Aerobic metabolic inhibition affects cancer cell proliferation.

<p>(A) Pancreatic cancer cells were treated with various concentrations of chidamide for indicated time, proliferation inhibition was determined by MTT assay. (B) Pancreatic cancer cells were treated with Mcl-1 siRNA for 48 h, proliferation inhibition of pancreatic cancer cells was determined by MTT assay. Data are shown as the means ± SD of three independent experiments. ** <i>P</i><0.01. (C) Primary pancreatic cancer cells were treated with 50 μM chidamide or Mcl-1 siRNA for indicated time, proliferation inhibition was determined by MTT assay. Data are shown as the means ± SD of three independent experiments. ** <i>P</i><0.01. (D) 50μM chidamide treated PANC-1 cells were transfected with empty vector or Mcl-1 plasmid for indicated time, proliferation inhibition was determined by MTT assay. Data are shown as the means ± SD of three independent experiments. ** <i>P</i><0.01.</p

Chidamide promotes Mcl-1 degradation via the ubiquitin-proteasome pathway.

<p>(A) Level of Mcl-1 protein in pancreatic cancer cells treated with chidamide for 24 h, determined by Western blot analysis. β-actin served as a loading control. (B) Transcript level of Mcl-1 in pancreatic cancer cells treated with chidamide for 24 h, determined by quantitative real-time PCR. T-tests, BxPC-3: <i>P</i> = 0.3097; PANC-1: <i>P</i> = 0.1599. (C) MG132 attenuates the degradation of Mcl-1 by chidamide treatment. Western blot was performed to analyze the status of Mcl-1 and β-actin. One-way ANOVA, ** <i>P</i><0.01.</p

Chidamide inhibits aerobic metabolism through Mcl-1degradation

<p>(A) ATP production in pancreatic cancer cells treated with 50 μM chidamide or Mcl-1 siRNA for 24 h, determined by CellTiter-Glo^® assay. Data are shown as the means ± SD of three independent experiments. ** <i>P</i><0.01. (B) O₂ consumption in pancreatic cancer cells treated with 50 μM chidamide or Mcl-1 siRNA for 24 h, determined by Seahorse XF96 analyzer. Data are shown as the means ± SD of three independent experiments. * <i>P</i><0.05, ** <i>P</i><0.01. (C) Interference efficiency of Mcl-1 siRNA was verified by quantitative real-time PCR and Western blot. (D) ATP production in primary pancreatic cancer cells treated with 50 μM chidamide or Mcl-1 siRNA for 24 h, determined by CellTiter-Glo^® assay. Data are shown as the means ± SD of three independent experiments. * <i>P</i><0.05, ** <i>P</i><0.01. (E) Overexpression of Mcl-1 attenuates the ATP production inhibition by chidamide in PANC-1 cells, determined by CellTiter-Glo^® assay. Data are shown as the means ± SD of three independent experiments. * <i>P</i><0.05. (F) Overexpression of Mcl-1 in PANC-1 cells. PANC-1 cells were transfected with pCMV6-Mcl-1 vector, and subjected to Western blot analysis of Mcl-1 protein level and quantitative real-time PCR analysis of Mcl-1 mRNA level.</p

HDACi promotes Mcl-1 protein acetylation and ubiquitination.

<p>(A) Acetylated level of Mcl-1 in BxPC-3 and PANC-1 cells treated with chidamide for 24 h, determined by Western blot analysis. (B) Ubiquitinated level of Mcl-1 in BxPC-3 and PANC-1 cells treated with chidamide for 24 h, determined by Western blot analysis. (C) Ubiquitinated level of Mcl-1 in BxPC-3 cells treated with TSA for 24 h, determined by Western blot analysis. (D) Level of Mcl-1 in BxPC-3 cells treated with TSA for 24 h, determined by Western blot analysis. β-actin served as a loading control. T-tests, ** <i>P</i><0.01.</p

PAEP gene knockdown in melanoma cells.

<p>Stable PAEP 624.38-Mel shRNA transfectant and its non-targeting negative control transfectant, shControl, were established with a corresponding shRNA lentivirus. PAEP gene expressions were assayed by semi-quantitative RT-PCR (A) and Western blotting (B). PAEP protein secreted by melanoma cells was quantitated by ELISA (C).</p

Activation of T helper lymphocytes by melanoma-derived PAEP protein.

<p>(A-B) The percent of CD3⁺ T cells in unfractionated PBLs from healthy donors was assayed for by direct staining with anti-CD3-FITC followed by flow cytometric analysis. (A) Negative control, (B) Healthy donor. Flow cytometry was applied to confirm the purity of CD4⁺ T cells before (C) and after (D) MACS isolation. (E) Levels of cytokines secreted by PHA-stimulated T helper cells co-cultured with PAEP-rich 624.38-Mel shControl or PAEP-poor 624.38-Mel shPAEP supernatant were determined. Melanoma-derived PAEP significantly inhibited both IL-2 and IFN- γ secretion by T helper cells (* p<0.05, Student’s <i>t</i>-test). (F) Levels of cytokines secreted by PHA- or anti-CD3 antibody-stimulated Th1 cells co-cultured with PAEP-rich 624.38-Mel shControl or PAEP-poor 624.38-Mel shPAEP supernatant were determined. Melanoma-derived PAEP significantly inhibited both IL-2 and IFN-γ secretion by Th1 cells (* p<0.05, Student’s <i>t</i>-test) (G-H) There were no differences in number of CD4/CD8 cells before or after PHA stimulation in the presence of PAEP (1 μg/ml). (G) PBLs; (H) CD3⁺ T cells. (I-J) The number of CD69/CD44 cells before and after activation in the absence or presence of PAEP was determined by direct staining with antibodies and flow cytometry. The number of CD69 cells significantly decreased co-cultured with PAEP-rich 624.38-Mel shControl supernatant. The experiment was repeated at least three times.</p

Proliferation and Apoptosis of lymphocyte affected by melanoma-derived PAEP protein.

<p>There was a significant difference of lymphocyte proliferation between PAEP-rich 624.38-Mel shControl group and PAEP-poor 624.38-Mel shPAEP group at concentrations of 1 μg/ml PAEP (A) and 2 μg/ml PAEP (B), indicating that proliferation of PBLs was significantly inhibited by PAEP (* P < 0.05, Student’s <i>t</i>-test). The early and late phase apoptosis of PBLs co-cultured with PAEP-rich 624.38-Mel shControl supernatant (equivalent to 1 μg/ml PAEP protein) for 36 h (C) was significantly increased compared with PBLs co-cultured with shPAEP supernatant (F). Similar results were obtained with purified CD8⁺ or CD4⁺ T cells (D vs. G, E vs. H). Results are presented from one representative experiment. The same experiment was repeated at least three times.</p