Figure 4. pA20-36 inhibits tumor growth in vivo. (A) Daily administration of pA20-36 results in inhibition of tumor growth in mice engrafted with A20 B lymphoma. Tumor volumes were measured in 6- to 8-week-old female BALB/c mice (n = 5 per group) that had been subcutaneously injected with tumor cells (5 × 106, left; 5 × 105, middle) and treated with daily intravenous administration of pA20-36 or scrambled pCNT (20 mg ≃ kg−1 ≃ d−1 in phosphate-buffered saline), or left untreated, beginning the day after tumor cell injection. As control, tumor volumes were also measured in 6- to 8-week-old C57BL/KaLwRij mice (n = 5 per group) that have been subcutaneously injected with 5T33MM tumor cells (5 × 106, right). One representative of 2 independent experiments with similar results is shown. Values are the mean volumes ± SEMs per group of animals (n = 5/group). Statistical analysis was performed by 2-way analysis of variance. In case of large tumor cell load (5 × 106), P < .001 for pA20-36 group versus untreated; P < .001 for pA20-36 group versus pCNT group. In the case of small tumor cell load (5 × 105), P < .001 for pA20-36 group versus untreated; P < .001 for pA20-36 group versus pCNT group. (B) The pA20-36 treatment enhances survival of mice engrafted with A20 B lymphoma. Kaplan-Meier survival curves of A20 B lymphoma–engrafted mice on peptide treatments. BALB/c mice (n = 5 per group) were subcutaneously inoculated with tumor cells and treated as described in panel A and killed when the tumor volume reached a size of 500 mm3, according to the ethical guidelines. Statistical analysis was performed by log-rank Mantel-Cox test; statistically significant difference was observed between pA20-36–treated and pCNT-treated groups (P < .001 for mice grafted with 5 × 105 tumor cells, or P < .003 for mice engrafted with 5 × 106 tumor cells). One representative of 2 independent experiments with similar results is shown. (C) Flow cytometry of apoptotic cells derived from pA20-treated and pCNT-treated mice. Apoptosis was evaluated at day 24 of peptide treatment by cell staining with annexin V–FITC and propidium iodide (PI). Apoptosis was measured as annexin V–positive/PI-negative cell population. Viable A20 cells from culture were used as control. A representative experiment of 4 independent experiments is shown. Quantitative analysis of annexin V–positive tumor cells from pA20-36– and pCNT-treated mice is also shown. Values are the mean ± SD (n = 4/group); statistical analysis was performed according to the Student t test. (D) Cell apoptosis in tumor masses of A20 B lymphoma. Tissue sections of A20 tumor masses were labeled by terminal deoxyuridine nick-end labeling (TUNEL) and anti-IgG to identify apoptotic DNA and B cells, respectively. Pictures were captured with a Leica TCS SP2 confocal microscope with a HC PL FLUOTAR 20×/0.50 oil UV objective (NA 0.50) in glycerol and acquired with Leica Confocal Software Version 2.61. Image manipulation was performed with Adobe Photoshop CS. Scale bar = 80 μm. (E) Cell-cycle profiles of A20 and 5T33MM tumor cells after peptide challenge in vivo. To track tumor cell in vivo, A20 (left) or 5T33MM (right) cells were stained with CFSE and injected intraperitoneally in BALB/c mice. After 3 days, mice were injected intraperitoneally with pCNT or pA20-36 peptide (100 μg/100 μL phosphate-buffered saline). Six hours later mice were killed, and single-cell suspensions were prepared from draining lymph nodes. Cell-cycle profile was analyzed by flow cytometry on CFSE-gated tumor population; data are the mean ± SD (n = 4/group); statistical analysis was performed according to the Student t test.
