Cimetidine and Ibuprofen Modulate T Cell Responses in a Mouse Model of Breast Cancer

Abstract

Cimetidine and ibuprofen exhibit immunomodulatory effects as an antagonist of histamine H2 receptor, and a cyclooxygenase inhibitor, respectively. Here, the effects of cimetidine and ibuprofen on some effector T cell-related parameters were investigated using a breast cancer (BC) model. BC was established in Balb/c mice using the 4T1 cell line. On day 10 after tumor induction, the BC-bearing mice were classified into four groups and treated with PBS, cimetidine (20 mg/kg), ibuprofen (20 mg/kg) or a combination of "cimetidine + ibuprofen" via intraperitoneal injection (daily from days 11 to 30). The mice were sacrificed on day 31 and the frequency of splenic Th1 and Treg cells, plasma IFN-γ and TGF-β levels, and intra-tumoral T-bet, GATA3, FOXP3 and RORγt expressions were detected using flowcytometry, ELISA and real-time-PCR, respectively. In untreated cancerous mice, the percentage of splenic Th1 cells and plasma IFN-γ levels were lower (P<0.003 and P<0.01, respectively), whereas the percentage of splenic Treg cells and plasma TGF-β levels were higher than in healthy mice (P<0.04 and P<0.005, respectively). Treatment of BC-bearing mice with cimetidine, ibuprofen or both drugs promoted the frequency of Th1 cells (P<0.05, P<0.007 and P<0.005, respectively) as well as IFN-γ levels (P<0.004, P<0.0001 and P<0.03, respectively), while reduced the frequencies of Treg cells (P<0.02, P<0.03 and P<0.01, respectively), TGF-β levels (P<0.006, P<0.02 and P<0.002, respectively), intra-tumoral expression of FOXP3 (P<0.006, P<0.005 and P<0.005, respectively), and intra-tumoral expression of RORγt (P<0.04, P<0.03 and P<0.05, respectively) compared with untreated BC mice. The "cimetidine + ibuprofen"-treated mice displayed greater T-bet expression than the un-treated mice (P<0.006). Cimetidine and/or ibuprofen-treated BC-bearing mice exhibited reduced intra-tumoral expression of GATA3 compared with the untreated BC mice, but the differences were not significant. Cimetidine and ibuprofen correct some effector T cell-related parameters in cancerous mice. Immunotherapeutic potentials cimetidine and ibuprofen in cancers need investigations.

Keywords: Cimetidine; Mice; T cells; breast cancer; ibuprofen.

Figure 1

Effects of Treatment with Cimetidine and Ibuprofen in Mice with Breast Cancer. The percentage increase in body weight in un-treated cancer bearing mice was lower than healthy mice and the treatment of cancer bearing mice with dose 20 mg/kg of cimetidine or/and ibuprofen enhanced the percentage increase in body weight as compared with un-treated cancerous mice

Figure 2

Effects of Treatment with Cimetidine and Ibuprofen on Tumor Sizes in Mice with Breast Cancer. The tumor sizes in cancerous mice treated with dose 20 mg/kg cimetidine and/or ibuprofen on days 16, 19, 22, 25, 28 and 31 after tumor induction were significantly lower than in non-treated cancer bearing mice

Figure 3

Effects of Treatment with Cimetidine and Ibuprofen on Tumor Weights in Mice with Breast Cancer. The tumor weights were reduced in breast cancer bearing mice treated with dose 20 mg/kg of cimetidine or/and ibuprofen as compared with un-treated cancerous mice

Figure 4

Effects of Treatment with Cimetidine and Ibuprofen on Survival Rate in Mice with Breast Cancer. The survival rate was enhanced in breast cancer bearing mice treated with dose 20 mg/kg of cimetidine or/and ibuprofen as compared with that in un-treated cancerous mice

Figure 5

Effects of Treatment with Cimetidine and Ibuprofen on Spleen Index in Mice with Breast Cancer. The spleen index was greater in un-treated cancer bearing mice compared with healthy group and the treatment of breast cancer bearing mice with dose 20 mg/kg of cimetidine or/and ibuprofen reduced the spleen index compared with that in un-treated cancerous mice

Figure 6

Effects of Treatment with Cimetidine and Ibuprofen on the Percentage of Splenic Th1 Cells in mice with Breast Cancer. The percentage of the splenic Th1 cells was reduced in untreated cancer bearing mice compared with healthy group and treatment of breast cancer bearing mice with dose 20 mg/kg of cimetidine or/and ibuprofen increased the percentage of the splenic Th1 cells compared with that in untreated cancerous mice

Figure 7

Effects of Treatment with Cimetidine and Ibuprofen on the Percentage of Splenic Treg Cells in Mice with Breast Cancer. The percentage of the splenic Treg cells was increased in untreated cancer bearing mice compared with healthy group. Treatment of breast cancer bearing mice with dose 20 mg/kg of cimetidine or/and ibuprofen reduced the percentage of the splenic Treg cells compared with that in untreated cancerous mice

Figure 8

Effects of Treatment with Cimetidine and Ibuprofen on the Plasma Levels of IFN-γ and TGF-β in Mice with Breast Cancer. The plasma IFN-γ levels were reduced, while the plasma TGF-β levels were increased in untreated cancer bearing mice compared with the healthy group. Treatment of breast cancer bearing mice with dose 20 mg/kg of cimetidine or/and ibuprofen increased the plasma IFN-γ levels, whereas reduced the TGF-β levels compared with those in untreated cancerous mice

Figure 9

Effects of Treatment with Cimetidine and Ibuprofen on the Intra-Tumoral T-bet, FOXP3, GATA3 and RORgt Expression in mice with Breast Cancer. Treatment of breast cancer-bearing mice with dose 20 mg/kg of cimetidine plus ibuprofen significantly increased the T-bet expression compared with untreated cancerous mice. Treatment of cancerous mice with dose 20 mg/kg of cimetidine or/and ibuprofen significantly reduced the FOXP3 and RORgt expression compared with that in untreated cancerous mice. Treatment of cancerous mice with dose 20 mg/kg of cimetidine or/and ibuprofen also reduced the GATA3 expression compared with that in untreated cancerous mice, but differences were not significant

Figure 10

Hematoxylin and Eosin Staining of Lung Sections. 10A shows lung pattern from normal mice. 10B shows the lung pattern from untreated breast cancer mouse. The alveolar space is reduced, while the thickness of alveoli wall was increased due to metastases formation. 10C, 10D and 10E indicate the lung patterns form ibuprofen-, cimetidine-, and “ibuprofen + cimetidine”-breast cancer treated mice, respectively. As indicated the alveolar spaces are increased, while the thicknesses of alveoli walls were reduced in treated breast cancer mice compared to untreated breast cancer mouse. The star and arrow symbols indicate the alveolar space and the thickness of alveoli wall, respectively