Fig 1.
Apigenin prevents Ikaros downregulation, in vitro.
(A) Western blot analysis of Ikaros in naïve splenocytes co-cultured in the absence or presence of Panc02 cells, treated with apigenin (API) and/or MG132 for four hours at 10μM and 20μM, in vitro. To control for equal protein loading the blot was reprobed with an antibody specific to GAPDH. (B) Representative quantification of normalized densitometric ratios of western blot data is shown. Graph represented is the mean ± S.E.M. of three independent experiments. Lanes 1–6 vs. Lane 1; Lane 7 vs. Lane 1; Lanes 8–12 vs. Lane 7 *p<0.05, **p<0.005; ***p<0.0001(by two-tailed Student’s t test).
Fig 2.
Apigenin increases survival and reduces tumor burden in TB mice, in vivo.
(A) Kaplan-Meier survival curve show that Control (CTRL) (n = 6), TB-API mice (n = 10) and TB mice (n = 10) per group. Survival curve graph represents at least three independent experiments. (B) Tumor weights of TB and TB-API mice on the last day of the study. (C) Body weights of CTRL, TB and TB-API mice on the last day of the study. (D) Spleen weights of CTRL, TB, and TB-API mice on the last day of the study. Graphs represented are the mean ± S.E.M of CTRL (n = 3), TB (n = 3) and TB-API (n = 3) mice of three independent experiments. *p<0.05 (by two-tailed Student’s t test).
Fig 3.
Apigenin partially stabilizes Ikaros expression, in vivo.
(A) Western blot analysis of Ikaros protein expression in splenocytes from Control (CTRL), TB and TB-API mice. To control for equal protein loading, the blot was reprobed with an antibody specific to β-actin. The arrows on the left indicate observed Ikaros isoforms. (B) Representative quantification of normalized densitometric ratios of western blot data is shown. Graph represented is the mean ± S.E.M. of CTRL (n = 3), TB (n = 3) and TB-API (n = 3) mice of three independent experiments. *p<0.05, (by two-tailed Student’s t test).
Fig 4.
Apigenin inhibits CK2 activity and improves PP1 expression, in vivo.
(A) Western blot analysis of CK2α protein expression in Control (CTRL), TB and TB-API splenocytes. (B) Counts per minute (C.P.M.) of CK2 activity in protein lysates from splenocytes from CTRL, TB and TB-API mice as assayed by an in vitro CK2 kinase assay (C) Western blot analysis of PP1 protein expression in CTRL, TB and TB-API splenocytes. (D) and (E) representative quantification of normalized densitometric ratios of western blots data of CK2 and PP1, respectively. To control for equal protein loading, the blots were reprobed with an antibody specific to β-actin. Graphs represented are the mean ± S.E.M. of CTRL (n = 3), TB (n = 3) and TB-API (n = 3) mice of three independent experiments. *p<0.05; (by two-tailed Student’s t test).
Fig 5.
Apigenin partially restores T cell homeostasis and immune responses, in vivo.
Flow cytometry analysis of (A) CD4+ T cells, (B) CD8+ T cells and (C) CD4+CD25+ Treg percentages in splenocytes from Control (CTRL), TB and TB-API mice. (D) Allogeneic CD8+ T cell proliferation responses of CFSE-labeled BALB/c splenocytes (responders) in response to CTRL, TB and TB-API splenocytes (stimulators) in a one-way mixed- leukocyte reaction (MLR), as analyzed by flow cytometry. (E) Flow cytometry analysis of IFN-γ production of CD8+ T cells in splenocytes from CTRL, TB and TB-API mice. Graphs represented are the mean ± S.E.M. of CTRL (n = 3), TB (n = 3) and TB-API (n = 3) mice of three independent experiments. *p<0.05; **p<0.005; ***p<0.0001(by two-tailed Student’s t test).
Fig 6.
Proposed model. Ikaros’ regulation by CK2.
(A) We propose that CK2 hyper-phosphorylates Ikaros, which facilitates its polyubiquitination and eventual protein degradation by the ubiquitin-proteasome system in PC. (B) We propose that API inhibits CK2 activity, stabilizing Ikaros expression by increasing PP1 activity and resulting in T cell homeostasis denoted by the broken arrows. We also propose that MG132 in vitro acts downstream by directly inhibiting that activity of the ubiquitin-proteasome system, preventing proteasomal degradation of Ikaros and thereby stabilizing its expression. Overall, our results suggest that in vivo API treatment increases T cell homeostasis and thus improves anti-tumor immune responses in PC microenvironment.