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Keywords:

  • α-GalCer;
  • 5-FU;
  • NK cells;
  • liver tumor

Abstract

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. References

α-Galactosylceramide (α-GalCer) has been reported to be therapeutic against metastatic liver tumors in mice. However, little is known regarding the efficacy of combined chemo-immunotherapy using α-GalCer and anticancer drugs. In this study, we evaluated the antitumor effect of the combination therapy of α-GalCer and 5-fluorouracil (5-FU) against liver tumors of MC38 colon cancer cells. The liver weights of tumor-bearing mice treated with the combination were significantly lower than those of nontreated mice and of mice treated with 5-FU or α-GalCer alone. No toxic effects on the liver and renal functions were observed in any of the treatment groups. α-GalCer treatment induced significant activation of liver NK cells in vivo, but 5-FU treatment did not. 5-FU treatment resulted in a significant upregulation of NKG2D activating molecules (Rae-1 and H60) and DNAM-1 ligands (CD112 and CD155) on MC38 cells, but α-GalCer did not. The cytolytic activity of α-GalCer-activated liver mononuclear cells against 5-FU-treated MC38 cells was significantly higher than that against nontreated cells. The increase of the cytolytic activity induced by 5-FU partially depended on NKG2D-Rae-1 or H60 signals. Depletion of NK cells significantly inhibited the antitumor efficacy of 5-FU against MC38 liver tumors, which suggested that the antitumor effect of 5-FU partially depended on the cytolytic activity of NK cells. These results demonstrated that the combination therapy of α-GalCer and 5-FU produced synergistic antitumor effects against liver tumors by increasing the expression of NK activating molecules on cancer cells. This study suggests a promising new chemo-immunotherapy against metastatic liver cancer.

Abbreviations
5-FU

5-fluorourcil

Alb

albumin

ALT

alanine aminotransferase

Cr

creatinine

IFN-α

interferon-α

MICA

major histocompatibility complex class I-related chain A

MNCs

mononuclear cells

PBS

phosphate buffered saline

T-Bil

total bilirubin

α-GalCer

α-galactosylceramide

Colon cancer is one of the most common cancers in the world. Despite recent progress in the development of treatment, the overall 5-year survival rate is only 50–60% due to local recurrence or distant metastasis.[1] In particular, patients with metastatic colon cancer have a median survival rate of only six months. 5-Fluorouracil (5-FU) remains key-drug in chemotherapy against colon cancer. However, colon cancer cells are becoming increasingly resistant to existing chemotherapies including 5-FU.[2] Therefore, novel strategies are needed especially for the treatment of advanced colon cancers including metastatic liver cancer.

A normal liver contains abundant lymphocytes that are usually enriched with NK and NKT cells in contrast to peripheral blood.[3, 4] Thus, the effective activation of innate immune cells might be beneficial in the treatment of metastatic liver cancer. To date, however, immunotherapy has not yet been established against metastatic liver cancer. α-Galactosylceramide (α-GalCer) induces the activation of NKT cells in a CD1d-dependent manner.[5, 6] Recently, α-GalCer has been attracting attention as a novel antitumor therapy. Systemic administration of α-GalCer has demonstrated antitumor effects against various tumors (including melanoma, sarcoma, colon carcinoma, and lymphoma) in vivo in animal models of hepatic and lung metastasis.[7, 8] We and others have demonstrated that sequential activation of both NKT and NK cells could be observed in the liver after α-GalCer administration.[8-10] Although most NKT cells had disappeared from the liver within 12 hr of α-GalCer administration, strong activation and proliferation of liver NK cells could be observed, and the antitumor effect of the α-GalCer treatment against liver tumors depended primarily on NK cells. Based on the promising results of preclinical studies, several Phase 1 clinical studies using intravenous administration of α-GalCer have been conducted, but clinical responses of α-GalCer have been limited.[11] In view of future α-GalCer treatment of metastatic liver cancer, new strategies should be explored. We have previously reported that anticancer drugs enhance the expression of the human NKG2D ligand, membrane-bound major histocompatibility complex class I-related chain A (MICA), and the NK sensitivity of human hepatocellular carcinoma cells in vitro.[12, 13] These findings suggest that the efficient activation of liver innate immunity after chemotherapy might represent a promising approach to the suppression of liver tumor growth.

In this study, we investigated the therapeutic potential of the combination of α-GalCer and 5-FU in the treatment of liver tumor of colon cancer cells. We found that 5-FU can enhance the NK sensitivity of colon cancer cells by increasing the expression of NK activating molecules. In addition, the combination therapy of α-GalCer and 5-FU showed synergistic antitumor effects against liver tumor of colon cancer cells. This study demonstrates a promising new therapeutic strategy for the treatment of metastatic liver cancer.

Material and Methods

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. References

Mice

Female C57BL/6 and BALB/c mice were purchased from Charles River Laboratories Japan, INC (Yokohama, Japan) and were used at 6–10 weeks of age. The mice were housed under conditions of controlled temperature and light with free access to food and water at the Institute of Experimental Animal Science, Osaka University Graduate School of Medicine. All animals received humane care and our study protocol complied with the institution's guidelines.

Cell lines

MC38, a mouse colon cancer cell line derived from C57BL/6 mice, was generously provided by Dr. Michio Imawari (Showa University School of Medicine, Tokyo, Japan). Colon26, a mouse colon cancer cell line derived from BALB/c mice, was kindly provided by Dr. Takashi Tsuruo (Institute of Molecular and Cellular Bioscience, University of Tokyo, Tokyo, Japan). This cell line was maintained in complete medium (CM, RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin, and 10 mM l-glutamine: all reagents from GIBCO/Life Technologies, Grand Island, NY) in a humidified incubator at 5% CO2 and 37°C.

Reagents

α-GalCer was purchased from Funakoshi (Tokyo, Japan) and prepared as previously described by Kawano et al.[5] 5-FU was purchased from Kyowa Hakko Kirin (Tokyo, Japan) and dissolved in phosphate buffered saline (PBS). MC38 cell viability was determined 24 hr after the addition of 5-FU (used at 10 nmol/l to 2 μmol/l) or PBS by the WST assay using the cell count reagent SF (Nacalai Tesque, Kyoto, Japan) as previously described (10).

Flow cytometry

MC38 cells were cultured with or without α-GalCer (100 ng/ml) or 5-FU (500 nmol/l) for 24 hr and evaluated for the expression of NK activating molecules. Treated and nontreated MC38 cells were incubated with PE-conjugated antibodies (Abs) against anti-Rae-1 (R&D Systems, Minneapolis, MN), H60 (R&D Systems), CD112 (Nectin-2) (Abcam, Cambridge, UK), and CD155 (BioLegend, San Diego, CA). Flow cytometric analysis was performed using a Canto II flow cytometer (Becton Dickinson, San Jose, CA).

Preparation of hepatic mononuclear cells from 5-FU- or α-GalCer-treated mice

C57BL/6 mice were administered 5-FU (20 mg/kg body weight)or PBS intraperitoneally (i.p.) for 3 consecutive days. Liver mononuclear cells (MNCs) were prepared as previously described.[8] In some experiments, C57BL/6 mice were administered α-GalCer (0.4 μg/mouse) or PBS i.p. on Day 0. On Day 3, hepatic MNCs were prepared. NK cells were identified as DX5+/TCRβ- by flow cytometry as previously described.[8] The expression levels of NKG2D and DNAM1 were evaluated with anti-NKG2D (R&D Systems) and anti-DNAM1 (BioLegend) Abs by flow cytometry.

Cytolytic assays

C57BL/6 mice were injected i.p. with α-GalCer (2 μg/mouse) for the preparation of activated NK cells as previously described.[8] Liver MNCs were prepared on Day 3 after α-GalCer injection. MC38 cells were cultured with or without 5-FU (500 nmol/l) for 1 day. α-GalCer-activated liver MNCs were subjected to a 4-hr 51Cr release assay against 5-FU-treated or nontreated MC38 cells as previously described.[12] The assays were performed in triplicate, and the spontaneous release of all assays did not exceed 25% of the maximum release. In some experiments, the cytolytic ability of activated NK cells was assessed by a 4-hr 51Cr-release assay with or without blocking Abs against Rae-1 (R&D Systems) or H60 (R&D Systems).

Animal experiments

C57BL/6 or BALB/c mice were injected in the liver with 3 × 105 MC38 cells or 5 × 105 Colon26 cells on Day 0. To evaluate the efficacy of the combination therapy of α-GalCer and 5-FU, the mice were treated with α-GalCer (0.4 μg/mouse) on Day 0 and/or 5-FU (C57BL/6, 10 mg/kg body weight; BALB/c, 20 mg/kg body weight respectively) for 5 consecutive days after tumor inoculation. Two weeks after the tumor injection, the liver weight was measured to examine the intrahepatic tumor growth. To evaluate the involvement of NK cells in the antitumor effect of 5-FU, mice were injected with an anti-asialo GM-1 (ASGM1) Ab (WAKO, Osaka, Japan) on Days −1, 4, and 9 after tumor inoculation. The efficiency of NK cell depletion was validated by flow cytometric analysis of splenocytes using PE-conjugated anti-DX5 mAbs (BD-Pharmingen) as previously described.[8] NK-depleted mice were treated with or without 5-FU (10 mg/kg body weight) for 5 consecutive days. Two weeks after the tumor injection, the livers of treated mice were removed, and the liver weight was measured to examine the intrahepatic tumor growth.

NKG2D lignads and DNAM1 ligands expression in MC38 tumor tissues and nontumor tissues in 5-FU-treated mice

C57BL/6 mice were injected in the liver with 3 × 105 MC38 cells on Day 0 and were treated with 5-FU on Day 4–8 after tumor inoculation. On Day 8, MC38 liver tumor or nontumor tissues were harvested and divided into single cells to evaluate the expression of NKG2D ligands (Rae-1 and H60) and DNAM1 ligands (CD112 and CD155) by flow cytometry.

Blood biochemistry test

Blood samples were obtained 24 hr after treatment. The levels of serum alanine aminotransferase (ALT), total bilirubin (T-Bil), albumin (Alb), and creatinine (Cr) were measured with a standard UV method using a Hitachi type 7170 automatic analyzer (Tokyo, Japan).

Statistics

All values are expressed as the mean and SD. Statistical analyses were performed by the unpaired Mann–Whitney U test or one-way ANOVA unless otherwise indicated. When ANOVA analyses were applied, differences in the mean values among groups were examined by the Scheffe post hoc correction. We defined statistical significance as p < 0.05.

Results

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. References

The combination therapy of α-GalCer and 5-FU showed a synergistic antitumor effect against MC38 liver tumors

We examined the antitumor effect of the combination therapy of α-GalCer and 5-FU against MC38 liver tumors. C57BL/6 mice were injected intrahepatically with MC38 cells. The mice were treated with α-GalCer on Day 0 and/or 5-FU for 5 consecutive days after tumor inoculation. As shown in Figure 1a, the liver weights of the mice treated with α-GalCer plus 5-FU were significantly lower than those of nontreated mice and mice treated with either 5-FU or α-GalCer alone. The liver weights of mice treated with 5-FU were significantly lower than those of nontreated mice, but treatment with α-GalCer did not produce this effect. We also examined the antitumor effect of α-GalCer plus 5-FU in a Colon26 liver tumor model. The liver weights of mice treated with α-GalCer plus 5-FU were significantly lower than those of nontreated mice and mice treated with either 5-FU or α-GalCer alone. The liver weights of mice treated with α-GalCer were significantly lower than those of nontreated and 5-FU-treated mice (Fig. 1b). Tumor rejection in the MC38 liver tumor model was observed in 2/8 of the α-GalCer plus 5-FU-treated mice, 0/8 of the 5-FU-treated mice, 1/8 of the α-GalCer-treated mice, and 0/7 of the PBS-treated mice (Fig. 1a). These results were consistent with those of another Colon26 liver tumor model in BALB/c mice, where tumor rejection was observed in 2/8 of the α-GalCer plus 5-FU-treated mice, 0/8 of the 5-FU-treated mice, 0/9 of the α-GalCer-treated mice, and 0/8 of the PBS-treated mice (Fig. 1b). These results demonstrated that the combination therapy of α-GalCer and 5-FU produced a synergistic antitumor effect against liver tumors in both the MC38 and Colon26 models. To evaluate the safety of this combination therapy, serum levels of ALT, T-Bil, Alb, and Cr were evaluated in C57BL/6 mice immunized with α-GalCer plus 5-FU, 5-FU, α-GalCer, or PBS. There was no toxic effect upon the ALT, T-Bil, Alb, or Cr levels for any of the treatment groups (Fig. 1c). These results demonstrated that the combination therapy of α-GalCer and 5-FU is not toxic to hepatocytes and does not harm the liver or kidney.

image

Figure 1. The antitumor effect of the α-GalCer and 5-FU combination therapy against MC38 liver tumors. (a, b) C57BL/6 mice or BALB/c mice were injected in the liver with 3 × 105 MC38 cells or 5 × 105 Colon26 cells on Day 0. To evaluate the efficacy of the α-GalCer and 5-FU combination therapy, the mice were treated with α-GalCer (0.4 μg/mouse) on Day 0 and/or 5-FU (C57BL/6, 10 mg/kg body weight; BALB/c, 20 mg/kg body weight) for 5 consecutive days after tumor inoculation. Two weeks after the tumor injection, the liver weight was measured to examine intrahepatic tumor growth. N = 7–9 mice/group. Each data point represents the mean liver weight ± SD. The fraction of mice achieving tumor rejection in each treatment group is shown in parentheses. *p < 0.05 versus PBS group, #p < 0.05 versus 5-FU group, †p < 0.05 versus α-GalCer group. (c) Blood samples from treated C57BL/6 mice were obtained 1 day after the final injection of each treatment. The serum levels of ALT, T-Bil, Alb, and Cr were examined. N = 3 /group. No significant differences were observed between any of the groups.

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α-GalCer, but not 5-FU, treatment induced NK activating receptors on NK cells

We examined the expression levels of activating (NKG2D and DNAM1) receptors on liver NK cells. C57BL/6 mice were treated with α-GalCer (0.4 μg/mouse) i.p. on Day 0 or 5-FU (10 mg/kg body weight) for 3 consecutive days and liver NK cells were isolated from 5-FU- and α-GalCer-treated mice. As shown in Figure 2, the expression levels of NKG2D and DNAM1 on liver NK cells from α-GalCer-treated mice were significantly higher than those from PBS-treated mice. In contrast, the expression of NKG2D and DNAM1 on liver NK cells from 5-FU-treated mice was similar to that of PBS-treated mice. These results demonstrated that α-GalCer, but not 5-FU, could activate liver NK cells.

image

Figure 2. Expression of NKG2D and DNAM1 on liver NK cells isolated from α-GalCer- or 5-FU-treated mice. C57BL/6 mice were treated with α-GalCer (0.4 μg/mouse) i.p. on Day 0 or with 5-FU (10 mg/kg body weight) for 3 consecutive days. Liver NK cells were isolated from α-GalCer or 5-FU-treated mice, and the expression levels of NKG2D and DNAM1 were evaluated by flow cytometry. Black bold line histograms: NKG2D or DNAM1 staining of NK cells from α-GalCer or 5-FU-treated mice; dotted line histograms: NKG2D or DNAM1 staining of NK cells from PBS-treated mice; shaded/gray histograms: control staining. The data are represented as the average of the MFI obtained from 3 separate experiments. *p < 0.05 versus PBS-treated group.

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5-FU, but not α-GalCer, treatment induced NK activating molecules on colon cancer cells

We next examined the expression of the NKG2D ligands (Rae-1 and H60) and DNAM1 ligands (CD112 and CD155) on MC38 colon cancer cells treated with α-GalCer and/or 5-FU. We first examined the cytotoxicity of 5-FU on MC38 cells by the WST-8 assay. The addition of more than 1 μmol/l of 5-FU resulted in a significant decrease in the growth of MC38 cells (data not shown). On the basis of these findings, we used 500 nmol/l of 5-FU to evaluate the biological effect on MC38 cells. MC38 cells were incubated with α-GalCer (100 ng/ml) and/or 5-FU (500 nmol/l) for 24 hr and the expression levels of NK activating molecules on MC38 cells were evaluated by flow cytometry. 5-FU induced the expression of Rae-1, H60, CD112, and CD155 on MC38 cells (Fig. 3). The expression of these molecules on 5-FU-treated MC 38 cells was significantly higher than that of nontreated MC38 cells. The induction of these NK activating molecules was dose-dependent (data not shown). In contrast, α-GalCer could not induce the expression of Rae-1, H60, CD112, or CD155 on MC38 cells. Even in the MC38 cells treated with this combination of α-GalCer and 5-FU, α-GalCer failed to induce additional expression of NK activating molecules. These results demonstrated that 5-FU, but not α-GalCer, could enhance the expression of NK activating molecules on colon cancer cells.

image

Figure 3. Expression of NKG2D ligands (Rae-1 and H60) and DNAM1 ligands (CD112 and CD155) on MC38 cells treated with α-GalCer and/or 5-FU. MC38 cells were cultured with or without α-GalCer (100 ng/ml) or 5-FU (500 nmol/l) for 24 hr. The treated cells were harvested and evaluated for the expression levels of NKG2D ligands (Rae-1 and H60) and DNAM1 ligands (CD112 and CD155) on MC38 cells by flow cytometry. Upper panel: representative data. Shaded/black histograms: NKG2D or DNAM1 ligand staining of α-GalCer or 5-FU-treated MC38 cells; shaded/gray histograms, control staining. Lower panel: data are represented as the average of MFI obtained from 3 separate experiments. *p < 0.05 versus PBS group, #p < 0.05 versus α-GalCer group.

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The cytolytic activity of α-GalCer activated liver MNCs against 5-FU-treated MC38 cells

We next examined the cytolytic activity of α-GalCer-activated liver MNCs against 5-FU-treated MC38 cells. We isolated liver MNCs from normal α-GalCer injected mice and the cytolytic activity of these α-GalCer-activated liver MNCs was measured. The cytolytic activity of liver MNCs against 5-FU-treated MC38 cells was significantly higher than that against nontreated cells (Fig. 4a). The cytolytic activity against 5-FU-treated MC38 cells decreased significantly following the addition of blocking Abs against Rae-1 or H60 (Fig. 4b).

image

Figure 4. The cytolytic activity of α-GalCer-activated MNCs against 5-FU-treated MC38 cells. C57BL/6 mice were injected i.p. with α-GalCer (2 μg/mice) to activate NK cells. Liver MNCs were prepared on Day 3 after α-GalCer injection. (a) MC38 cells were cultured with or without 5-FU (500 nmol/l) for 24 hr. α-GalCer-activated liver MNCs were subjected to a 4-hr 51Cr release assay against 5-FU-treated (■) or nontreated (●) MC38 cells. (b) In some experiments, the cytolytic ability of activated NK cells was assessed by a 4-hr 51Cr-release assay with or without blocking Abs against Rae-1 or H60 at an E/T ratio of 30:1. Similar results were obtained from 3 independent experiments. *p < 0.05 versus the cytolytic activity of activated NK cells against nontreated cells, #p < 0.05 versus the cytolytic activity of activated NK cells against 5-FU-treated cells.

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5-FU treatment induced the expression of NK activating molecules in MC38 liver tumor tissues but not in MC38 nontumor tissues

We examined the induction of NKG2D ligand (Rae-1 and H60) and DNAM1 ligand (CD112 and CD155) expression in MC38 liver tumor or nontumor tissues of 5-FU-treated mice. As shown in Figure 5, the expression of Rae-1 in liver tumor tissues of 5-FU-treated mice was significantly higher than that of liver tumor and nontumor tissues of PBS-treated mice and that of nontumor tissues of 5-FU-treated mice. The expression of H60 and CD112 was similar to that of Rae-1. The expression of CD155 in liver tumor tissues of 5-FU-treated mice tended to be higher than that of PBS-treated mice, although the difference was not statistically significant. These results demonstrated that 5-FU treatment induced the expression of NK activating molecules in liver tumor tissues but not in nontumor tissues consistent with the in vitro results.

image

Figure 5. Expression of NKG2D ligands (Rae-1 and H60) and DNAM1 ligands (CD112 and CD155) on MC38 liver tumor tissues of mice treated with 5-FU. C57BL/6 mice were injected in the liver with 3 × 105 MC38 cells on Day 0 and were treated with 5-FU on Day 4–8 after tumor inoculation. On Day 8, MC38 liver tumor or nontumor tissues were harvested and divided into single cells to evaluate the expression of NKG2D ligands (Rae-1 and H60) and DNAM1 ligands (CD112 and CD155) by flow cytometry. N = 3/group. *p < 0.05 versus nontumor tissues of PBS group, #p < 0.05 versus tumor tissues of PBS group, †p < 0.05 versus nontumor tissues of 5-FU group.

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The antitumor effect of 5-FU depended on both direct cytotoxicity and the cytolytic activity of NK cells in mouse colon cancer

The above results suggested that 5-FU could enhance the NK sensitivity of MC38 cells. To confirm that NK activity played a role in the antitumor effect of 5-FU, we examined the antitumor effect of 5-FU against MC38 liver tumors in NK depleted mice. As shown in Figure 6, the liver weights of 5-FU-treated mice were significantly lower than those of vehicle-treated mice. Depletion of NK cells significantly inhibited the antitumor efficacy of 5-FU against MC38 liver tumors. These results suggested that the antitumor effect of 5-FU depended on not only on the direct cytotoxic effect of 5-FU but also on the cytolytic activity of NK cells. Therefore, NK activity plays a role in the antitumor effect of 5-FU in the liver which contains abundant NK cells.

image

Figure 6. The antitumor effect of 5-FU against MC38 liver tumors in NK-depleted mice. To evaluate the involvement of NK cells in the antitumor effect of 5-FU, mice were injected with an anti-ASGM1 Ab. NK-depleted mice were treated with or without 5-FU (10 mg/kg body weight) for 5 consecutive days. Two weeks after the tumor injection, the livers of the treated mice were removed, and the weight was measured to examine the intrahepatic tumor growth. *p < 0.05 versus PBS group, #p < 0.05 versus 5-FU group. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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Discussion

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. References

The lymphocytes in the liver are typically enriched with a higher number of NK cells than that found in the peripheral blood in a normal mouse.[3, 4] Efficient activation of the abundant NK cells in the liver might be important in antitumor defense against liver tumors. Interferon-α (IFN-α) could activate liver NK cells efficiently.[14] Bui et al.[15] reported that IFN-α reduced the expression of H60 on MCA sarcoma cells, suggesting that IFN-α treatment may reduce the NK sensitivity of cancer cells. We and others have previously demonstrated that the systemic administration of α-GalCer can lead to antitumor effects against metastatic liver tumors through the efficient activation of liver NK cells.[8, 16] Although α-GalCer has not yet been officially accepted for clinical application in cancer treatment, these previous results encouraged us to evaluate the antitumor effect of the combination of α-GalCer and 5-FU against MC38 liver tumors. In most reports, high dose (2 μg/mouse) α-GalCer was applied for the treatment of liver tumors. However, administration of these high dose resulted in liver injury.[9, 17, 18] In the present study, we used low dose (0.4 μg/mouse) α-GalCer in the combination therapy. The administration of low dose α-GalCer is enough to activate liver NK cells and did not affect the expression of NK activating molecules on MC38 cells. Importantly, the administration of this low dose α-GalCer did not cause liver injury. The antitumor effect of the combination therapy of α-GalCer and 5-FU against MC38 and Colon26 liver tumors was stronger than that of 5-FU alone or α-GalCer alone. The antitumor effect of the combination therapy of low dose (0.4 μg/mouse) α-GalCer and 5-FU was equal to that of the combination therapy of high dose (2 μg/mouse) α-GalCer and 5-FU (Aketa et al., unpublished data). Our results might offer new chemo-immunotherapy strategies, especially for those patients with advanced stages of cancer.

In this study, we demonstrated that 5-FU treatment enhanced the expression of both NKG2D ligands (Rae-1 and H60) and DNAM1 ligands (CD112 and CD155) on MC38 cells. In contrast, 5-FU treatment did not affect the activating molecules on NK cells. Both pathways involving NKG2D and DNAM1 play critical roles in the activation of NK cells and have been implicated in tumor surveillance.[19] The expression of NKG2D ligands has been associated with a good prognosis in patients with colon cancer.[20] Thus, these results suggest that the upregulation of NKG2D ligand expression might improve the prognosis of patients with colon cancer. Gasser et al.[21] previously reported that DNA-damaging agents and DNA-synthesis inhibitors including 5-FU could induce the expression of NKG2D ligands on tumor cells. We also demonstrated that 5-FU treatment could induce the expression of NK activating molecules in MC38 liver tumor tissues but not in nontumor tissues, which was consistent with the in vitro results. Our present results suggest that 5-FU treatment might have strong immune-editing potential to enhance the NK sensitivity of colon cancer cells by regulating DNAM1 and NKG2D ligands.

In this study, we demonstrated that 5-FU treatment enhanced the susceptibility of MC38 cells to the cytolytic activity of liver MNCs via the NKG2D-NKG2D ligand pathway. Because the blocking antibody of the DNAM1-DNAM1 ligand is not commercially available, we could not evaluate the involvement of this pathway. We have previously demonstrated that membrane-bound MICA, an activating molecule of NK cells, on HCC cells is essential in the NK sensitivity of HCC cells.[12, 13] The addition of both epirubicin and sorafenib enhanced the NK sensitivity of HCC cells by increasing the membrane-bound MICA.[12, 13] This finding is consistent with this study of a colon cancer model. Interestingly, the expression of death receptors, such as FAS and TRAIL receptors, on MC38 cells was significantly increased by 5-FU treatment (Aketa et al., unpublished data). This result may also explain the enhancement of the susceptibility of MC38 cells to the cytolytic activity of liver MNCs. We previously demonstrated that α-GalCer administration resulted in rapid and strong activation of liver NK cells and that the cytolytic activity of liver MNCs early after α-GalCer administration mainly depended primarily on liver NK cells and not on NKT or T cells.[8, 10] Taken together, these results suggest that the addition of 5-FU enhanced the NK sensitivity of MC38 cells by increasing the expression of Rae-1 or H60 on MC38 cells. Therefore, 5-FU treatment might be expected to enhance the susceptibility of MC38 cells to the cytolytic activity of NK cells by modifying the expression of NKG2D and DNAM1 ligands.

NK depletion decreased the antitumor effect of 5-FU against MC38 liver tumors, demonstrating that the antitumor effect of 5-FU depends on NK activity in addition to direct cytotoxicity. We also examined the antitumor effect of 5-FU against the Colon26 liver tumor model, derived from BALB/c colon cancer. The liver weights of 5-FU-treated mice were significantly lower than those of vehicle-treated mice. The depletion of NK cells also significantly inhibited the antitumor efficacy of 5-FU against Colon26 liver tumors in BALB/c mice. A significant upregulation of Rae-1, H60, CD112, and CD155 could also be observed in 5-FU-treated Colon26 cells derived from BALB/c mice (Aketa et al., unpublished data). These results were consistent with the results of C57BL/6 mice and suggest that the antitumor effect of 5-FU may always depend on NK activity in the liver. The liver contains abundant NK cells. In cancer tissues that are rich in NK cells, the combination therapy of α-GalCer and 5-FU might have a potential as a new chemo-immunotherapeutic strategy.

The liver is the most common site of metastasis of gastrointestinal cancers (i.e., colorectal, gastric, and pancreatic cancers). Thus, new therapeutic approaches of cancer immunotherapy for metastatic liver cancer need to be developed. We have shown here that 5-FU can enhance the NK sensitivity of cancer cells by inducing the expression of NK activating molecules in addition to the direct cytotoxicity of 5-FU to the cancer cells. In addition, the combination therapy of α-GalCer and 5-FU showed sufficient antitumor effects against MC38 liver tumors. These findings indicate that this new combination chemo-immunotherapy might represent a particularly promising approach for patients with metastatic liver cancer.

References

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. References