anti-PD-1 therapy. We have found that lycopene is a promising natural chemical that could synergistically function with anti-PD-1 to prevent tumorigenesis. Its immunomodulatory role assists anti-PD-1 to elevate the levels of IL-1 and IFNγ but reduce the levels of IL-4 and IL-10 in the spleen. In addition to its effects on the CD4+/CD8+ ratio, lycopene also increases the number of IFNγ expressing CD8+ T cells in tumor tissues. Upon IFNγ stimulation, lycopene represses PD-L1 expression via AKT signaling. These results demonstrate that lycopene could be applied to synergistically improve the efficiency of anti-PD-1 therapy.
Conception and design: XJ, HW, WZ, XD and QY. Collection and assembly of data: FZ and MQ. Data analysis and interpretation: XJ and PY. Contribution of reagents, materials, and analysis tools wrote the paper: XJ, LS and XG. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
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This work was supported by Fund Program: Wuxi Municipal Health Bureau Project (MS201701) and Major Projects of Wuxi Municipal Health Committee (Z201811).
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