Effects of anticancer agents on cell viability, proliferative activity and cytokine production of peripheral blood mononuclear cells

Abstract

We investigated the effects of anticancer agents on peripheral blood mononuclear cells for the purpose of providing data to support new translational chemoimmunotherapy regimens. Peripheral-blood mononuclear cells were treated with one of four anticancer agents (5-fluorouracil, irinotecan, cisplatin, and gemcitabine) for 2 h, after which cell viability was determined. For assessment of effects of each drug on proliferation and cytokine production, cells were stimulated with phytohemagglutinin for 48 h. As a result, the anticancer agents did not affect cell viability. Cell proliferation was unaffected by 5-fluorouracil and irinotecan but inhibited by cisplatin and gemcitabine. Treatment with gemcitabine enhanced the production of IFN-γ and decreased the number of regulatory T cells. gemcitabine treatment increased IFN-γ production among CD4 T cells but not among CD8 T cells. The results indicated that GEM had immunoregulatory properties that might support immune response against cancer. This finding has implications for designing chemoimmunotherapy strategies.

Keywords: 5-fluorouracil (5-FU); chemoimmunotherapy; cisplatin (CDDP); gemcitabine (GEM); irinotecan (CPT-11).

Fig. 1

Effects of anticancer agents on cell viability of PBMCs. PBMCs and incremental concentrations of each anticancer agent were incubated at 37°C for 2 h. After stimulation of anticancer agents, cell viability of PBMCs was determined by WST-8 assay. Data are expressed as the mean ± standard error of the mean (SEM).

Fig. 2

Effects of anticancer agents on proliferative activity of PBMCs. After stimulation with incremental concentrations of anticancer agents for 2 h, PBMCs were then stimulated with PHA (5 µg/ml) for 48 h. Cells were pulsed with bromodeoxyuridine (BrdU) solution and measured using a microplate reader. Data are expressed as the mean ± SEM. *p<0.05, **p<0.0001, vs PHA(+), no anticancer agent.

Fig. 3

Effects of 5-FU on PHA-induced cytokine production. After 2 h chemical stimulation with 5-FU, PBMCs were then incubated with PHA for 48 h. Cell culture supernatant was assayed for TNF-α(a), IL-2(b), IL-4(c), IL-10(d) and IFN-γ(e) by using a cytokine-specific solid phase sandwich ELISA. In all panels, data are expressed as the mean ± SEM. *p<0.05, vs PHA(+), no anticancer agent.

Fig. 4

Effects of CDDP on PHA-induced cytokine production. After 2 h chemical stimulation with CDDP, PBMCs were then incubated with PHA for 48 h. Cell culture supernatant was assayed for TNF-α(a), IL-2(b), IL-4(c), IL-10(d) and IFN-γ(e) by using a cytokine-specific solid phase sandwich ELISA. In all panels, data are expressed as the mean ± SEM. *p<0.05, **p<0.0001, vs PHA(+), no anticancer agent.

Fig. 5

Effects of CPT-11 on PHA-induced cytokine production. After 2 h chemical stimulation with CPT-11, PBMCs were then incubated with PHA for 48 h. Cell culture supernatant was assayed for TNF-α(a), IL-2(b), IL-4(c), IL-10(d) and IFN-γ(e) by using a cytokine-specific solid phase sandwich ELISA. In all panels, data are expressed as the mean ± SEM. *p<0.05, **p<0.0001, vs PHA(+), no anticancer agent.

Fig. 6

Effects of GEM on PHA-induced cytokine production. After 2 h chemical stimulation with GEM, PBMCs were then incubated with PHA for 48 h. Cell culture supernatant was assayed for TNF-α(a), IL-2(b), IL-4(c), IL-10(d) and IFN-γ(e) by using a cytokine-specific solid phase sandwich ELISA. In all panels, data are expressed as the mean ± SEM. *p<0.05, **p<0.0001, vs PHA(+), no anticancer agent.

Fig. 7

A representative result of CD4 and Foxp3 dual staining and flow cytometry. After 2 h stimulation with GEM, PBMCs were incubated with PHA for 48 h. CD4 and intracellular staining for Foxp3 were evaluated by flow cytometry.

Fig. 8

Effects of GEM on lymphocyte populations. After 2 h stimulation with GEM, PBMCs were incubated with PHA for 48 h. Cell types (CD4 and CD8) and intracellular staining for Foxp3 were evaluated by flow cytometry. In all panels, data are expressed as the mean ± SEM. *p<0.05, vs PHA(+), GEM(−).

Fig. 9

A representative result of CD4 and IFN-γ dual staining and flow cytometry. After 2 h stimulation with GEM, PBMCs were incubated with PHA for 48 h. CD4 and intracellular staining for IFN-γ were evaluated by flow cytometry.

Fig. 10

Effects of GEM on the frequency of IFN-γ-producing cells. After 2 h stimulation with GEM, PBMCs were incubated with PHA for 48 h. Cell types (CD4 and CD8) and intracellular staining for IFN-γ were evaluated by flow cytometry. In all panels, data are expressed as the mean ± SEM. *p<0.01, vs PHA(+), GEM(−).