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. 2018 May 21;16(1):132.
doi: 10.1186/s12967-018-1508-5.

CD8+ T cells mediate the antitumor activity of frankincense and myrrh in hepatocellular carcinoma

Chun Xu  1 , Xian Lu  1   2 , Wei Liu  1 , Anxian Chen  1 , Gang Meng  1 , Hailin Zhang  1 , Binghua Li  1   2 , Yonghui Zhang  1 , Junhua Wu  3 , Jiwu Wei  4   5
Affiliations

CD8+ T cells mediate the antitumor activity of frankincense and myrrh in hepatocellular carcinoma

Chun Xu et al. J Transl Med. .

Abstract

Background: Tumor-promoting inflammation is an emerging hallmark of cancer, which participates in both cancer progression and immune escape. Hepatocellular carcinoma (HCC) is a typical inflammation-related cancer with an extremely poor prognosis. Frankincense and myrrh are anti-inflammation agents commonly used in clinic. The purpose of this study is to investigate whether extract of frankincense and myrrh (FM) downregulates inflammatory microenvironment of HCC and thereby restores antitumor immune responses.

Methods: The water-decocting FM was obtained and quantified. HCC cell lines HCCLM3 and Hepa1-6 were used to evaluate the efficacy of FM _targeting NF-κB and STAT3 signaling with western blot and qRT-PCR analysis. CD8+NKG2D+ cells were derived from human peripheral blood and were used for evaluation of immune cells-mediated inflammation and oncolysis on HCCLM3 cells. The antitumor efficacy of FM was investigated both in immune compromised and immune competent mice bearing subcutaneous HCC. Mice received daily oral gavage of FM at 60 mg/kg. Immune activity within tumor microenvironment (TME) was assessed by ELISpot assay and flow cytometry, respectively. Depletion of CD8+ T cells or NK cells was achieved by intraperitoneal injection of respective neutralizing antibody.

Results: FM significantly inhibited the activation of NF-κB and STAT3 signaling in HCC cells induced by cytokines (TNF-α or IL-6) and in co-culture system with CD8+NKG2D+ cells. Furthermore, FM sensitized HCC cells to CD8+NKG2D+ cells-mediated oncolysis. In HCC-bearing mice, FM at a non-toxic dose failed to reduce tumor growth in immune compromised mice, whereas it significantly inhibited tumor growth and prolonged life span in immune competent mice. While the number of IFN-γ-producing cells within TME was increased in mice treated with FM, the infiltration of CD8+ T cells and NK cells was not increased. Finally, we identified that depletion of CD8+ T cells rather than NK cells abrogated the antitumor activity of FM.

Conclusions: Our results show for the first time that CD8+ T cells mediate the antitumor activity of FM at a non-toxic dose. This may provide new insights to this ancient mysterious prescription in cancer therapy, which offers a novel and practical therapeutic strategy and the possibilities of combined immunotherapy for HCC as well as other inflammation-related cancers in clinic.

Keywords: Antitumor immunity; Cancer-related inflammation; Frankincense; Hepatocellular carcinoma; Myrrh; NF-κB/STAT3 signaling.

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Figures

Fig. 1
Fig. 1
FM reduces the activation of NF-κB and STAT3 in HCC cells. a Human HCCLM3 or mouse Hepa1-6 HCC cells were seeded in 96-well plates overnight. Then cells were treated with serial concentrations of FM. 24 and 48 h later, cells were subjected to MTT assay. b–e 2 × 105 HCCLM3 or Hepa1-6 cells were seeded into 12-well plates. Cells were treated with FM at indicated concentrations for 24 h and then stimulated with recombinant TNF-α (human, 15 ng/μl; mouse, 80 ng/μl) or recombinant IL-6 (both at 25 ng/μl). Total protein or mRNA was extracted and subjected to western blot or qRT-PCR analysis. b The protein level of total/phosphorylated IκBα, c mRNA level of TNF-α and IL-6, and d protein level of total/phosphorylated STAT3 after TNF-α stimulation are shown. GAPDH was used as loading control. e Phosphorylation of STAT3 after IL-6 stimulation is shown. The results of qRT-PCR are shown as Mean ± SD. Similar results were obtained in three independent experiments. #p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001
Fig. 2
Fig. 2
FM mitigates CD8+NKG2D+ cell-activated inflammatory signaling and enhances the oncolysis of CD8+NKG2D+ cells. a CD8+NKG2D+ cells were generated as described in methods. Cells were identified by flow cytometry with anti-CD3, anti-CD8, anti-NKG2D, anti-CD27 and anti-CD57 antibodies. b–d HCCLM3 cells stably expressing luciferase (HCCLM3-luciferase) (_target cells, T) were pre-treated with FM at a non-toxic dose of 0.5 mg/ml for 12 h or left untreated, then medium was discarded. CD8+NKG2D+ cells (effector cells, E) were added at a ratio of 5:1 (E: T) in fresh medium for 12 h. Then, cells were harvested for western blot, qRT-PCR analysis, or luciferase activity measurement. b The protein level of IκBα, phosphorylated IκBα, STAT3 and phosphorylated STAT3, c mRNA level of TNF-α (left panel) and IL-6 (right panel), and d luciferase activity reflecting the cell viability are shown. GAPDH was used as loading control. Similar results were obtained in two independent experiments. Mean ± SD of each group are shown. ***p < 0.001, ****p < 0.0001
Fig. 3
Fig. 3
An intact immune system is indispensable to the antitumor efficacy of FM. 4–6-week-old male (a) Balb/c nude mice received subcutaneous injection of 1 × 107 HCCLM3 cells, or (d) C57BL/6 mice received 5 × 106 Hepa1-6 cells in the right flank. When tumor size reached about 4–6 mm in diameter, mice were randomly divided into two groups. Then the mice were either treated daily with oral gavage of FM at a dose of 60 mg/kg, or left untreated as a control (n = 8 in each group). Tumor volumes (a, d) were measured by caliper and body weight (b, e) were monitored every 2–3 days during the treatment. Mean ± SD of each group are shown. Mice were sacrificed when tumor volume reached 2 cm3, or when mice appeared moribund. c, f Survival was determined and plotted for Kaplan–Meier survival analysis and analyzed by log-rank test. #p > 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001
Fig. 4
Fig. 4
FM increases amount of antitumor cytokine IFN-γ within TME. C57BL/6 mice bearing subcutaneous HCC received oral gavage of FM (60 mg/kg/day, n = 4) or left untreated (n = 4) 0.18 days later, tumors were dissected. Single cell suspensions were obtained from tumor tissue and cells were counted after trypan blue staining using CountStar. a Tumor volumes and body weight were monitored every 3 days. Mean ± SD of each group are shown. b Immune activity in the TME was detected by the mouse IFN-γ ELISpot assay kit (left panel) and means of immune activity in each group are shown (right panel). c Single cell suspensions were stained with anti-CD8-PerCp or anti-NK1.1-FITC antibodies and then subjected to flow cytometry to determine the percentage of CD8+ T cells (upper panel) or NK cells (lower panel). Representative flow plots (left panel) and mean ± SD of each group (right panel) are shown. #p > 0.05, ***p < 0.001
Fig. 5
Fig. 5
CD8+ T cells mediate antitumor activity of FM in vivo. 4- to 6-week-old male C57BL/6 mice received subcutaneous injection of 5 × 106 Hepa1-6 cells. When tumor volumes reached about 4–6 mm in diameter, mice were randomized to four groups. Anti-CD8 or anti-NK neutralizing antibodies were injected intraperitoneally (500 μg/mouse) 1 day prior to and 13 days after FM treatment (60 mg/kg/day, n = 8). Mice in other two groups received oral gavage of FM alone (60 mg/kg/day, n = 8), or left untreated (n = 8). a The scheme depicts the schedules of the interventions. b Peripheral blood was obtained from each mouse, stained with anti-CD8-PerCp or anti-NK1.1-PerCp antibodies and then subjected to flow cytometry to monitor the depletion of CD8 or NK cells 1 and 13 days after first injection of neutralizing antibodies. c–e Tumor volume and body weight were monitored every 3 days during the treatment. c Tumor volume of each mouse in each group is shown at each monitoring time point. d An overview of tumor growth e and body weight are also shown. Mean ± SD of each group are shown. #p > 0.05, *p < 0.05, **p < 0.01, ****p < 0.0001
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