Korean J Physiol Pharmacol.  2016 May;20(3):261-268. 10.4196/kjpp.2016.20.3.261.

Immunotherapy with methyl gallate, an inhibitor of Treg cell migration, enhances the anti-cancer effect of cisplatin therapy

• Kim H
• Lee G
• Sohn SH
• Lee C
• Kwak JW
• Bae H

• KMID: 2285598
• DOI: http://doi.org/10.4196/kjpp.2016.20.3.261

Abstract

Foxp3+ CD25+CD4+ regulatory T (Treg) cells are crucial for the maintenance of immunological self-tolerance and are abundant in tumors. Most of these cells are chemo-attracted to tumor tissues and suppress anti-tumor responses inside the tumor. Currently, several cancer immunotherapies targeting Treg cells are being clinically tested. Cisplatin is one of the most potent chemotherapy drugs widely used for cancer treatment. While cisplatin is a powerful drug for the treatment of multiple cancers, there are obstacles that limit its use, such as renal dysfunction and the development of cisplatin-resistant cancer cells after its use. To minimize these barriers, combinatorial therapies of cisplatin with other drugs have been developed and have proven to be more effective to treat cancer. In the present study, we evaluated the eff ect of the combination therapy using methyl gallate with cisplatin in EL4 murine lymphoma bearing C57BL/6 mice. The combinatorial therapy of methyl gallate and cisplatin showed stronger anti-cancer eff ects than methyl gallate or cisplatin as single treatments. In Treg cell-depleted mice, however, the eff ect of methyl gallate vanished. It was found that methyl gallate treatment inhibited Treg cell migration into the tumor regardless of cisplatin treatment. Additionally, in both the normal and cisplatin-treated tumor-bearing mice, there was no renal toxicity attributed to methyl gallate treatment. These findings suggest that methyl gallate treatment could be useful as an adjuvant method accompanied with cisplatin therapy.

Keyword

Cisplatin; EL4 lymphoma; Immunotherapy; Methyl gallate; Regulatory T cell

MeSH Terms

Animals
Cisplatin*
Drug Therapy
Immunotherapy*
Lymphoma
Mice
T-Lymphocytes, Regulatory*
Cisplatin

Figure

• 

  Fig. 1 Methyl gallate treatment enhances the anti-tumor effect of cisplatin therapy.All mice received injections of EL4 cells (5×104/0.1 ml) s.c. into the right flank. Methyl gallate (20 mg/kg) was injected i.p. every other day for 14 days from day 5 of tumor inoculation (n=10). Cisplatin (1 mg/kg) was injected i.p. 3 times (on days 5, 11 and 17). All mice were sacrificed at day 21, and the tumor was fully separated from the mice and weighed. The results represent the means±SEM. *p<0.05, **p<0.01, ***p<0.001 vs. each respective saline-treated control. #p<0.05 vs. indicated control. MG, methyl gallate; Cis, cisplatin; MG+Cis, methyl gallate+cisplatin.

• 

  Fig. 2 Methyl gallate treatment inhibits the migration of Treg cells into tumors.Foxp3EGFPC57BL/6 Treg cells were detected in tumors using EGFP signal detected with confocal microscope imaging. The results are presented as the mean±SEM. *p<0.05, **p<0.01 vs. each respective saline-treated control. #p<0.05 vs. indicated control. NS, not significant; MG, methyl gallate; Cis, cisplatin; MG+Cis, methyl gallate+cisplatin.

• 

  Fig. 3 The anti-cancer effect of methyl gallate depends on Treg cells.All mice received injections of EL4 cells (5×104/0.1 ml) s.c. into the right flank. Methyl gallate (20 mg/kg) was injected every other day for 14 days from day 5 of tumor inoculation (n=10). Cisplatin (1 mg/kg) was injected 3 times (on days 5, 11 and 17). All mice were sacrificed at day 21, and the tumor was fully separated from the mice. To deplete Treg cells, all group of mice was injected anti-CD25 antibody. The results are presented by the mean±SEM. NS, not significant; MG, methyl gallate; Cis, cisplatin; MG+Cis, methyl gallate+cisplatin.

• 

  Fig. 4 Methyl gallate has no effect on the cell cycle and the viability of EL4 cells.EL4 cells were treated methyl gallate or cisplatin for 24 h. (A) The cell cycle was measured by flow cytometry. (B) Cell viability was measured using the MTS. Value was normalized against PBS control (pentaplicate for each concentration). The data was calculated with mean values obtained from three independent experiments and presented as the mean±SEM.**p<0.01, ***p<0.001 vs. each respective PBS-treated control. NS, not significant; MG, methyl gallate; Cis, cisplatin.

• 

  Fig. 5 Methyl gallate has no effect on immune cell proportions in the spleen.Splenocytes were prepared from Foxp3EGFP mice injected with methyl gallate (20 mg/kg, every day for 5 days). The cells were stained with anti-CD3, anti-CD4, and anti-CD19 antibodies for 30 minutes on ice. The results are presented as the mean±SEM. NS, not significant vs. each respective saline-treated control. MG, methyl gallate.

• 

  Fig. 6 Methyl gallate does not cause renal toxicity.(A, B) The serum levels of creatinine and BUN were measured in the normal mice injected with methyl gallate (20 mg/kg) for 5 days everyday (n=5). (C, D) The mice received injections of EL4 cells (5×104/0.1 ml) s.c. into the right flank. Methyl gallate (20 mg/kg) was injected every other day for 14 days from day 5 of the tumor inoculation (n=10). Cisplatin (1 mg/kg) was injected 3 times (on days 5, 11 and 17). The level of creatinine and BUN were measured in the serum. The results are presented as the mean±SEM. *p<0.05, ***p<0.001 vs. each respective saline-treated control. NS, not significant; MG, methyl gallate; Cis, cisplatin; MG+Cis, methyl gallate+cisplatin.

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