Jill A. Trendel

Vimentin expression in human squamous carcinoma cells: relationship with phenotypic changes and cadherin-based cell adhesion.

Phenotypic changes resembling an epithelial‐to‐mesenchymal transition often occur as epithelial cells become tumorigenic. Two proteins that have been implicated in this process are vimentin and N‐cadherin. In this study, we sought to establish a link between expression of vimentin and N‐cadherin as oral squamous epithelial cells undergo a morphologic change resembling an epithelial‐to‐mesenchymal transition. We found that N‐cadherin and vimentin did not influence the expression of one another. J. Cell. Biochem. 78:141–150, 2000.

The Interferon-γ-induced GTPase, mGBP-2, Inhibits Tumor Necrosis Factor α (TNF-α) Induction of Matrix Metalloproteinase-9 (MMP-9) by Inhibiting NF-κB and Rac Protein

Matrix metalloproteinase-9 (MMP-9) is important in numerous normal and pathological processes, including the angiogenic switch during tumor development and tumor metastasis. Whereas TNF-α and other cytokines up-regulate MMP-9 expression, interferons (IFNs) inhibit MMP-9 expression. We found that IFN-γ treatment or forced expression of the IFN-induced GTPase, mGBP-2, inhibit TNF-α-induced MMP-9 expression in NIH 3T3 fibroblasts, by inhibiting MMP-9 transcription. The NF-κB transcription factor is required for full induction of MMP-9 by TNF-α. Both IFN-γ and mGBP-2 inhibit the transcription of a NF-κB-dependent reporter construct, suggesting that mGBP-2 inhibits MMP-9 induction via inhibition of NF-κB-mediated transcription. Interestingly, mGBP-2 does not inhibit TNF-α-induced degradation of IκBα or p65/RelA translocation into the nucleus. However, mGBP-2 inhibits p65 binding to a κB oligonucleotide probe in gel shift assays and to the MMP-9 promoter in chromatin immunoprecipitation assays. In addition, TNF-α activation of NF-κB in NIH 3T3 cells is dependent on Rac activation, as evidenced by the inhibition of TNF-α induction of NF-κB-mediated transcription by a dominant inhibitory form of Rac1. A role for Rac in the inhibitory action of mGBP-2 on NF-κB is further shown by the findings that mGBP-2 inhibits TNF-α activation of endogenous Rac and constitutively activate Rac can restore NF-κB transcription in the presence of mGBP-2. This is a novel mechanism by which IFNs can inhibit the cytokine induction of MMP-9 expression.

The interferon-γ-induced GTPase, mGBP-2, inhibits TNF-α-induction of matrix metalloproteinase-9 (MMP-9) by inhibiting NF-κB and Rac.

Matrix metalloproteinase-9 (MMP-9) is important in numerous normal and pathological processes, including the angiogenic switch during tumor development and tumor metastasis. Whereas TNF-α and other cytokines up-regulate MMP-9 expression, interferons (IFNs) inhibit MMP-9 expression. We found that IFN-γ treatment or forced expression of the IFN-induced GTPase, mGBP-2, inhibit TNF-α-induced MMP-9 expression in NIH 3T3 fibroblasts, by inhibiting MMP-9 transcription. The NF-κB transcription factor is required for full induction of MMP-9 by TNF-α. Both IFN-γ and mGBP-2 inhibit the transcription of a NF-κB-dependent reporter construct, suggesting that mGBP-2 inhibits MMP-9 induction via inhibition of NF-κB-mediated transcription. Interestingly, mGBP-2 does not inhibit TNF-α-induced degradation of IκBα or p65/RelA translocation into the nucleus. However, mGBP-2 inhibits p65 binding to a κB oligonucleotide probe in gel shift assays and to the MMP-9 promoter in chromatin immunoprecipitation assays. In addition, TNF-α activation of NF-κB in NIH 3T3 cells is dependent on Rac activation, as evidenced by the inhibition of TNF-α induction of NF-κB-mediated transcription by a dominant inhibitory form of Rac1. A role for Rac in the inhibitory action of mGBP-2 on NF-κB is further shown by the findings that mGBP-2 inhibits TNF-α activation of endogenous Rac and constitutively activate Rac can restore NF-κB transcription in the presence of mGBP-2. This is a novel mechanism by which IFNs can inhibit the cytokine induction of MMP-9 expression.

Biomimetic Syntheses and Antiproliferative Activities of Racemic, Natural (−), and Unnnatural (+) Glyceollin I

A 14-step biomimetic synthetic route to glyceollin I (1.5% overall yield) was developed and deployed to produce the natural enantiomeric form in soy, its unnatural stereoisomer, and a racemic mixture. Enantiomeric excess was assessed by asymmetric NMR shift reagents and chiral HPLC. Antiproliferative effects were measured in human breast, ovarian, and prostate cancer cell lines, with all three chiral forms exhibiting growth inhibition (GI) in the low to mid μM range for all cells. The natural enantiomer, and in some cases the racemate, gave significantly greater GI than the unnatural stereoisomer for estrogen receptor positive (ER(+)) versus ER(-) breast/ovarian cell lines as well as for androgen receptor positive (AR(+)) versus AR(-) prostate cancer cells. Surprisingly, differences between ER(+) and ER(-) cell lines were not altered by media estrogen conditions. These results suggest the antiproliferative mechanism of glyceollin I stereoisomers may be more complicated than strictly ER interactions.

Catalytically Active Peptidylglycine α-Amidating Monooxygenase in the Media of Androgen-Independent Prostate Cancer Cell Lines

Peptidylglycine α-amidating monooxygenase (PAM) converts inactive terminal-glycine prohormones into their activated α-amidated forms. PAM is thought to play a role in the development of antiandrogen drug resistance in prostate cancer (CaP) through PAMactivated autocrine growth. On the basis of the previous finding that many lung cancer cell lines excrete PAM into their culture media, this study investigates PAM levels in media collected from human CaP cell line cultures. Androgen-independent DU145 and PC-3 prostate cancer cell lines exhibited readily detectable levels of PAM activity in extracts and media, whereas the androgen-dependent LNCaP cell line showed little or no activity. Because of the much larger volume of media versus cell extracts, more than 90% of the total PAM activity was located in the media for both the PC-3 and DU145 cell lines, providing a readily accessible source of CaP PAM. A simple, scalable method to obtain PAM from the culture media of androgen-independent human prostate cancer cell lines is described in this article. This approach provides a much easier means of collecting CaP-derived PAM than previously described cell fractionation procedures and should facilitate the investigations of the role and targeting of PAM in hormone-independent CaP. (Journal of Biomolecular Screening 2008:804-809)

Rapid and sensitive HPLC assay for simultaneous determination of procaine and para-aminobenzoic acid from human and rat liver tissue extracts

A sensitive and rapid high-performance liquid chromatography method has been developed for simultaneous determination of procaine and its metabolite p-aminobenzoic acid (PABA) from human and rat liver tissue extracts. The method has been validated according to ICH guidelines in terms of selectivity, linearity, lower limit of detection, lower limit of quantitation, accuracy, precision and recovery from human and rat liver tissue extracts. Chromatography was carried out on a Discovery C(18) column using 10mM ammonium acetate at pH 4.0 and acetonitrile as mobile phase. Retention times for procaine and PABA were 6.6 and 5.3 min, respectively. Linearity for each calibration curve in both tissue extracts was observed across a range from 10 microM to 750 microM for procaine and PABA. The lower limit of detection for both procaine and PABA was 5 microM and the lower limit of quantitation was 10 microM in both tissue extracts. The intra- and inter-day relative standard deviations (R.S.D.) for both procaine and PABA were <6%. Recoveries of procaine and PABA from human and rat liver tissue extracts were determined by two different methods with a single-step protein precipitation technique being employed in both methods. Recoveries for both procaine and PABA were greater than 80% from both human and rat liver tissue extracts.

The hurdle of antiandrogen drug resistance: drug design strategies

Introduction: Prostate cancer is the second most common cancer death in men after lung cancer, due to distant metastases. While distant prostate cancer is typically castrate resistant, it is not necessarily androgen independent. For this reason, a review of the literature regarding the pathways involved in androgen signaling and therapeutic regimens to treat distant metastases is beneficial to increasing the survival rate of prostate cancer patients. Areas covered: In this article, the author reviews the literature from the past decade covering metastatic hormone refractory prostate cancer with the aim to examine and identify pathways, therapeutic targets and current therapies for treating castrate-resistant disease. As this area is lacking, the author aims to provide the reader with knowledge of the molecular consequences of castrate resistant prostate cancer, the current treatment paradigms and future directions. Expert opinion: While there have been advances in the treatment of castrate resistant prostate cancer, only minimal advances have been made in overall survival rate. Due to aberrant mutations and activation in the androgen receptor gene, and the complexity of cell signaling within prostate cancer, the androgen receptor should remain a main target for drug discovery efforts. This author believes that designing compounds that will reduce the activation of the androgen receptor may hold the key to a cure in the future.

Early Stage Efficacy and Toxicology Screening for Antibiotics and Enzyme Inhibitors

The rise in organisms resistant to existing drugs has added urgency to the search for new antimicrobial agents. Aspartate β-semialdehyde dehydrogenase (ASADH) catalyzes a critical step in an essential microbial pathway that is absent in mammals. Our laboratory is using fragment library screening to identify efficient and selective ASADH inhibitors. These preliminary agents are then tested to identify compounds with desired antimicrobial properties for further refinement. Toward this end, we have established a microplate-based, dual-assay approach using a single reagent to evaluate antibiotic activity and mammalian cell toxicity during early stage development. The bacterial assay uses nonpathogenic bacteria to allow efficacy testing without a dedicated microbial laboratory. Toxicity assays are performed with a panel of mammalian cells derived from representative susceptible tissues. These assays can be adapted to target other microbial systems, such as fungi and biofilms, and additional mammalian cell lines can be added as needed. Application of this screening approach to antibiotic standards demonstrates the ability of these assays to identify bacterial selectivity and potential toxicity issues. Tests with selected agents from the ASADH inhibitor fragment library show some compounds with antibiotic activity, but as expected, most of these early agents display higher than desired mammalian cell toxicity.

hGBP-1 Expression Predicts Shorter Progression-Free Survival in Ovarian Cancers, While Contributing to Paclitaxel Resistance

Ovarian cancer is the gynecological cancer with the poorest prognosis. One significant reason is the development of resistance to the chemotherapeutic drugs used in its treatment. The large GTPase, hGBP-1, has been implicated in paclitaxel resistance in ovarian cell lines. Forced expression of hGBP-1 in SKOV3 ovarian cancer cells protects them from paclitaxel-induced cell death. However, prior to this study, nothing was known about whether hGBP-1 was expressed in ovarian tumors and whether its expression correlated with paclitaxel resistance. hGBP-1 is expressed in 17% of ovarian tumors from patients that have not yet received treatment. However, at least 80% of the ovarian tumors that recurred after therapies that included a tax-ane, either paclitaxel or docetaxel, were positive for hGBP-1. In addition, hGBP-1 expression predicts a significantly shorter progression-free survival in ovarian cancers. Based on these studies, hGBP-1 could prove to be a potential biomarker for paclitaxel resistance in ovarian cancer.