Fig 2: ZQT selectively induced G-MDSC apoptosis by activating the STAT3/S100A9/Bcl-2/caspase-3 signaling pathway. (a) MDSCs from the tumor tissue of tumor-bearing mice treated with normal saline, ZQT, STAT3 inhibitor (S3I-201), or ZQT combined with S3I-201 were isolated using the MojoSort Mouse Isolation Kit. The protein expression of Ki67, STAT1, p-STAT1, STAT3, p-STAT3, S100A9, Arg-1, Bcl-2, Bax, cl-PARP, cyt c, and cl-caspase-3 in MDSCs from both normal saline and ZQT-treated mice was confirmed by Western blotting. (b) Lung tumor tissues from normal saline or ZQT-treated tumor-bearing mice were stained using S100A9, Ly6G, Ly6C, CD4, and CD8 antibodies for IHC assay (scale bar: 50 µM). (c) T-lymphocyte proliferation assay was used to show dose-dependent suppression of T cell proliferation by G-MDSCs isolated from tumor tissue of tumor-bearing mice treated with normal saline or ZQT. CD3+ T cells isolated from tumor stained with 5 µM CFSE were incubated with G-MDSCs for 72 h. Meanwhile, CD3 and CD28 antibodies were added for CD3+ T cell stimulation. Flow cytometry was used to quantify 72 h CFSE dilution. (d, e) G-MDSCs from tumor tissue of tumor-bearing mice treated with normal saline or ZQT were isolated using flow cytometry. The purity (CD11b+ Ly6G+) was >90% as assessed, and the protein expression of STAT3 and cl-caspase-3 was determined by IF (scale bar: 20 µM). n = 5. Data are expressed as the mean ± SD. n/s: nonstatistical significance. *p < 0.05; **p < 0.01; ***p < 0.001.

Fig 3: CMA3 suppresses CCA growth and induces apoptosis in the xenograft mouse model. KKU-213 cells were subcutaneously injected into both flanks of Balb/c RJ mice. CMA3 was administered at 0.5 mg/kg into the tail vein once a week for 3 times. Control mice were treated with DMSO. Mice were euthanized on day 22 of the experiment. (A) Tumor volumes were measured every 3 days and are represented as the mean ± SD. ***P<0.001. (B) The numbers of Ki-67-positive nuclei per pancytokeratin-positive areas were compared between the control and CMA3-treated groups (n=4/group, 3 images/tumor, total=12 images/group). Data are presented as the mean ± SD. ***P<0.001. (C) Representative Ki-67, pancytokeratin and a-SMA immunohistochemistry staining of the control and CMA3-treated tumors are shown. Scale bar=100 µm. (D) Cytokeratin 19, caspase-9 and Bax expression in representative areas from the control and CMA3-treated groups. Scale bar=50 µm. SD, standard deviation; CMA3, Chromomycin A3; CCA, cholangiocarcinoma; SMA, smooth muscle actin.

Fig 4: Combined treatment of Andro and TRAIL induces caspase-dependent apoptosis in 786-0 cells. (A) Cell apoptosis was determined by Annexin V-FITC after DMSO, TRAIL (50 ng/ml), and/or Andro (5 µM) treatment for 24 h. (B) Indicated protein levels in 786-0 cells treated with TRAIL (50 ng/ml) and/or Andro (5 µM) for 24 h as detected by immunoblotting. (C) Cells were treated with DMSO, TRAIL (50 ng/ml), and Andro (5 µM), pan-caspase inhibitor Z-VAD (0.05 µM), and cell-necrosis inhibitor necrostatin-1 (Nec-1) (0.05 µM). Then cell viability was determined by MTS assay (one-way ANOVA, Tukey). (D) Images (magnification, ×400) show the apoptotic cells following treatment under different conditions. Data are shown as mean ± SD; n.s. (not significant), P>0.05, ***P<0.001, n=3). Andro, andrographolide; TRAIL, tumor necrosis factor-related apoptosis-inducing ligand; PI, propidium iodide; PARP1, poly(ADP ribose) polymerase 1; Bax, Bcl-2 associated X, apoptosis regulator, DR, death receptor.

Fig 5: CMA3 induces apoptosis in CCA cells via downregulation of Sp1-related anti-apoptotic proteins. KKU-213 was treated with 40 nM CMA3 for 0-24 h. (A) The expression of Sp1-related anti-apoptotic proteins: FLIPs, cIAP2, Mcl-1, survivin and XIAP was demonstrated by western blotting. (B) The expression of Sp1 transcription factor and (C) pro-apoptotic proteins; Bax, Bim and Noxa, is revealed. Intensities of protein bands were normalized with Hsc70 and compared relative to those without CMA3 (0 h=1). (D) Normalized intensities of Sp1 and Sp1-related protein expression from three independent experiments were shown. For FLIPS/L, only the FLIPS isoform was quantitated. CMA3, Chromomycin A3; CCA, cholangiocarcinoma; Sp1, specificity protein 1; FLIP, FADD-like IL-1ß-converting enzyme-inhibitory protein; cIAP2, cellular inhibitor of apoptosis; XIAP, X-linked inhibitor of apoptosis protein; Mcl-1, myeloid cell leukemia-1. *P<0.05, **P<0.01, ***P<0.001 vs. 0 h.