Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Shikonin Mediates Apoptosis through G Protein-Coupled Estrogen Receptor of Ovarian Cancer Cells

doi: 10.1155/2022/6517732

Figure Lengend Snippet: Regulation of EGFR/PI3K/p-AKT signaling pathway mediated by SK via GPER in SKOV3 and A2780 cells. (a) The expression of EGFR, p-EGFR, PI3K, AKT and p-AKT following G-1 and G-15 treatment together with 8.6 μ M SK in SKOV3 cells. (b) The expression of EGFR, p-EGFR, PI3K, AKT, and p-AKT following G-1 and G-15 treatment together with 0.56 μ M SK in A2780 cells. The results are means of three independent replicates ± S.D. ∗∗ P < 0.01 or ∗ P < 0.05 vs. control group, △△ P < 0.01 or △ P < 0.05 vs. SK treatment group were considered as statistically significant.

Article Snippet: GPER-specific agonists G-1 and G-15 were obtained from MCE (Shanghai, China).

Techniques: Expressing, Control

Journal: Advanced Science

Article Title: Calcium Channel Blocker Lacidipine Promotes Antitumor Immunity by Reprogramming Tryptophan Metabolism

doi: 10.1002/advs.202409310

Figure Lengend Snippet: Lacidipine attenuates the enzymatic activity and expression of IDO1. A) Chemical structure of lacidipine. B) MDA‐MB‐231 and MCF‐7 cells were treated with lacidipine (5 × 10 −6 , 10 × 10 −6 , 20 × 10 −6 , 40 × 10 −6 , and 80 × 10 −6 m ) for 2 h, and then treated with IFN γ (100 ng mL −1 ) for 24 h. The concentration of kynurenine in the cell supernatants was determined. Bars, ± standard error of mean (SEM). The curves were plotted using a variable slope (four‐parameter) non‐linear fit. C) Effect of lacidipine on the IDO1 expression. MDA‐MB‐231 cells were pre‐treated with lacidipine (10 × 10 −6 , 20 × 10 −6 , and 40 × 10 −6 m ) and JAK inhibitor I (5 × 10 −6 m ) for 2 h, then stimulated with IFN γ (100 ng mL −1 ) for 24 h. The expression of IDO1, IDO2, and TDO2 was analyzed by western blotting. GAPDH was used as the loading control. D) MDA‐MB‐231 and MCF‐7 cells were pre‐treated with lacidipine (10 × 10 −6 , 20 × 10 −6 , and 40 × 10 −6 m ) and JAK inhibitor I (5 × 10 −6 m ) for 2 h, then stimulated with IFN γ (100 ng mL −1 ) for 24 h. The mRNA expressions of IDO1 examined by qRT‐PCR. The mRNA levels of these genes were normalized against GAPDH expression levels. Bars, ± SEM. * p < 0.05, ** p < 0.01, and *** p < 0.001 versus IFN γ treatment group (unpaired two‐tailed Student's t ‐test). E) HEK293A cells are transfected with pCMV3‐IDO1 plasmids for 48 h, and then treated with lacidipine (10 × 10 −6 , 20 × 10 −6 , and 40 × 10 −6 Μ) and epacadostat (25 × 10 −9 m ) for 6 h. Cell supernatants were used to detect the activity of IDO1. Bars, ± SEM. * p < 0.05, ** p < 0.01 versus pCMV3‐IDO1 group (unpaired two‐tailed Student's t ‐test). The cell lysates were immunoblotted with IDO1 antibody. GAPDH was used as the loading control. All experiments were conducted with three independent replicates. F) The catalytic activity of the recombinant expressed IDO1 protein was measured. G) The binding location of lacidipine in the IDO1 protein. H) Detailed interactions between lacidipine and the active binding site of the IDO1 protein.

Article Snippet: [ ] In brief, the IDO activity was measured with the standard reaction mixture (250 L) containing potassium phosphate buffer (50 × 10 −3 m , pH = 6.5), L‐tryptophan (200 × 10 −6 m ), methylene blue (10 × 10 −6 m ) (#7220‐79‐3, Macklin), ascorbic acid (20 × 10 −3 m ) (#A8100, Solarbio), catalase (100 µg mL −1 ) (#C8070, Solarbio), and recombinant expressed IDO1 enzyme (10 µg mL −1 ) (#11650‐H70E, SinoBiological).

Techniques: Activity Assay, Expressing, Concentration Assay, Western Blot, Control, Quantitative RT-PCR, Two Tailed Test, Transfection, Recombinant, Binding Assay

Journal: Advanced Science

Article Title: Calcium Channel Blocker Lacidipine Promotes Antitumor Immunity by Reprogramming Tryptophan Metabolism

doi: 10.1002/advs.202409310

Figure Lengend Snippet: Lacidipine exerts potential anti‐tumor effects in vivo. A) Tumor volumes were recorded in the DOX only, and DOX + lacidipine (0.5 or 1 mg kg −1 ) groups ( n = 6, each group) and are presented as the mean ± SEM. B) Image of the tumors in the DOX only, and DOX + lacidipine (0.5 or 1 mg kg −1 ) groups ( n = 6, each group). C) Tumor mass was recorded in the DOX only, and DOX + lacidipine (0.5 or 1 mg kg −1 ) groups ( n = 6, each group) and the results are presented as the mean ± SEM; * p < 0.05 versus control group and # p < 0.05, ### p < 0.001 versus DOX‐treated group (unpaired two‐tailed Student's t ‐test). D) Tumor volumes were recorded in the DOX only, lacidipine (10 or 20 mg kg −1 ) only, and DOX + lacidipine (20 mg kg −1 )/1‐MT groups ( n = 12, each group) and are presented as the mean ± SEM. E) Image of the tumors in the DOX only, lacidipine (10 or 20 mg kg −1 ) only, and DOX + lacidipine (20 mg kg −1 )/1‐MT groups ( n = 6, each group). F) Tumor mass was recorded in the DOX only, lacidipine (10 or 20 mg kg −1 ) only, and DOX + lacidipine (20 mg kg −1 )/1‐MT groups ( n = 6, each group) and the results are presented as the mean ± SEM; ** p < 0.01, *** p < 0.001, and **** p < 0.0001 versus control group and #### p < 0.0001 versus DOX‐treated group (unpaired two‐tailed Student's t ‐test). G) The ratio of Kyn/Try in mouse serum was determined by HPLC at the end of the treatment. * p < 0.05, ** p < 0.01 and *** p < 0.001 versus control group and # p < 0.05, ## p < 0.01 versus DOX‐treated group (unpaired two‐tailed Student's t ‐test). H,I) The expression of IDO1, IDO2 and TDO2 were analyzed by western blotting in mouse tumor tissue homogenate of at the end of treatment. GAPDH was used as the loading control. The experiments were conducted with three independent replicates.

Article Snippet: [ ] In brief, the IDO activity was measured with the standard reaction mixture (250 L) containing potassium phosphate buffer (50 × 10 −3 m , pH = 6.5), L‐tryptophan (200 × 10 −6 m ), methylene blue (10 × 10 −6 m ) (#7220‐79‐3, Macklin), ascorbic acid (20 × 10 −3 m ) (#A8100, Solarbio), catalase (100 µg mL −1 ) (#C8070, Solarbio), and recombinant expressed IDO1 enzyme (10 µg mL −1 ) (#11650‐H70E, SinoBiological).

Techniques: In Vivo, Control, Two Tailed Test, Expressing, Western Blot

Journal: Advanced Science

Article Title: Calcium Channel Blocker Lacidipine Promotes Antitumor Immunity by Reprogramming Tryptophan Metabolism

doi: 10.1002/advs.202409310

Figure Lengend Snippet: Lacidipine inhibits the IDO1 expression through suppression of JAK/STAT and NF‐κB signaling pathways. A) Gene set enrichment analysis (GSEA) for JAK/STAT and NF‐κB signaling pathways. Rank statistics (bottom; see y axis) and normalized enrichment scores (top; see y axis) indicate downregulation and upregulation, respectively. JAK/STAT signaling pathways, normalized enrichment score (NES) = −1.37, p < 0.001, FDR q = 0.11; NF‐κB signaling pathways, NES = ‐1.64, p = 0.01, FDR q = 0.17. B) Heat map hierarchical clustering displays the differentially expressed genes in the JAK/STAT and NF‐κB signaling pathways with a p < 0.05 ( n = 3, each group). La, Lacidipine. C) MDA‐MB‐231 cells were pre‐treated with lacidipine (10 × 10 −6 , 20 × 10 −6 , and 40 × 10 −6 m ) and JAK inhibitor I (5 × 10 −6 m ) for 2 h, then stimulated with IFN γ (100 ng mL −1 ) for 12 h. The expression of key proteins of the JAK/STAT signaling pathway was analyzed by western blotting. GAPDH was used as the loading control. D) MDA‐MB‐231 cells were pre‐treated with lacidipine (10 × 10 −6 , 20 × 10 −6 , and 40 × 10 −6 m ) and BAY11‐7082 (10 × 10 −6 m ) for 2 h, then stimulated with IFN γ (100 ng mL −1 ) for 12 h. The expression of key proteins of the NF‐κB signaling pathway was analyzed by western blotting. GAPDH was used as the loading control. E,F) The the key proteins expression of the JAK/STAT and NF‐κB signaling pathways were analyzed by western blotting in mouse tumor tissue homogenate of at the end of treatment. GAPDH was used as the loading control. All western blot experiments were conducted with three independent replicates.

Article Snippet: [ ] In brief, the IDO activity was measured with the standard reaction mixture (250 L) containing potassium phosphate buffer (50 × 10 −3 m , pH = 6.5), L‐tryptophan (200 × 10 −6 m ), methylene blue (10 × 10 −6 m ) (#7220‐79‐3, Macklin), ascorbic acid (20 × 10 −3 m ) (#A8100, Solarbio), catalase (100 µg mL −1 ) (#C8070, Solarbio), and recombinant expressed IDO1 enzyme (10 µg mL −1 ) (#11650‐H70E, SinoBiological).

Techniques: Expressing, Western Blot, Control

Journal: Advanced Science

Article Title: Calcium Channel Blocker Lacidipine Promotes Antitumor Immunity by Reprogramming Tryptophan Metabolism

doi: 10.1002/advs.202409310

Figure Lengend Snippet: Ca 2+ participates in lacidipine regulation of IDO1 expression. A) Effect of IFN γ only and lacidipine + IFN γ on intracellular Ca 2+ concentration. MDA‐MB‐231 cells were pre‐treated with lacidipine (20 × 10 −6 m ) for 2 h, and then stimulated with IFN γ (100 ng mL −1 ) for different durations (0, 0.5, 1, 2, and 4 h). The cells were loaded with 5 × 10 −6 m fluo‐3 AM, and intracellular Ca 2+ levels were analyzed using flow cytometry. B–E) MDA‐MB‐231 cells were pre‐treated with lacidipine (20 × 10 −6 m ) and diltiazem (1 × 10 −6 , 10 × 10 −6 , 25 × 10 −6 m ) for 2 h, then stimulated with IFN γ (100 ng mL −1 ) for 24 h. The expression of B,C) IDO1 proteins and D) the key proteins of the JAK/STAT and E) NF‐κB signaling pathways were analyzed by western blotting. GAPDH was used as the loading control. F,G) MDA‐MB‐231 cells were pre‐treated with lacidipine (20 × 10 −6 m ) and BAY K 8644 (0.5 × 10 −6 , 1 × 10 −6 , and 5 × 10 −6 m ) for 2 h, then stimulated with IFN γ (100 ng mL −1 ) for 24 h. The expression of IDO1 proteins were analyzed by western blotting. GAPDH was used as the loading control. H) MDA‐MB‐231 cells were pre‐treated with lacidipine (20 × 10 −6 m ) and BAPTA‐AM (10 × 10 −6 , 25 × 10 −6 , and 50 × 10 −6 m ) for 2 h, then stimulated with IFN γ (100 ng mL −1 ) for 24 h. The expression of IDO1 proteins was analyzed using western blotting. GAPDH was used as the loading control. All experiments were conducted with three independent replicates.

Article Snippet: [ ] In brief, the IDO activity was measured with the standard reaction mixture (250 L) containing potassium phosphate buffer (50 × 10 −3 m , pH = 6.5), L‐tryptophan (200 × 10 −6 m ), methylene blue (10 × 10 −6 m ) (#7220‐79‐3, Macklin), ascorbic acid (20 × 10 −3 m ) (#A8100, Solarbio), catalase (100 µg mL −1 ) (#C8070, Solarbio), and recombinant expressed IDO1 enzyme (10 µg mL −1 ) (#11650‐H70E, SinoBiological).

Techniques: Expressing, Concentration Assay, Flow Cytometry, Western Blot, Control

Journal: Advanced Science

Article Title: Calcium Channel Blocker Lacidipine Promotes Antitumor Immunity by Reprogramming Tryptophan Metabolism

doi: 10.1002/advs.202409310

Figure Lengend Snippet: Ca V 1.2/1.3 regulates IDO1 expression via Pyk2 and calmodulin. A,B) MDA‐MB‐231 cells were pre‐treated with lacidipine (10 × 10 −6 , 20 × 10 −6 , and 40 × 10 −6 m ) and tyrphostin A9 (5 × 10 −6 m ) or W‐7 (35 × 10 −6 m ) for 2 h, then stimulated with IFN γ (100 ng mL −1 ) for 24 h. The expression of Pyk2 phosphorylated at A) Tyr402, Pyk2 and B) calmodulin (B) proteins was analyzed by western blotting. GAPDH was used as the loading control. MDA‐MB‐231 cells were pre‐treated with lacidipine (20 × 10 −6 m ) and C) tyrphostin A9 (5 × 10 −6 m ) or D) W‐7 for 2 h, then stimulated with IFN γ (100 ng mL −1 ) for 24 h. The expression of IDO1 proteins was analyzed by western blotting. GAPDH was used as the loading control. MDA‐MB‐231 cells were pre‐treated with E) lacidipine (20 × 10 −6 m ) and tyrphostin A9 (5 × 10 −6 m ) or F) W‐7 for 2 h, then stimulated with IFN γ (100 ng mL −1 ) for 24 h. The expression of phosphorylated JAK at Tyr1022, phosphorylated STAT1 at Tyr701, phosphorylated IKK α/β, phosphorylated IκBα, and IκBα proteins was analyzed by western blotting. GAPDH was used as the loading control. G) MDA‐MB‐231 cells were treated with lacidipine (20 × 10 −6 m ) for 3 h and then co‐immunoprecipitated with Pyk2 antibodies or control immunoglobulin G (IgG) and analyzed for antibody‐specific JAK1, calmodulin, and Pyk2. The MDA‐MB‐231 cells were transfected with the indicated H) Ca V 1.2 and I) Ca V 1.3 siRNA. After transfection for 48 h, cells were treated with IFN γ (100 ng mL −1 ) for 24 h and cell lysates were collected for western blot analysis using the indicated antibodies to assess the effect of Ca V 1.2 and Ca V 1.3. GAPDH was used as the loading control. All experiments were conducted with three independent replicates.

Article Snippet: [ ] In brief, the IDO activity was measured with the standard reaction mixture (250 L) containing potassium phosphate buffer (50 × 10 −3 m , pH = 6.5), L‐tryptophan (200 × 10 −6 m ), methylene blue (10 × 10 −6 m ) (#7220‐79‐3, Macklin), ascorbic acid (20 × 10 −3 m ) (#A8100, Solarbio), catalase (100 µg mL −1 ) (#C8070, Solarbio), and recombinant expressed IDO1 enzyme (10 µg mL −1 ) (#11650‐H70E, SinoBiological).

Techniques: Expressing, Western Blot, Control, Immunoprecipitation, Transfection

Journal: Advanced Science

Article Title: Calcium Channel Blocker Lacidipine Promotes Antitumor Immunity by Reprogramming Tryptophan Metabolism

doi: 10.1002/advs.202409310

Figure Lengend Snippet: The expression levels of IDO1 and Pyk2 phosphorylation correlate with human breast cancer development. A) Statistical analysis of IDO1 expression in the breast tumor tissues ( n = 136). B,C) Expression of IDO1 in different subtypes of breast cancer ( n = 136). D) Statistical analysis of Pyk2 phosphorylation expression in the breast tumor tissues ( n = 136). E,F) Expression levels of Pyk2 phosphorylation in different subtypes of breast cancer ( n = 136). G) The correlation of IDO1 and Pyk2 phosphorylation, R = 0.3, p = 0.00042. H) Expression levels of IDO1 in the breast tumor tissues, adjacent normal breast tissues, and normal breast tissue based on TCGA data ( n = 1034). I) Expression levels of IDO1 in different subtypes of breast cancer based on TCGA data, p < 0.001 ( n = 1034).

Article Snippet: [ ] In brief, the IDO activity was measured with the standard reaction mixture (250 L) containing potassium phosphate buffer (50 × 10 −3 m , pH = 6.5), L‐tryptophan (200 × 10 −6 m ), methylene blue (10 × 10 −6 m ) (#7220‐79‐3, Macklin), ascorbic acid (20 × 10 −3 m ) (#A8100, Solarbio), catalase (100 µg mL −1 ) (#C8070, Solarbio), and recombinant expressed IDO1 enzyme (10 µg mL −1 ) (#11650‐H70E, SinoBiological).

Techniques: Expressing