Tumor selective G2/M cell cycle arrest and apoptosis of epithelial and hematological malignancies by BBL22, a benzazepine

Abstract

Two distinct benzodiazepine binding sites have been identified, (i) a central site restricted to brain and (ii) a ubiquitously expressed mitochondrial binding site, the so-called peripheral-type benzodiazepine receptor (PBR). In this paper, we show that a benzazepine referred to as BBL22 (2-amino 9-chloro-7-(2-fluorophenyl)-5H-pyrimidol[5,4-d][2]benzazepine), which is classified as a PBR ligand based on structure, induces arrest in G(2)/M phase of the cell cycle in human tumor cell lines of both epithelial and hematopoietic cellular origin. After G(2)/M arrest, several tumor types, notably prostate and certain breast cancer lines exhibited significant apoptosis. Ideally, cancer therapies should selectively target tumor cells while sparing normal cell counterparts. BBL22 exhibited such selectivity, as it did not affect the growth and survival of nonmalignant breast and prostate epithelial lines. Moreover, BBL22 demonstrated structural requirements for this selective antitumor activity as 11 structurally related PBR ligands, including high-affinity ligands Ro5-4864 and PK11195, failed to induce tumor cell growth arrest or apoptosis. The in vivo antitumor activity of BBL22 was examined in a human xenograft model of androgen-independent prostate cancer where BBL22 significantly reduced the growth of PC3 prostate tumors without eliciting overt toxicity. Identification of BBL22 represents a tumor selective therapeutic strategy for a variety of human tumors.

Figure 1

BBL22 induces human tumor cell apoptosis. DNA histograms indicate cell cycle kinetics of androgen-dependent LNCaP prostate cancer cells (A–C), androgen-independent PC3 prostate cancer cells (D–F), and the estrogen-receptor-negative HBL100 breast carcinoma line (G and H) before (0 h) and at various time points (24 h and 72 h) after treatment with 50 μM BBL22. The percentage of cells in G₁, S phase, and G₂/M are indicated. The sub-G₁ peak represents the apoptotic population. Data shown is representative of five independent experiments.

Figure 2

Activation of caspase-3 by BBL22. (A) Immunoblot analysis of caspase-3 in LNCaP cells before (lane 1) and 72 h after (lane 2) treatment with 50 μM BBL22. The 32-kDa procaspase-3 form (arrow) is the predominant species in untreated cells. After treatment with BBL22, procaspase-3 is cleaved to the active 17-kDa product (arrowhead). (B) Proteolytic cleavage of procaspase-3 (arrow) to the active caspase-3 form (arrowhead) after treatment of HBL100 cells with BBL22 (as described previously). Untreated controls (lane 1), 25 μM BBL22 for 72 h (lane 2), and 50 μM BBL22 for 72 h (lane 3). Equal amounts of total cell lysate were loaded to each lane in both A and B.

Figure 3

Antitumor activity of BBL22 in PC3 xenografts. Palpable tumors were established by inoculating nude athymic mice s.c. with 5 × 10⁶ PC3 cells. At day 17 after tumor cell inoculation, animals were injected s.c. with vehicle alone (filled diamonds), BBL22 50 mg/kg per day (open squares), or BBL22 250 mg/kg per day (filled circles) for 4 consecutive days (days 17–20, indicated by arrows). Tumor volumes were determined on days 14, 18, 20, 24, and 32. Horizontal lines indicate mean values for each treatment group.