Fig. 7: Combined inhibition of thymidylate synthase and BCL-XL is effective in vivo.

A LNCaP xenografts were developed in immunodeficient male mice. Mice were randomized to control, raltitrexed (50 mg/kg q4 days), navitoclax (50 mg/kg q2 days), or the combination as indicated in schema. Actual tumor size measurements are shown on left. Individual tumor size changes at 14 days relative to baseline (tumor size at randomization) are shown on right panel. Five (5) biological replicates are shown. Data were analyzed by Wilcoxon Rank Sum Test ***p < 0.001. Upper panel was created in Biorender. Yuan, X. (2025) https://BioRender.com/n41n675B Tumor-bearing mice were treated as in (A) for 14 days were monitored for tumor growth and euthanized when tumors reached 2.0 cm³. Graph shows Kaplan-Meier curve for mice survival from randomization. Data were analyzed by logrank test. C Tumors in another cohort were collected 6 h after the second raltitrexed dose (combination treated mice received 2 navitoclax doses). Apoptosis markers (cleaved PARP and cleaved caspase 3, CC3) were assessed with immunoblotting. D Tumors were collected 6 h after second raltitrexed dose and survivin protein expression was assessed with immunoblotting. Fold change in survivin is quantified. E Immunoblotting for MCL-1 and survivin in A549 (lung adenocarcinoma) and ZR75 (breast cancer) cells treated with 5-FU. Fold change in survivin is quantified. F ZR75 cells were treated with 5-FU for 48 h followed by navitoclax for 6 h, and apoptosis was assessed by CaspaseGlo 3/7 assay. Mean and SEM for 3 biological replicates are shown. Data at each 5-FU concentration were analyzed by unpaired t-test, *p < .05. Analysis by two-way ANOVA showed raltitrexed significantly enhanced apoptosis, p < .001. G ZR75 xenografts were developed in immunodeficient mice. Mice were randomized to control, capecitabine (200 mg/kg daily), navitoclax (50 mg/kg q2 days), or combination therapy as indicated in schema. Actual tumor size measurements are shown in the left panel. Individual tumor size changes at 28 days relative to baseline are shown in right panel. Five biological replicates are shown. Data were analyzed by Wilcoxon Rank Sum Test, ***p < 0.001. Upper panel was created in Biorender. Yuan, X. (2025) https://BioRender.com/obtzw73H Tumor-bearing mice were treated for 28 days. After that, mice were monitored for tumor growth as described above. Graph shows Kaplan-Meier curve for mice survival from randomization. Data were analyzed by logrank test. I Tumors were collected 6 h after the fourth capecitabine dose, and survivin protein expression was assessed with immunoblotting. Fold change in survivin is quantified. Source data are provided as a Source Data file.