Figure 1.
Wogonin induced calreticulin (CRT) translocation to cell surface membrane is dependent on the PERK and PI3K/AKT.
Gastric carcinoma cell line MFC cells were transfected with scramble siRNA (Ctrl, 200 nM) or PERK siRNA (200 nM), after 48 hours, expression level of PERK was detected by Western blots to verify PERK level after siRNA treatment. Successfully PERK knockdown cells and their control cells (Ctrl siRNA treated cells) were treated with wogonin (100 µM) for 2 and 4 hours. Cell surface proteins in the plasma membrane fraction were than biotinylated and tested for CRT, EGFR and actin. Total cell lysate were also obtained to test CRT, PERK and actin (A). MFC cells were pretreated with AKT specific inhibitor X (AKTi 100 nM) or PI3K/AKT inhibitor LY294002 (100 nM) for 2 hours, followed by wogonin (100 µM) treated for 2 and 4 hours, CRT, EGFR and actin in the plasma membrane protein fraction were detected by Western blots. CRT, p-AKT (ser 473), AKT1/2 and actin in total cell lysate were also detected by Western blots (B).CRT translocation to cell surface after wogonin treatment was also confirmed by confocal immune-fluorescence microscopy, the translocation was inhibited by Akt inhibitor X (AKTi 100 nM), doxorubicin (Dox, 1 µM, 2 hrs treatment) was used here as positive controls. (C). WT and AKT1/2 double knockout MEFs were treated with wogonin (100 µM) for 4 hours; CRT, EGFR and actin in the plasma membrane protein fraction were detected by Western blots. P-S6 (S235/236), p-AKT (S473), AKT1/2, CRT and actin in whole cell lysate were detected by Western blots(D). Experiments in this figure were repeated at least 3 times and similar results were obtained. Bar = 10 µm.
Figure 2.
Wogonin induces ROS dependent ER stress response, severing as upstream signal for the activation of AKT.
Gastric carcinoma cell line MFC cells were treated with wogonin (100 µM) for indicated time points, AKT and downstream mTORC1 activation were detected by western blots using indicated antibodies, AKT1/2, S6K, S6 and β-actin were also tested as equal loadings (A). AKT phosphorylation was quantified using Image J software after normalized to AKT1/2 (B). Note that Wogonin induced an early activation but later inhibition of AKT. MFC cells were treated with wogonin (100 µM) for indicated time points, p-PERK (Thr980), p-eIF2-α (Ser51), p-PKR (Thr446/451), their corresponding non-phospho proteins and AKT1/2 were detected by Western blots (C). MFC cells were transfected with PERK siRNA for 48 hours, successfully transfected cells (confirmed by western blot) were treated with wogonin (100 µM) for 1 hour, p-PERK (Thr980), p-eIF2-α (Ser51), p-AKT (Ser 473), p-4E-BP1 (Ser 65), PERK, their corresponding non-phospho proteins and β-actin were detected by Western blots(D), AKT phosphorylation was quantified using Image J software after normalized to AKT1/2 (E). MFC Cells were pre-treated with anti-oxidant N-Acetyl-Cysteine (NAC, 400 µM) for 2 hour, followed by wogonin (100 µM) treatment for 30 minutes, ROS production were detected by both fluorescence (F) and FACS (G) assay respectively as described above. MFC Cells were pre-treated with anti-oxidant N-Acetyl-Cysteine (NAC, 400 µM) for 2 hour, followed by wogonin (100 µM) for 30 and 60 minutes, p-PERK (Thr980), p-PKR (Thr446/451), p-4E-BP1 (Ser 65), p-AKT (Ser 473) and their corresponding non-phospho proteins were detected by Western blots(H). Experiments in this figure were repeated at least 3 times and similar results were obtained. #P<0.05 vs. Control siRNA group. *P<0.05 vs. group without NAC pre-treatment. Bar = 10 µm.
Figure 3.
DNA-PKcs, forms a complex with PERK, mediates AKT activation by Wogonin.
Gastric carcinoma cell line MFC Cells were pre-treated with a DNA-PKcs inhibitor (Nu 7062, 10 µM) for 2 hour, followed by wogonin (100 µM) treatment for 1 and 2 hours, AKT phosphorylation were detected by Western blot and DNA-PKcs and AKT1/2 were detected as equal loadings (A). MCF-7 cell were transfected with scramble (Ctrl, 200 nM) or DNA-PKcs (200 nM) for 48 hours by using transfection methods motioned above, successful transfected cells were used for testing AKT signaling in wogonin treated MFC cells (B). MFC Cells were pre-treated with a DNA-PKcs inhibitor (Nu 7062, 10 µM) for 2 hour, followed by wogonin (100 µM) treatment for 1 h, the precleared 600-µg aliquots of cell lysates were incubated with anti-DNA-PKcs, followed by Western blotting analysis with anti-DNA-PKcs, AKT, PERK, Ku80, IgG and β-actin respectively(C). Experiments in this figure were repeated at least 3 times and similar results were obtained. *P<0.05 vs. group without Nu7062, *P<0.05 vs. group without DNA-PKcs knockdown.
Figure 4.
Indentify p22 and Annexin A1 as possible targets of Wogonin.
Gastric carcinoma cell line MKN-45 cells were either left untreated or treated with wogonin 50, 100, 200 µM, respectively (A), Two-dimensional gel electrophoresis was performed and proteins were identified by mass spectrometry. Note a obvious induction of p22, Annexin A1 and HMGB1 after 24 hours, expression level of those proteins was quantified (B). MFC cells were treated with wogonin (100 µM) for 1, 2 and 4 hours, followed by immunoblot detection of CRT, p22, Annexin 1, EGFR and actin in the plasma membrane protein fraction and in whole cell lysate (C), pre-cleared 600-µg aliquots of cell lysates for the 4 hour time point were incubated with anti-CRT, followed by Western blotting analysis with anti-CRT, p22, Annexin A1 and IgG respectively (D). MFC cells transfected with control or p22 siRNA for 48 hours, p22 expression in whole cell lysate were detected by Western blot. Successful p22 knockdown cells were then treated with wogonin (100 µM) for 2 hours, followed by immunoblot detection of CRT, p22, Annexin A1, EGFR and actin in the plasma membrane protein fraction, p22, Annexin A1 and actin in cell lysate were also tested (E). The effect of p22 siRNA on CRT translocation was also confirmed by confocal immune-fluorescence microscopy assay(F). MCFs cells transfected with control or PERK siRNA (200 nM, for 48 hours) were treated with wogonin (100 µM) for 2 and 4 hours, followed by immunoblot detection of p22 and Annexin A1 in the plasma membrane protein fraction and in total cell lysate (G). WT and AKT1/2 double knockout MEFs were treated with wogonin (100 µM) for 4 hours, p22 and Annexin A1 in both plasma membrane protein fraction and total cell lysate were tested (H). MFC cells were pretreated with AKT specific inhibitor X (AKTi 100 nM) or PI3K/AKT inhibitor LY294002 (100 nM) for 2 hours, followed by wogonin (100 µM) treated for 2 and 4 hours, p22 and Annexin A1 in both plasma membrane protein fraction and total cell lysate were tested (I). *P<0.05 vs. group without wogonin treatment.
Figure 5.
Wogonin induces the release of HMGB1 and ATP and activation of dendritic cells (DCs).
Gastric carcinoma cell line MFC cells were treated with indicated concentration of wogonin (0, 50, 100 and 200 µM) for 36 hours, medium were collected and HMGB1 release (A) was detected by the ELISA assay and eATP was measured by Molecular Probes' ATP Determination Kit (B). Medium of MFC cells with or without 24 hours of wogonin (100 µM) treatment was then transferred to MoDCs, its signaling activation (PI3K/AKT/mTOR/MAPK/NF-kB) were detected by Western blots using indicated antibodies(C), TNF-α and IL-6 release from MoDCs were detected by ELISA assay 24 hours after indicated medium treatment (D and E). To detect the changes of phagocytic activities by treatment with wogonin, MoDCs were incubated with MFC cells in 0.1% DMSO or wogonin with 50 µM, 100 µM, 200 µM wogonin, respectively (Fa, b, c, d and G). Experiments in this figure were repeated at least 3 times and similar results were obtained. *P<0.05 and **P<0.01 vs. group without wogonin treatment.
Figure 6.
In vivo anti-tumor cell vaccination experiment.
3×106 MFC cells, suspended in 200 ml PBS, either left untreaed or treated with wogonin (100 µM) for 4 h. Wogonin treated MFC cells were inoculated subcutaneously into the lower flank of 615 mice, whereas 5×105 untreated control cells were inoculated into the contralateral flank 7 days later. Two weeks later, the tumor free mice were calculated (A) and the numbers of lymphocytes, leucocytes and monocytes in the blood were measured by flow cytometry (B). Experiments in this figure were repeated at least 3 times and similar results were obtained. n = 20. *P<0.05 vs. group without wogonin treatment. Hypothetical scheme of the signaling pathway for wogonin-induced immunity enhancement of cancer cell vaccine. Wogonin induces ROS production, which elicits ER stress response, the phosphorylation of ER stress proteins PERK and/or PKR activates downstream signal AKT in a DNA-PKcs dependent manner, mediates CRT/Annexin A1/p22 translocation and/or HMGB1/ATP release, acting as an “eat me” signaling and conferring immunogenicity(C).