Deborah J. Kuhn ¹, Yang Wang ², Vesna Minic ¹, Cristina Coates ², G. Suresh Kumar Reddy ², Kenyon G. Daniel ¹, Jeung-Yeop Shim ², Di Chen ¹, Kristin R. Landis-Piwowar ¹, Fred R. Miller ³, Edward Turos ² and Q. Ping Dou ¹

1 The Prevention and ³ Breast Cancer Programs, Barbara Ann Karmanos Cancer Institute, and Department of Pathology, School of Medicine, Wayne State University, Detroit, Michigan, USA, ² Department of Chemistry, College of Arts and Sciences, University of South Florida, Tampa, Florida, USA.

FIGURES

Figure 1. Chemical structures of N-methylthiolated beta-lactams.

Figure 2. Structure-activity relationship (SAR) analysis of N-thiolated beta-lactams. MCF-7 cells were plated in a 96-well plate and grown to 70-80% confluency followed by addition of 50 mM beta-lactam for 24 h. Cells were then incubated with 1 mg/ml MTT for 3 h and proliferation rates were determined using a muli-label plate reader (Victor3, Perkin Elmer; ±SD).

Figure 3. Lactam 18 induces caspase activity associated with Hsp70 expression and p38 phosphorylation. A. Jurkat T cells were treated with 20 mM lactam 1 or lactam 18 for 24 h. Following the treatment, the cells were then incubated with a FITC-conjugated marker that binds to activated caspases. Cell suspension was then transferred to glass slides in the presence of Vector Shield mounting medium with DAPI. Images were captured using AxioVision 4.1 and adjusted using Adobe Photoshop 6.0 software. B, Jurkat cells treated with 25 or 50 mM of lactam 1 or lactam 18 for 16 h, followed by Western blot analysis using specific antibodies to HSP70, p-p38, and Actin. Data shown are representative from three independent experiments.

Figure 4. (+)-Lactam 19 effects proliferation and cell death in a dose-dependent manner. A, MCF10AT1Kcl.2 cells were plated in a 96-well plate and grown to 70-80% confluency followed by addition of 50 mM of indicated beta-lactams for 24 h. Cells were then incubated with 1 mg/ml MTT for 4 h and proliferation rates were determined using a muli-label plate reader (Victor3, Perkin Elmer; ± SD). B, Jurkats T cells treated with lactam 1, (+)-lactam 19, (-)-lactam 19 at indicated doses and assayed for cell death by trypan blue incorporation (± SD).

Figure 5. beta-Lactams induce apoptosis in a tumor cell-specific manner. A, Jurkat T and YT cells were treated with lactam 1 and lactam 18 at indicated concentration for 16 h, followed by measurement of cell-free caspase-3 activity by incubating whole cell extracts with caspase-3 substrate and measuring free AMCs. B, (+)-lactam 19 is 2-fold more potent that lactam 1 at inducing apoptosis in a tumor cell specific manner. Jurkat T and YT cells were treated for 24 h with 25 and 50 mM of lactam 1 versus 25 mM of (+)-lactam 19 and (-)-lactam 19. Cell-free caspase-3 activity was then determined by incubating whole cell extracts with caspase-3 substrate and measuring free AMCs.

Figure 6. (+)-Lactam 18 induce apoptosis selectively in tumorigenic cells. A, Leukemic Jurkat T and non-transformed YT cells were treated with lactam 1 or isomers of lactam 18 at 50 mM for 24 h. Cell death is given as a percent of dead cells over total cell population (± SD). B, Both detached and attached VA-13 and WI-38 fibroblast cell populations were collected and stained with the nuclear staining dye Hoechst 33342. Each sample was then analyzed by fluorescence microscopy for nuclear morphology.