Protein kinase C iota as a therapeutic target in alveolar rhabdomyosarcoma.

Alveolar rhabdomyosarcoma is an aggressive pediatric cancer exhibiting skeletal-muscle differentiation. New therapeutic targets are required to improve the dismal prognosis for invasive or metastatic alveolar rhabdomyosarcoma. Protein kinase C iota (PKCι) has been shown to have an important role in tumorigenesis of many cancers, but little is known about its role in rhabdomyosarcoma. Our gene-expression studies in human tumor samples revealed overexpression of PRKCI. We confirmed overexpression of PKCι at the mRNA and protein levels using our conditional mouse model that authentically recapitulates the progression of rhabdomyosarcoma in humans. Inhibition of Prkci by RNA interference resulted in a dramatic decrease in anchorage-independent colony formation. Interestingly, treatment of primary cell cultures using aurothiomalate (ATM), which is a gold-containing classical anti-rheumatic agent and a PKCι-specific inhibitor, resulted in decreased interaction between PKCι and Par6, decreased Rac1 activity and reduced cell viability at clinically relevant concentrations. Moreover, co-treatment with ATM and vincristine (VCR), a microtubule inhibitor currently used in rhabdomyosarcoma treatment regimens, resulted in a combination index of 0.470-0.793 through cooperative accumulation of non-proliferative multinuclear cells in the G2/M phase, indicating that these two drugs synergize. For in vivo tumor growth inhibition studies, ATM demonstrated a trend toward enhanced VCR sensitivity. Overall, these results suggest that PKCι is functionally important in alveolar rhabdomyosarcoma anchorage-independent growth and tumor-cell proliferation and that combination therapy with ATM and microtubule inhibitors holds promise for the treatment of alveolar rhabdomyosarcoma.

Collagen-based Tissue Engineering as Applied to Heart Valves.

Using the method of directed collagen gel shrinkage, we have been fabricating heart valves and mitral valve chordae [1,2,3]. The principle involves mixing solubilized collagen with the appropriate cells. When the collagen-cell mixture is neutralized, soluble collagen reassembles into fibrils and a gel is created. When the gel is mechanically constrained, the collagen fibrils align in the direction of constraint. The generation of tensile force during contraction is crucial for the formation of highly aligned, compacted collagenous constructs. So far, inappropriate mechanical properties have been one of the main limitations of most collagen-based tissue equivalents. In this study, we focused on providing both biomechanical and biochemical stimuli to increase cellular proliferation, matrix synthesis, and hence improve the mechanical properties of the collagen constructs. We explored a number of holder materials and configurations, with an objective to maximize the lateral compaction of our constructs. We designed a bioreactor that can provide controlled static tension to our collagen constructs. We also developed a nutrition-fortified medium that includes trace elements (Zn2+, Cu2+, Fe2+ and Mn2+), various amino acids, and vitamins (A, B complex, and C). Our ultimate goal was to combine biomechanical and biochemical stimuli, and enhance the mechanical strength of our collagen constructs.

Progress in developing a composite tissue-engineered aortic valve.

This paper reviews the rationale for developing a tissue-engineered aortic valve by building up the complex microstructure from its basic components, and presents recent progress towards that goal. Over the past 4 years, we have been working on engineering the functional components of the composite valve the collagen fiber bundles, the elastin sheets, and the hyaluronan matrix that keeps the tissue hydrated. Most recently, we have been working on optimizing the geometry and material properties of the collagen constructs, by varying their size and aspect ratio, and the types of loading protocols the constructs experience during the culture process.