Energy Band Gap Investigation of Biomaterials: A Comprehensive Material Approach for Biocompatibility of Medical Electronic Devices.

Over the past ten years, tissue engineering has witnessed significant technological and scientific advancements. Progress in both stem cell science and additive manufacturing have established new horizons in research and are poised to bring improvements in healthcare closer to reality. However, more sophisticated indications such as the scale-up fabrication of biological structures (e.g., human tissues and organs) still require standardization. To that end, biocompatible electronics may be helpful in the biofabrication process. Here, we report the results of our systematic exploration to seek biocompatible/degradable functional electronic materials that could be used for electronic device fabrications. We investigated the electronic properties of various biomaterials in terms of energy diagrams, and the energy band gaps of such materials were obtained using optical absorption spectroscopy. The main component of an electronic device is manufactured with semiconductor materials (i.e., Eg between 1 to 2.5 eV). Most biomaterials showed an optical absorption edge greater than 2.5 eV. For example, fibrinogen, glycerol, and gelatin showed values of 3.54, 3.02, and 3.0 eV, respectively. Meanwhile, a few materials used in the tissue engineering field were found to be semiconductors, such as the phenol red in cell culture media (1.96 eV energy band gap). The data from this research may be used to fabricate biocompatible/degradable electronic devices for medical applications.

Nanosensors for therapeutic drug monitoring: implications for transplantation.

The number of patients requiring organ transplantations is exponentially increasing. New organs are either provided by healthy or deceased donors, or are grown in laboratories by tissue engineers. Post-surgical follow-up is vital for preventing any complications that can cause organ rejection. Physiological monitoring of a patient who receives newly transplanted organs is crucial. Many efforts are being made to enhance follow-up technologies for monitoring organ recipients, and point-of-care devices are beginning to emerge. Here, we describe the role of biosensors and nanosensors in improving organ transplantation efficiency, managing post-surgical follow-up and reducing overall costs. We provide an overview of the state-of-the-art biosensing technologies and offer some perspectives related to their further development.

Induction of heme oxygenase I (HMOX1) by HPP-4382: a novel modulator of Bach1 activity.

Oxidative stress is generated by reactive oxygen species (ROS) produced in response to metabolic activity and environmental factors. Increased oxidative stress is associated with the pathophysiology of a broad spectrum of inflammatory diseases. Cellular response to excess ROS involves the induction of antioxidant response element (ARE) genes under control of the transcriptional activator Nrf2 and the transcriptional repressor Bach1. The development of synthetic small molecules that activate the protective anti-oxidant response network is of major therapeutic interest. Traditional small molecules targeting ARE-regulated gene activation (e.g., bardoxolone, dimethyl fumarate) function by alkylating numerous proteins including Keap1, the controlling protein of Nrf2. An alternative is to target the repressor Bach1. Bach1 has an endogenous ligand, heme, that inhibits Bach1 binding to ARE, thus allowing Nrf2-mediated gene expression including that of heme-oxygenase-1 (HMOX1), a well described target of Bach1 repression. In this report, normal human lung fibroblasts were used to screen a collection of synthetic small molecules for their ability to induce HMOX1. A class of HMOX1-inducing compounds, represented by HPP-4382, was discovered. These compounds are not reactive electrophiles, are not suppressed by N-acetyl cysteine, and do not perturb either ROS or cellular glutathione. Using RNAi, we further demonstrate that HPP-4382 induces HMOX1 in an Nrf2-dependent manner. Chromatin immunoprecipitation verified that HPP-4382 treatment of NHLF cells reciprocally coordinated a decrease in binding of Bach1 and an increase of Nrf2 binding to the HMOX1 E2 enhancer. Finally we show that HPP-4382 can inhibit Bach1 activity in a reporter assay that measures transcription driven by the human HMOX1 E2 enhancer. Our results suggest that HPP-4382 is a novel activator of the antioxidant response through the modulation of Bach1 binding to the ARE binding site of target genes.

3D in vitro tissue models and their potential for drug screening.

INTRODUCTION: The development of one standard, simplified in vitro three-dimensional tissue model suitable to biological and pathological investigation and drug-discovery may not yet be feasible, but standardized models for individual tissues or organs are a possibility. Tissue bioengineering, while concerned with finding methods of restoring functionality in disease, is developing technology that can be miniaturized for high throughput screening (HTS) of putative drugs. Through collaboration between biologists, physicists and engineers, cell-based assays are expanding into the realm of tissue analysis. Accordingly, three-dimensional (3D) micro-organoid systems will play an increasing role in drug testing and therapeutics over the next decade. Nevertheless, important hurdles remain before these models are fully developed for HTS. AREAS COVERED: We highlight advances in the field of tissue bioengineering aimed at enhancing the success of drug candidates through pre-clinical optimization. We discuss models that are most amenable to high throughput screening with emphasis on detection platforms and data modeling. EXPERT OPINION: Modeling 3D tissues to mimic in-vivo architecture remains a major challenge. As technology advances to provide novel methods of HTS analysis, so do potential pitfalls associated with such models and methods. We remain hopeful that integration of biofabrication with HTS will significantly reduce attrition rates in drug development.

Genomic and phenotypic analysis reveals a key role for CCN1 (CYR61) in BAG3-modulated adhesion and invasion.

Chaperone protein quantity may regulate the balance of proteins involved in invasion and malignancy. BAG3 is a co-chaperone and pro-survival protein that has been implicated in adhesion, migration, and metastasis. We reported that BAG3 overexpression in MDA435 human breast cancer cells results in a significant decrease in migration and adhesion to matrix molecules that is reversed upon deletion of the BAG3 proline-rich domain (dPXXP). We now hypothesize that transcriptional analysis would identify proteins involved in matrix-related processes that are regulated by BAG3 and/or its PXXP domain mutant. Expression array analysis of MDA435 cells overexpressing either wild-type BAG3 (FL) or dPXXP identified CCN1 as a BAG3 target protein. CCN1 is a known AP-1 target. Increased AP-1 transcriptional activity and AP-1 DNA-binding was found in MDA435 dPXXP cells. Consistent with these findings, CCN1 quantity and secretion were increased in dPXXP mutants but suppressed in FL cells; both BAG3 forms resulted in up-regulated CCN1 in HeLa cells. CCN1 silencing in the BAG3 FL overexpressors reduced the already low phospho-integrin beta1 in response to attachment on collagen IV. Matrigel invasion of HeLa cells engineered with the BAG3 constructs was enhanced in FL cells and minimal in dPXXP cells. CCN1 silencing blocked a greater percentage of the serum-induced invasion in FL cells than in dPXXP cells. This implies a context-dependent function of BAG3 on CCN1 and thus mesenchymal behaviour. CCN1 may be necessary for adhesion and matrix-related signalling in FL cells, abrogating a negative signal of the PXXP domain when BAG3 is intact. We propose that BAG3 regulates CCN1 expression to regulate tumour cell adhesion and migration.

The anti-apoptotic activity of BAG3 is restricted by caspases and the proteasome.

BACKGROUND: Caspase-mediated cleavage and proteasomal degradation of ubiquitinated proteins are two independent mechanisms for the regulation of protein stability and cellular function. We previously reported BAG3 overexpression protected ubiquitinated clients, such as AKT, from proteasomal degradation and conferred cytoprotection against heat shock. We hypothesized that the BAG3 protein is regulated by proteolysis. METHODOLOGY/PRINCIPAL FINDINGS: Staurosporine (STS) was used as a tool to test for caspase involvement in BAG3 degradation. MDA435 and HeLa human cancer cell lines exposed to STS underwent apoptosis with a concomitant time and dose-dependent loss of BAG3, suggesting the survival role of BAG3 was subject to STS regulation. zVAD-fmk or caspase 3 and 9 inhibitors provided a strong but incomplete protection of both cells and BAG3 protein. Two putative caspase cleavage sites were tested: KEVD (BAG3(E345A/D347A)) within the proline-rich center of BAG3 (PXXP) and the C-terminal LEAD site (BAG3(E516A/D518A)). PXXP deletion mutant and BAG3(E345A/D347A), or BAG3(E516A/D518A) respectively slowed or stalled STS-mediated BAG3 loss. BAG3, ubiquitinated under basal growth conditions, underwent augmented ubiquitination upon STS treatment, while there was no increase in ubiquitination of the BAG3(E516A/D518A) caspase-resistant mutant. Caspase and proteasome inhibition resulted in partial and independent protection of BAG3 whereas inhibitors of both blocked BAG3 degradation. STS-induced apoptosis was increased when BAG3 was silenced, and retention of BAG3 was associated with cytoprotection. CONCLUSIONS/SIGNIFICANCE: BAG3 is tightly controlled by selective degradation during STS exposure. Loss of BAG3 under STS injury required sequential caspase cleavage followed by polyubiquitination and proteasomal degradation. The need for dual regulation of BAG3 in apoptosis suggests a key role for BAG3 in cancer cell resistance to apoptosis.

CAIR-1/BAG-3 modulates cell adhesion and migration by downregulating activity of focal adhesion proteins.

CAIR-1/BAG-3 is a stress and survival protein that has been shown to bind SH3 domain-containing proteins through its proline-rich (PXXP) domain. Because stress and survival pathways are active during invasion and metastasis, we hypothesized that CAIR-1 is a regulator of signaling pathways that modulate cell adhesion and migration. MDA-435 human breast carcinoma cells were stably transfected with full-length CAIR-1 (FL) or a proline-rich domain deleted mutant (dPXXP). FL cells migrated poorly through collagen IV-coated filters to serum (14% of control, p=0.0004), whereas migration of dPXXP cells was more robust (228%, p=0.00001). Adhesion to collagen IV-coated surfaces was reduced in FL cells and augmented in dPXXP cells (FL 64%, p=0.03; dPXXP 138%, p=0.01). Rhodamine-phalloidin staining highlighted more stress fibers and thicker filopodial protrusions in dPXXP cells. Fewer focal adhesions were also seen in FL cells. A reduction in tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin occurred in FL cells under these conditions. In contrast, increased FAK and paxillin phosphorylation was documented in dPXXP cells. Differential FAK phosphorylation occurred at the major autophosphorylation site Y(397) and Src phosphorylation site Y(861). Concordant with these findings, there was decreased interaction between FAK and its downstream partners p(130)Cas and Crk observed in FL cells but not in dPXXP cells. These results collectively indicate that CAIR-1 may negatively regulate adhesion, focal adhesion assembly, signaling, and migration via its PXXP domain.

Tumor microenvironment: what can effusions teach us?

Malignant effusions, which are composed of malignant pleural and peritoneal fluid, are an unusual manifestation of cancer and frequently portend a poor prognosis. Neoplastic cells that disseminate into cavities containing effusions are highly metastatic and possess a strong autonomous proliferative drive while concurrently being stimulatory of exudative effusions. Most effusions will respond to transient therapeutic intervention, including the obliteration of potential space via pleurodesis. Cure, however, is rare, thus making effusions a biologically, biochemically, and clinically important topic of study. The local microenvironment that supports malignant growth, invasion, and dissemination of the solid primary tumor into the vasculature is composed of activated stroma that includes scaffolding consisting of materials that promote the tumor function, and vascular structures to provide conduits for travel and nutrient delivery. Less is understood about the tumor-cell microenvironment in malignant effusions. The fluid nature of such a microenvironment when compared with the solid primary tumor may have significant implications for disease dissemination and progression. Dissecting the signaling activity and components of such microenvironments will improve our understanding and ultimately our ability to provide better patient care.

DU145 human prostate carcinoma invasiveness is modulated by urokinase receptor (uPAR) downstream of epidermal growth factor receptor (EGFR) signaling.

Tumor cell motility and invasion have been linked to upregulated signaling from both the epidermal growth factor receptor (EGFR) and that for urokinase-type plasminogen activator (uPAR). However, we do not know whether these events are interdependent or unrelated, despite the obvious diagnostic and therapeutic implications. Gene microarray analyses have suggested that EGFR signaling via phospholipase C-gamma (PLCgamma) induces uPAR transcription. We utilized two sublines of the DU145 human prostate carcinoma cell line that are genetically engineered to differentially activate the EGFR/PLCgamma cascade and are variously invasive in vitro and in vivo. uPAR protein levels in these cells were found to be dependent on PLC signaling, pharmacologic inhibition of PLC signaling reduced uPAR expression. To determine whether uPAR was a required element in EGFR-mediated invasion, we stably expressed uPAR cDNA in either sense or antisense orientation in the two DU145 sublines. Interestingly, uPA production was modulated in parallel, although to a lesser degree, with uPAR in these sublines. Antisense to uPAR significantly restricted invasion of the highly invasive DU145 WT cells through Matrigel and reduced aggressiveness of tumors in nude mice. Up-regulation of uPAR significantly increased the invasiveness of the moderately invasive DU145 parental (DU145 P) cells through Matrigel, but this increased invasiveness was not seen in mice. uPA activity appears to contribute to invasiveness at least through Matrigel, as antibody to uPA or amiloride limited the transmigration. These results support a model of tumor invasion promoted by autocrine EGFR signaling involving reinforcing altered gene expression, of uPAR at least, that further induces cell motility. Herein, a number of key molecules whose expression levels are interrelated, including both EGFR and uPAR, are required but none are sufficient in the absence of other keys molecules in promoting tumor progression.

Epidermal growth factor receptor-stimulated activation of phospholipase Cgamma-1 promotes invasion of head and neck squamous cell carcinoma.

Lymph node metastasis and local invasion of head and neck squamous cell carcinoma (HNSCC) is associated with a poor prognosis. However, little is known about the factors governing tumor cell invasion in HNSCC. Phospholipase Cgamma-1 (PLCgamma-1) contributes to tumor cell invasion in experimental systems when activated by the epidermal growth factor receptor (EGFR). We hypothesized that EGFR overexpression in HNSCC mediates invasion via PLCgamma-1. On EGFR ligand stimulation, phosphorylation of PLCgamma-1 increased in all of the HNSCC cell lines tested (4 of 4). In the presence of EGFR-specific tyrosine kinase inhibitor (PD153035) or an anti-EGFR antibody (C225), PLCgamma-1 activation was abrogated indicating that PLCgamma-1 was downstream of EGFR. Blocking cellular PLC with an inhibitor (U73122) reduced inositol phosphate turnover in all of the HNSCC cell lines examined, and treatment with the PLC inhibitor or antisense oligonucleotides targeting PLCgamma-1 significantly reduced in vitro invasiveness of HNSCC cell lines through Matrigel. To determine the clinical relevance of these findings, we compared levels of PLCgamma-1 in tumor and paired normal tissue from 33 patients with HNSCC. PLCgamma-1 levels were significantly higher (P < 0.0001) in the tumors compared with the normal mucosa of HNSCC patients. Levels of activated PLCgamma-1 were analyzed in 20 patients. Tumors expressed higher levels of phosphorylated PLCgamma-1 compared with normal adjacent mucosa (P = 0.05). Thus, PLCgamma-1 may mediate invasion and metastasis downstream of EGFR in HNSCC.

Calcium as a molecular target in angiogenesis.

The plethora of cellular pathways and events involved in angiogenesis are a prime example of the widespread role of calcium ion flux in biological functions. Indeed, calcium is a main point of intersection for many distinct molecular signaling pathways that promote and modulate angiogenesis. Here, we illustrate some of the important aspects of calcium induction, function, downstream effects, and resulting cellular changes that ensue. We describe some of the main mechanisms of calcium regulation in cells as well as intracellular and cross-membrane flux, highlighting key players that are known to facilitate these events. We review some of the major signaling pathways that tie into angiogenesis, and also describe how cellular phenotypic changes that occur during angiogenesis require processes rich in calcium ion stimulation of gradient shifts. Lastly, we hypothesize on current thinking of the role of calcium as a whole in angiogenic cellular function and propose new insight into calcium as a universal effector molecule and a prime target for therapeutic intervention.

EBV-expressing AGS gastric carcinoma cell sublines present increased motility and invasiveness.

Tumor invasion marks a critical point in cancer progression; it is a harbinger of morbidity and mortality. Thus, the cellular events that enable the invasive phenotype are under intense investigation. Epstein-Barr virus (EBV) is associated with a number of cancers, including Burkitt lymphoma (BL) and nasopharyngeal carcinoma (NPC) and is suspected to contribute to their tumorigenesis. On average, 8% of gastric carcinomas have been shown to carry this virus. To explore whether the presence of EBV in gastric carcinoma contributes to tumor progression in this predominantly invasive carcinoma, we examined a panel of 2 in vitro EBV-infected human gastric cancer cell line sublines and their mock-infected AGS parental control line. We found EBV infection caused a marked increase in transmigration of a Matrigel barrier (415% and 303%, p < 0.05, for the 2 infected lines). This correlated with increased motility of these sublines (233% and 140%, p < 0.05). As this pattern of increased motility leading to a more pronounced enhancement of invasion has been noted in other tumor cells, we explored the roles of autocrine signaling pathways previously implicated in carcinoma motility and invasion. Inhibitors to the epidermal growth factor receptor (EGFR) (PD153035), phospholipase C (PLC) (U73122), extracellular-signal regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) (PD089035) and PI-3 kinase (Wortmannin) were not informative. These data suggest that EBV increases migration of AGS cells by a mechanism independent of these autocrine growth factor-induced pathways. Instead, we found that the EBV-infected cells presented increased focal adhesion kinase (FAK) phosphorylation. These findings suggest a role for integrin-mediated signaling in promoting EBV-associated invasiveness.

Growth factor-induced cell motility in tumor invasion.

Tumor progression to the invasive and metastatic states dramatically enhances the morbidity and mortality of cancer. Rational therapeutic interventions will only be possible when we understand the molecular mechanisms governing the cell behavior underlying this transformation. For invasion, a subpopulation of tumor cells must recognize the extracellular matrix barrier, modify the barrier, migrate through the barrier, and then proliferate in the adjacent but ectopic locale. Prevention of any one of these steps would prevent invasion, but determining the most sensitively dysregulated step should provide the most promising therapeutic index. In many invasive tumors, upregulation of active motility is stimulated by growth factor receptor signaling, the EGF receptor being the most frequently implicated. Two key downstream molecular switches, PLCgamma and m-calpain, are required for growth factor-induced motility but not basal, matrix-stimulated motility. Inhibition of either of these enzymes blocks in vitro and in vivo invasion of prostate, breast, and bladder carcinomas and glioblastomas. These represent novel and potentially selective targets for drug development. Future advances in the imaging of tumors in animals and ex vivo organ culture systems should provide additional new targets.

Luteinizing hormone-releasing hormone agonist limits DU-145 prostate cancer growth by attenuating epidermal growth factor receptor signaling.

PURPOSE: Advanced prostate cancer is treated initially by central suppression of androgen production by luteinizing hormone-releasing hormone (LHRH) agonists. Intriguingly, even hormone-independent cancers often show some, if only slight, growth retardation when these agonists are delivered in pharmacological doses. Previous studies have shown in cell lines and animal xenograft models that activation of peripheral LHRH receptors on prostate carcinoma cells lead to growth suppression. In parallel, there is a decrease of epidermal growth factor receptors (EGFRs) and activity. Because autocrine EGFR stimulation exists in most, if not all, prostate carcinomas and is required for cell proliferation, we asked whether LHRH signaling cross-attenuated EGFR to limit tumor growth. One possible mechanism was suggested by LHRH receptors triggering phospholipase-C (PLC) to activate protein kinase C (PKC) because PKC activation limits EGFR tyrosine kinase activity by phosphorylating EGFR at threonine 654. EXPERIMENTAL DESIGN: To determine the role of this cross-attenuation mechanism, we mutated the threonine 654 amino acid to an alanine (A654) to abrogate this inhibition. DU-145 cells stably expressing wild-type and A654 EGFR were grown as xenografts in the s.c. space of athymic mice. RESULTS: DU-145 cells, overexpressing wild-type EGFR, formed tumors in athymic mice that were inhibitable by goserelin acetate (Zoladex). Tumors expressing the A654 EGFR were resistant to this growth inhibition. These results paralleled in vitro studies in which goserelin acetate blocked proliferation of the WT DU-145 but not A654 DU-145 cells. CONCLUSIONS: These data support the model of LHRH agonists preventing EGFR-mediated tumor growth through a PKC pathway. This suggests new targets of modulatory intervention to limit the growth of androgen-independent prostate carcinomas.

Motility is rate-limiting for invasion of bladder carcinoma cell lines.

Induced migration of tumor cells is generally considered to be one critical step in cancer progression to the invasive and metastatic stage. The implicit caveat of studies that show this is that other, unknown, signaling pathways and biophysical events are actually the operative rate-limiting steps, and not motility per se. Thus, to examine the hypothesis that motility is a single, but overall rate-limiting function required for invasion, disparate motility processes need be blocked with concordant effects on tumor invasion. Recently, we and others have described two signaling pathways that are critical to growth factor-induced motility but not mitogenesis. The key molecular switches are phospholipase C-gamma (PLCgamma) and calpain for cytoskeletal reorganization and rear detachment, respectively. We examined this hypothesis in a highly invasive tumor, bladder carcinoma. Three different human tumor cell lines, 253J-B-V, UMUC and T-24, were tested for invasiveness in vitro by transmigration of a Matrigel barrier. Inhibiting PLCgamma with the pharmacologic agent U73122 or the molecular dominant-negative PLCz construct reduced both invasiveness and motility. The same was noted when calpain was blocked using calpain inhibitor I (ALLN). These results demonstrate that one interventional target for limiting invasion is not necessarily an individual motility pathway but rather cell migration per se.