Signaling pathways in osteogenesis and osteoclastogenesis: Lessons from cranial sutures and applications to regenerative medicine.

One of the simplest models for examining the interplay between bone formation and resorption is the junction between the cranial bones. Although only roughly a quarter of patients diagnosed with craniosynostosis have been linked to known genetic disturbances, the molecular mechanisms elucidated from these studies have provided basic knowledge of bone homeostasis. This work has translated to methods and advances in bone tissue engineering. In this review, we examine the current knowledge of cranial suture biology derived from human craniosynostosis syndromes and discuss its application to regenerative medicine

Signaling pathways in osteogenesis and osteoclastogenesis: Lessons from cranial sutures and applications to regenerative medicine.

One of the simplest models for examining the interplay between bone formation and resorption is the junction between the cranial bones. Although only roughly a quarter of patients diagnosed with craniosynostosis have been linked to known genetic disturbances, the molecular mechanisms elucidated from these studies have provided basic knowledge of bone homeostasis. This work has translated to methods and advances in bone tissue engineering. In this review, we examine the current knowledge of cranial suture biology derived from human craniosynostosis syndromes and discuss its application to regenerative medicine

Clinical Applications of the Pedicled Anterolateral Thigh Flap in Complex Abdominal-Pelvic Reconstruction

Background: The free anterolateral thigh (ALT) flap has become a reconstructive workhorse with great versatility throughout the body. However, the utility of the pedicled ALT flap is less described for complex defects. A skin paddle with reliable blood circulation and wide range of reach, low donor site morbidity along with the avoidance of many of the complications plaguing free flaps, are just some of the several benefits offered by the pedicled ALT flap. We investigated specific clinical examples within our ALT flap database where the pedicled ALT flap was used for coverage of complex wounds and highlight its advantages. Methods: We conducted a retrospective chart review on those patients in whom a pedicled ALT flap was used for complex wound reconstruction over a 7-year period between July 2002 and October 2009 at The R Adams Cowley Shock Trauma Center performed by a single surgeon. Results: Four patients underwent a pedicled ALT flap as part of their reconstruction. Flaps ranged in size from 75 to 648 cm(2), and all but one were cutaneous in nature with the other one being fasciocutaneous. The flaps averaged 2 perforators (range, 1-3) and the donor sites were all closed primarily except for one. Conclusions: When faced with a complex defect in the abdominal-pelvic region, we propose that the pedicled ALT flap has several advantages to other types of tissue coverage and is an excellent option for the reconstructive surgeon

Induction of apoptosis of lung and esophageal cancer cells treated with the combination of histone deacetylase inhibitor (trichostatin A) and protein kinase C inhibitor (calphostin C)

Histone deacetylase inhibitors mediate a potent growth-inhibitory effect in cancer cells through induction of cell-cycle arrest and apoptosis. Moreover, these agents significantly induce transcriptional activation of nuclear factor κB, as well as p21 regulated by protein kinase C, and are thought to negatively influence the ability of histone deacetylase inhibitor to effectively mediate apoptosis. This study aimed to evaluate the effect of calphostin C (a protein kinase C inhibitor) on trichostatin A (a histone deacetylase inhibitor)–mediated upregulation of nuclear factor κB and p21 promotor transcriptional activity, as well as induction of apoptosis in lung and esophageal cancer cells. Cultured lung and esophageal cancer cells were treated with calphostin C and trichostatin A. Nuclear factor κB transcriptional activity was quantitated by using the nuclear factor κB–luciferase assay. Transcription of p21 gene and p21 protein levels was evaluated by using the p21 promoter–luciferase assay and the p21 enzyme-linked immunoassay, respectively. Apoptosis was evaluated by using the terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling–based ApoBrdU assay. Levels of expression of nuclear factor κB–dependent antiapoptotic and proapoptotic proteins were evaluated by means of Western blotting. Exposure of lung or esophageal cancer cells to trichostatin A resulted in a dose- and cell-dependent 2-fold to greater than 20-fold increase of nuclear factor κB and p21 transcriptional activity. Treatment with trichostatin A and calphostin C led to a 50% to 90% decrease of trichostatin A– mediated upregulation of nuclear factor κB and p21 activation. Inhibition of nuclear factor κB activity resulted in significant reduction (30% to >99%) of trichostatin A– mediated activation of not only nuclear factor κB transcription but also p21 promotor activity. Importantly, 90% to 96% of thoracic cancer cells under-went apoptosis after exposure to the combination of trichostatin A plus calphostin C. Inhibition of protein kinase C abrogates trichostatin A–mediated upregulation of nuclear factor κB transcriptional activity and p21 expression that is associated with profound induction of apoptosis in lung or esophageal cancer cells. Protein kinase C might be a novel target for enhancing the efficacy of histone deacetylase inhibitor in cancer therapy

Rapid and profound potentiation of Apo2L/TRAIL-mediated cytotoxicity and apoptosis in thoracic cancer cells by the histone deacetylase inhibitor Trichostatin A: the essential role of the mitochondria-mediated caspase activation cascade

Apo2L/TRAIL is actively investigated as a novel targeted agent to directly induce apoptosis of susceptible cancer cells. Apo2L/TRAIL-refractory cells can be sensitized to the cytotoxic effect of this ligand by cytotoxic chemotherapeutics. The aim of this study was to evaluate the in vitro tumoricidal activity of the Apo2L/TRAIL + Trichostatin A in cultured thoracic cancer cells and to elucidate the molecular basis of the synergistic cytotoxicity of this combination. Concurrent exposure of cultured cancer cells to sublethal concentrations of Apo2L/TRAIL and Trichostatin A resulted in profound enhancement of Apo2L/TRAIL-mediated cytotoxicity in all cell lines regardless of their intrinsic susceptibility to this ligand. This combination was not toxic to primary normal cells. While Apo2L/TRAIL alone or Trichostatin A alone mediated < 20% cell death, 60 to 90% of cancer cells were apoptotic following treatment with TSA + Apo2L/TRAIL combinations. Complete translocation of Bax from the cytosol to the mitochondria compartment was mainly observed in combination-treated cells and this was correlated with robust elevation of caspase 9 proteolytic activity indicative of activation of the mitochondria apoptogenic effect. Profound TSA + Apo2L/TRAIL–mediated cytotoxicity and apoptosis were completely abrogated by either Bcl2 over-expression or by the selective caspase 9 inhibitor, highlighting the essential role of mitochondria-dependent apoptosis signaling cascade in this process. Moreover, increased caspase 8 activity observed in cells treated with the TSA + Apo2L/TRAIL combination was completely suppressed by Bcl-2 over-expression or by the selective caspase 9 inhibitor indicating that the elevated caspase 8 activity in combination-treated cells was secondary to a mitochondria-mediated amplification feedback loop of caspase activation. These finding form the basis for further development of HDAC inhibitors + Apo2L/TRAIL combination as novel targeted therapy for thoracic malignancies

The selective epidermal growth factor receptor tyrosine kinase inhibitor PD153035 suppresses expression of prometastasis phenotypes in malignant pleural mesothelioma cells in vitro

ObjectiveMalignant pleural mesothelioma is notoriously refractory to aggressive multimodality therapy. Epidermal growth factor receptor expression has been observed on malignant pleural mesothelioma cells. Epidermal growth factor receptor-mediated signaling promotes tumorigenesis and metastasis of cancer cells. The purpose of this study is to evaluate the ability of the epidermal growth factor receptor tyrosine kinase inhibitor PD153035 to abrogate the expression of prometastasis phenotypes in malignant pleural mesothelioma cells in vitro.MethodsEpidermal growth factor receptor expression of malignant pleural mesothelioma cells and primary normal cells was quantitated by means of flow cytometry. PD153035-mediated growth inhibition was determined by means of 1-(4,5-Dimethylthiazol-2-yl)-3,5-diphenylformazan and clonogenic assays. Cell motility and invasion of extracellular matrix was evaluated with in vitro wound-healing and Matrigel invasion assays, respectively. Vascular epidermal growth factor levels in conditioned media were measured by using enzyme-linked immunosorbent assay.ResultsEpidermal growth factor receptor expression was detected on all 6 cultured malignant pleural mesothelioma cells, with 4 of 6 having normal receptor expression and 2 of 6 overexpressing the receptor. PD153035 suppressed cell motility and cell invasion through a Matrigel membrane, regardless of the baseline epidermal growth factor receptor expression. Decreased vascular epidermal growth factor production and significant inhibition of growth only occurred in malignant pleural mesothelioma cells that overexpress epidermal growth factor receptor.ConclusionsEpidermal growth factor receptor tyrosine kinase inhibitor PD153035 significantly inhibited motility and invasion in malignant pleural mesothelioma cells in vitro, regardless of their epidermal growth factor receptor expression levels. Inhibition of epidermal growth factor receptor-dependent signaling might be a useful strategy to diminish malignant pleural mesothelioma recurrence after aggressive cytoreductive surgery

Cisplatin enhances apoptosis induced by a tumor-selective adenovirus expressing tumor necrosis factor–related apoptosis-inducing ligand

Cancer cells frequently exhibit resistance to the cytotoxic effect of tumor necrosis factor–related apoptosis-inducing ligand (TRAIL). Pretreatment of TRAIL-resistant cells with cisplatin sensitizes them to this ligand. Cisplatin also has been shown to enhance adenoviral transgene expression. This study aims to evaluate the ability of cisplatin to enhance the expression and the cytotoxic effect of the tumor-specific adenoviral vector Ad/gTRAIL, which expresses a green fluorescent protein–TRAIL fusion protein. Cultured cancer cells and normal human cells were infected with Ad/gTRAIL with or without cisplatin pretreatment. Adenoviral transgene expression was determined by using flow cytometry to measure green fluorescent protein fluorescence. Cytotoxicity was measured by using thiazolyl blue tetrazolium bromide assays and an apoptosis enzyme-linked immunosorbent assay kit. Green fluorescent protein–TRAIL fusion protein expression was significantly enhanced by cisplatin pretreatment in cancer cells. Cisplatin treatment before Ad/gTRAIL infection resulted in a 2- to 12-fold increase in green fluorescent protein fluorescence intensity across cancer lines. Although Ad/gTRAIL induced mild cytotoxicity in all cancer lines (inhibitory concentration of 50% values of >500 pfu/cell), pretreatment with cisplatin resulted in a dose-dependent enhancement of Ad/gTRAIL-mediated cytotoxicity, as indicated by the drastic reduction of inhibitory concentration of 50% values to 4 to 42 pfu/cell in all cell lines. There was no cytotoxicity noted in normal cells treated with both cisplatin and Ad/gTRAIL. Cisplatin pretreatment enhances Ad/gTRAIL cytotoxicity in malignant cells while not affecting normal cells. The mechanisms underlying this effect might include both enhancement of the susceptibility of cisplatin-treated cells to TRAIL and cisplatin-mediated enhancement of TRAIL expression in Ad/gTRAIL infected cells. These findings provide a rationale for development of Ad/gTRAIL-based therapy for thoracic malignancies