Jeffrey C. Mai

Targeted inhibition of Stat3 with a decoy oligonucleotide abrogates head and neck cancer cell growth

The transcription factor signal transducer and activator of transcription 3 (Stat3) is constitutively activated in a variety of cancers including squamous cell carcinoma of the head and neck (SCCHN). Previous investigations have demonstrated that activated Stat3 contributes to a loss of growth control and transformation. To investigate the therapeutic potential of blocking Stat3 in cancer cells, we developed a transcription factor decoy to selectively abrogate activated Stat3. The Stat3 decoy was composed of a 15-mer double-stranded oligonucleotide, which corresponded closely to the Stat3 response element within the c-fos promoter. The Stat3 decoy bound specifically to activated Stat3 and blocked binding of Stat3 to a radiolabeled Stat3 binding element. By contrast, a mutated version of the decoy that differed by only a single base pair did not bind the activated Stat3 protein. Treatment of head and neck cancer cells with the Stat3 decoy inhibited proliferation and Stat3-mediated gene expression, but did not decrease the proliferation of normal oral keratinocytes. Thus, disruption of activated Stat3 by using a transcription factor decoy approach may serve as a novel therapeutic strategy for cancers characterized by constitutive Stat3 activation.

Efficiency of Protein Transduction Is Cell Type-dependent and Is Enhanced by Dextran Sulfate

Protein transduction domains (PTDs), both naturally occurring and synthetic, have been increasingly utilized to deliver biologically active agents to a variety of cell types in vitro and in vivo. We report that in addition to previously characterized arginine-rich PTDs, including TAT, lysine homopolymers were able to mediate transduction of a wide variety of cell types, as measured by flow cytometric and enzymatic assays. The efficiency of PTD-mediated transduction was influenced by the cell type tested, although polylysine homopolymers demonstrate levels of internalization that consistently exceeded those of TAT and arginine homopolymers. Transduction of arginine/lysine-rich PTDs occurred at 4u2009°C and following depletion of cellular ATP pools, albeit generally at reduced levels. Although transduction was reduced in Chinese hamster ovary mutant lines deficient in either heparan sulfate or glycosaminoglycan synthesis, uptake was restored to wild-type levels by incubating target cells with dextran sulfate. The enhancement of transduction by dextran sulfate suggests that electrostatic interactions play an important first step in the process by which PTDs and their cargoes traverse the plasma membrane.

Identification of a synovial fibroblast-specific protein transduction domain for delivery of apoptotic agents to hyperplastic synovium.

Synovial hyperplasia, resulting in erosion of cartilage and bone, represents one of the major pathologies associated with rheumatoid arthritis. To develop an approach for efficient delivery of proteins or agents to synovium to induce targeted apoptosis of hyperplastic synovial tissue, we have screened an M13 peptide phage display library for synovial-specific transduction peptides. We identified a novel synovial-targeted transduction peptide, HAP-1, which is able to facilitate specific internalization of protein complexes into human and rabbit synovial cells in culture and rabbit synovial lining in vivo. HAP-1 and a non-tissue-specific cationic protein transduction domain, PTD-5, were fused to an antimicrobial peptide, (KLAK)(2), to generate two proapoptotic peptides termed DP2 and DP1, respectively. Administration of these peptides was able to induce apoptosis of rabbit and human synovial cells in culture, with DP2 inducing synovial cell-specific apoptosis. Intra-articular injection of DP1 and DP2 into arthritic rabbit joints with synovial hyperplasia induced extensive apoptosis of the hyperplastic synovium, while reducing the leukocytic infiltration and synovitis. These results suggest that proapoptotic peptides and, in particular, DP2 can be clinically useful for treatment of synovial hyperplasia, as well as inflammation. Moreover, the results demonstrate the feasibility of identifying tissue-specific transduction peptides capable of mediating efficient transduction in vivo.

Evaluation of peptide-mediated transduction in human CD34+ cells.

Protein transduction domains (PTDs) have been used increasingly to deliver biologically active agents to a variety of cell types in vitro and in vivo. To define the most effective PTDs for transducing hematopoietic cells, we have screened a panel of PTD peptides in human CD34(+) cells for delivery of a 60-kd marker protein and assessed its impact on phenotypic maintenence in vitro. Compared to the HIV-TAT peptide, most peptide complexes displayed high efficiency in transducing the CD34(+) cells, except for those based on shorter peptides (4R, 4K, and 5RQ). In particular, the arginine homopolymers including 8R, 10R, and 12R, were internalized by the cells to a greater extent than the other PTDs. Transduction was significantly potentiated by preincubation of cells with dextran sulfate. Importantly, colony forming ability and CD34(+) CD38(-) primitive phenotype were not significantly altered in the presence of these peptides during a short-term liquid culture. Together, these data suggest the potential usefulness of arginine homopolymers in hematopoietic stem and progenitor cell manipulations.

294. Adenoviral-TRAIL and a Smac34-8K Fusion Peptide Act Synergistically to Induce Apoptosis in Low CAR-Expressing Human Glioma Cells

Gliomas, the most common type of brain tumor, remain refractory to conventional treatment. One potential therapy involves the use of adenoviral vectors (Ad) to deliver Apo2L/tumor necrosis factor-related apoptosis inducing ligand (TRAIL), a protein shown to specifically induce apoptosis in cancer cells. However, gliomas exhibit resistance to infection by adenovirus. Indeed, the three glioma cell lines investigated, U-118MG, CCF-SSTG1 and U-138MG lacked surface expression of the adenovirus receptor, coxsackie-adenovirus receptor (CAR). Deceased CAR expression correlated with diminished adenovirus entry into these cells. Recent evidence has demonstrated that peptides containing protein transduction domains (PTDs) enhance adenovirus infectivity. Thus, Ad.EGFP was incubated with a panel of PTDs prior to infection of glioma cells. Measurement of the mean fluorescent intensity and the percent EGFP positive cells revealed that the PTDs promoted viral entry, but in a cell-type dependent manner. A protein transduction peptide comprised of 8 lysines (8K) proved the most effective for enhancing adenoviral infection. However, infection of CCF-SSTG1 cells with adenovirus encoding soluble, trimerized TRAIL (Ad.stTRAIL) pre-incubated with 8K did not greatly induce apoptosis. In an attempt to circumvent resistance to TRAIL, Ad.stTRAIL was pre-incubated with a fusion peptide containing 8K and first 34 amino acids of Smac (Smac34-8K). This region of Smac has been demonstrated to enhance TRAIL-mediated apoptosis by interacting with anti-apoptotic proteins. Smac34-8K significantly enhanced Ad.stTRAIL-induced apoptosis in CCF-SSTG1 cells, likely by increasing both virus infectivity and TRAIL action. These results demonstrate that the combination of pro-apoptotic cell permeable peptides with adenovirus expressing therapeutic proteins may be an effective strategy in the fight against cancer.