Fig. 1

Discovery of a novel non-RGD αvβ3 inhibitor for castration resistant prostate cancer (CRPC) treatment. a Sensorgram of C19 interacted with αvβ3 measured by SPR method, and data was analyzed by General Electric Company(GE)‘s BIAevaluation software. The KD value was 2.68 μM. b The synergistic effect on inhibition of 22RV1 cell proliferation of C19 and enzalutamide. c The binding affinity of C19-9 with αvβ3 tested by SPR. d The result of superposition of molecular dynamics (MD) simulations stable structure and the crystal structure of integrin avβ3. The crystal structure is shown in light blue, and the MD stable structure is shown in grayish-white. e The 3D binding mode of integrin avβ3 with C19-9. The backbone and residue of the integrin avβ3 alpha chain are colored in white, and the backbone and residue of the integrin avβ3 beta chain are colored in light blue. C19-9 is colored in yellow, and the Mn²⁺ in MIDAS site is shown as a purple sphere. The surrounding residues in the binding pockets are shown as sticks. The yellow dashes represent hydrogen bond interaction, the magentas represent metal contact. f The Celltiter-Glo assay of evaluation of anti-tumor proliferation ability of C19-9 on prostate cancer organoid at 24 h. g-i The effects of C19-9 on the expression of VEGFA, p-ERK, ERK, p-PI3K, PI3K, p-AKT, AKT, AR and JAK/STAT3/IDO signaling pathway were detected by Western blot assay in 22RV1 cell line. j 1 × 10⁶ 22RV1 cells in suspension were subcutaneously injected into the right flank of BALB/c-nude mice. After the volume of tumor nodules reached about 75 mm³, tumor-bearing BALB/c-nude mice were randomly assigned to four groups (n = 7 per group) and treated with C19-9, enzalutamide, or vehicle as indicated. The control group was injected with DMSO. Tumor volume was measured twice per week. Tumor weight in different groups treated with C19-9 and enzalutamide after three weeks. k In the 22RV1-derived subcutaneous tumor growth xenograft model, tumor tissue was obtained after treatment for three weeks. The expression of JAK/STAT3 signaling pathway related proteins were tested by Western blot assay. l Therapeutic effects of C19-9 on castration-resistant xenografts. Murine prostate cancer TRAMP-C1 cells (1 × 10⁶) were injected subcutaneously into C57BL/6 mice. The tumor-bearing mice were castrated and randomly assigned to four groups (n = 10 per group). Animals were intraperitoneally injected with C19-9 (3 mg/kg and 6 mg/kg) or vehicle, and enzalutamide (10 mg/kg) by intragastric administration. The tumor volume weight was measured every 2 days. Tumor weight in different groups was measured following the termination of the experiment. m Image of tumors in different groups following the termination of TRAMP-C1 castration-resistant xenografts experiment. n Polychromatic immunofluorescence staining showing the expression of CD4 and FOXP3 in tumor tissues of C19-9 and vehicle. Bars: 50 μm. o The mechanism of integrin αvβ3 inhibitor C19-9 for overcoming enzalutamide resistant in CRPC. Data are expressed as mean ± SD. Statistical analyses were performed using the two-tailed Student’s t test to detect differences between the groups, *P-value < 0.05, **P-value < 0.01, ***P-value < 0.001