Ingalill Avis

Five-lipoxygenase inhibitors can mediate apoptosis in human breast cancer cell lines through complex eicosanoid interactions.

Many arachidonic acid metabolites function in growth signaling for epithelial cells, and we previously reported the expression of the major arachidonic acid enzymes in human breast cancer cell lines. To evaluate the role of the 5‐lipoxygenase (5‐LO) pathway on breast cancer growth regulation, we exposed cells to insulinlike growth factor‐1 or transferrin, which increased the levels of the 5‐LO metabolite, 5(S)‐hydrooxyeicosa‐6E,8C,11Z,14Z‐tetraenoic acid (5‐HETE), by radioimmunoassay and high‐performance liquid chromatography. Addition of 5‐HETE to breast cancer cells resulted in growth stimulation, whereas selective biochemical inhibitors of 5‐LO reduced the levels of 5‐HETE and related metabolites. Application of 5‐LO or 5‐LO activating protein‐directed inhibitors, but not a cyclooxygenase inhibitor, reduced growth, increased apoptosis, down‐regulated bcl‐2, up‐regulated bax, and increased G1 arrest. Exposure of breast cancer cells to a 5‐LO inhibitor up‐regulated peroxisome proliferator‐activated receptor (PPAR)α and PPARγ expression, and these same cells were growth inhibited when exposed to relevant PPAR agonists. These results suggest that disruption of the 5‐LO signaling pathway mediates growth arrest and apoptosis in breast cancer cells. Additional experiments suggest that this involves the interplay of several factors, including the loss of growth stimulation by 5‐LO products, the induction of PPARγ, and the potential activation of PPARγ by interactions with shunted endoperoxides.

Inhibitors of the Arachidonic Acid Pathway and Peroxisome Proliferator–Activated Receptor Ligands Have Superadditive Effects on Lung Cancer Growth Inhibition

Arachidonic acid (AA) metabolizing enzymes and peroxisome proliferator-activated receptors (PPARs) have been shown to regulate the growth of epithelial cells. We have previously reported that exposure to the 5-lipoxygenase activating protein-directed inhibitor MK886 but not the cyclooxygenase inhibitor, indomethacin, reduced growth, increased apoptosis, and up-regulated PPARalpha and gamma expression in breast cancer cell lines. In the present study, we explore approaches to maximizing the proapoptotic effects of PPARgamma on lung cancer cell lines. Non-small-cell cancer cell line A549 revealed dose-dependent PPARgamma reporter activity after treatment with MK886. The addition of indomethacin in combination with MK886 further increases reporter activity. We also show increased growth inhibition and up-regulation of apoptosis after exposure to MK886 alone, or in combination with indomethacin and the PPAR ligand, 15-deoxy-Delta12,14-prostaglandin J2 compared with single drug exposures on the adenocarcinoma cell line A549 and small-cell cancer cell lines H345, N417, and H510. Real-time PCR analyses showed increased PPAR mRNA and retinoid X receptor (RXR)alpha mRNA expression after exposure to MK886 and indomethacin in a time-dependent fashion. The results suggest that the principal proapoptotic effect of these drugs may be mediated through the known antiproliferative effects of the PPARgamma-RXR interaction. We therefore explored a three-drug approach to attempt to maximize this effect. The combination of low-dose MK886, ciglitazone, and 13-cis-retinoic acid interacted at least in a superadditive fashion to inhibit the growth of lung cancer cell lines A549 and H1299, suggesting that targeting PPARgamma and AA action is a promising approach to lung cancer growth with a favorable therapeutic index.

Differential expression of the early lung cancer detection marker, heterogeneous nuclear ribonucleoprotein-A2/B1 (hnRNP-A2/B1) in normal breast and neoplastic breast cancer.

Heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNP-A2/B1) is highly expressed during critical stages of lung development and carcinogenesis. To determine if the expression of hnRNP-A2/B1 is an informative biomarker in breast carcinogenesis, we analyzed hnRNP-A2/B1 overexpression by immunohistochemistry in archived specimens. Expression was detected in 48/85 (56.5%) primary invasive breast cancers and 7/72 (9.7%) specimens of normal breast tissue. Northern analysis of breast cancer cells also demonstrated higher level of hnRNP-A2/B1 expression compared to normal or transformed breast cells. Expression of hnRNP-A2/B1 in breast cancer cells was decreased by exposure to retinoids coordinately with decreased cell growth. These results warrant further evaluation of hnRNP-A2/B1 as a marker of breast carcinogenesis.

Ingenuity Network-Assisted Transcription Profiling: Identification of a New Pharmacologic Mechanism for MK886

The small molecular inhibitor MK886 is known to block 5-lipoxygenase-activating protein ALOX5AP and shows antitumor activity in multiple human cell lines. The broad antitumor therapeutic window reported in vivo for MK886 in rodents supports further consideration of this structural class. Better understanding of the mode of action of the drug is important for application in humans to take place. Affymetrix microarray study was conducted to explore MK886 pharmacologic mechanism. Ingenuity Pathway Analysis software was applied to validate the results at the transcriptional level by putting them in the context of an experimental proteomic network. Genes most affected by MK886 included actin B and focal adhesion components. A subsequent National Cancer Institute-60 panel study, RT-PCR validation followed by confocal microscopy, and Western blotting also pointed to actin B down-regulation, filamentous actin loss, and disorganization of the transcription machinery. In agreement with these observations, MK886 was found to enhance the effect of UV radiation in H720 lung cancer cell line. In light of the modification of cytoskeleton and cell motility by lipid phosphoinositide 3-kinase products, MK886 interaction with actin B might be biologically important. The low toxicity of MK886 in vivo was modeled and explained by binding and transport by dietary lipids. The rate of lipid absorbance is generally higher for tumors, suggesting a promise of a targeted liposome-based delivery system for this drug. These results suggest a novel antitumor pharmacologic mechanism.

Randomized, Double-Blind, Placebo-Controlled Phase IIB Trial of the Cyclooxygenase Inhibitor Ketorolac as an Oral Rinse in Oropharyngeal Leukoplakia

Purpose: Nonselective cyclooxygenase (COX) inhibitors have been reported to decrease the frequency of upper aerodigestive cancers. Ketorolac tromethamine oral rinse has been shown to resolve another COX-dependent process, periodontal disease, without incurring gastrointestinal side effects. This trial evaluated if a topically delivered oral rinse containing ketorolac was as safe as and more effective than oral rinse alone in reducing the area of oral leukoplakia. Experimental Design: 57 patients were randomized (2:1 ratio) in a double-blind, placebo-controlled study of ketorolac (10 ml of a 0.1% ketorolac rinse solution; n = 38) or placebo (10 ml of rinse solution; n = 19) given twice daily for 30 s over 90 days. Primary end point was evaluated visually obtaining bidimensional measurement of the size of leukoplakia lesion(s) at entry and at 90 days. Secondary end point was histological assessment of the leukoplakia as sampled by serial punch biopsy and independently reviewed by three pathologists. Results: The patients included 67% males, 11% non-Caucasian, and 86% used tobacco with no significant differences between the two arms. Both rinses were well tolerated with good compliance, and there was no significant difference in adverse events (P = 0.27). Major response rate (complete response and partial response) was 30% for ketorolac and 32% for the placebo arm. There was no significant difference in change in histology between the two arms. Conclusion: Local delivery of a COX-containing oral rinse was well tolerated but produced no significant reduction in the extent of leukoplakia compared with the placebo. However, the favorable response rate to placebo arm remains unexplained and additional investigation of the tissue penetration with ketorolac is warranted.

Clinical use of a monoclonal antibody to bombesin-like peptide in patients with lung cancer.

Small cell lung cancer (SCLC) is one of four major types of lung cancer and constitutes roughly 25% of all the new cases. Unfortunately, despite major improvements in overall patient survival as a result of combination chemotherapy, over 90% of all SCLC patients die of their tumor.’,’ More effective treatments are clearly needed. Drug development programs based on empiric screening of various compounds have been successful for treatment of certain tumors, but not of lung cancer, despite massive investments of time and money.’*‘ Many cancer investigators feel that therapeutic progress for lung cancer will be made by exploiting the increased understanding of tumor cell biology in order to use rationally based therapies. The observation that a monoclonal antibody which binds to gastrin-releasing

Growth Factor Effects on Small Cell Lung Cancer Cells Using a Colorimetric Assay: Can a Transferrin-Like Factor Mediate Autocrine Growth?

A semiautomated colorimetric assay (MTT assay), based on the ability of live cells to reduce a tetrazolium-based compound, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), to a purplish colored formazan product that can be measured spectrophotometrically, has recently been adapted for use in drug sensitivity analysis of cultured human tumor cell lines. We report the application of this assay for the evaluation of the growth factor requirements of human small cell lung cancer (SCLC) cell lines. Specifically, the growth stimulation of each constituent of a previously reported serum-free defined medium system for SCLC including various concentrations of hydrocortisone, insulin, transferrin, 17 beta-estradiol, and selenium (HITES) was evaluated. The optimal concentrations for insulin, transferrin, and selenium derived in the previously reported experiments with direct counting of viable cells were similar to optimal concentrations determined for the growth of three SCLC cell lines (NCI-H82, NCI-N417, NCI-H526) using the MTT assay. In contrast to the previous report, the growth-stimulating effects of hydrocortisone and 17 beta-estradiol were negligible. Using the MTT we have shown that a SCLC cell line, NCI-H345 (which has been previously reported to produce a transferrin-like molecule), was growth-inhibited by an anti-transferrin receptor antibody, when grown in transferrin-free media. The conditioned media from this cell line is stimulatory to other transferrin-sensitive cell lines, suggesting the possibility of an autocrine role for this transferrin-like molecule at least in that cell line. With carefully defined conditions for a given cell line in which cell density and other parameters are within a range of constant MTT metabolism, the assay is well suited for precise analysis of growth factor effects.

Scientific basis for cancer prevention. Intermediate cancer markers.

Promising cancer clinical trials results involving the disruption of early stages of cancer with intervention agents such as tamoxifen or retinoids have led to significant new research interest in developing preventative strategy for the control of epithelial cancers. Key to the efficient progress in this field is a clear understanding of the complex biology of the early stages of cancerization that proceed on the epithelial surface. Systematic analysis of the biology of strategic targets such as growth factors is one approach to this problem. Gastrin‐releasing peptide is an autocrine growth factor for certain types of lung cancer cells. Mechanisms involved in the production and activation of this peptide are discussed as an example of how rational approaches to neutralization of cancer promotion biology can be achieved. The tools to monitor the success of this type of intervention also emerge from the understanding of the biology of growth factors, and intermediate end point markers that determine the presence or effects of a growth factor are attractive candidates for evaluation. Additional biologic tools reflecting the early stages of the cancer process need to be validated for use in serially evaluating the status of the relevant epithelium so that the ongoing success of a cancer intervention procedure can be established. Through this type of translational research, important applications of molecular biology may greatly improve the success of preventative strategies for cancer control.

Novel Phenotypic Fluorescent Three-Dimensional Platforms for High-throughput Drug Screening and Personalized Chemotherapy

We have developed novel phenotypic fluorescent three-dimensional co-culture platforms that efficiently and economically screen anti-angiogenic/anti-metastatic drugs on a high-throughput scale. Individual cell populations can be identified and isolated for protein/gene expression profiling studies and cellular movement/interactions can be tracked by time-lapse cinematography. More importantly, these platforms closely parallel the in vivo angiogenic and metastatic outcomes of a given tumor xenograft in the nude mouse model but, unlike in vivo models, our co-culture platforms produce comparable results in five to nine days. Potentially, by incorporating cancer patient biopsies, the co-culture platforms should greatly improve the effectiveness and efficiency of personalized chemotherapy.

Solid-phase peptide quantitation assay using labeled monoclonal antibody and glutaraldehyde fixation☆

A solid-phase radioimmunoassay utilizing iodinated peptide-specific monoclonal antibody as a detection system instead of labeled peptide has been developed. Regional specific monoclonal antibodies to either gastrin-releasing peptide or gastrin were used as models to validate the general application of our modified assay. Conditions for radioactive labeling of the monoclonal antibody were determined to minimize oxidant damage, which compromises the sensitivity of other reported peptide quantitation assays. Pretreatment of 96-well polyvinyl chloride test plates with a 5% glutaraldehyde solution resulted in consistent retention of sufficient target peptide on the solid-phase matrix to allow precise quantitation. This quantitative method is completed within 1 h of peptide solid phasing. Pretreatment of assay plates with glutaraldehyde increased binding of target peptide and maximized antibody binding by optimizing antigen presentation. The hypothesis that glutaraldehyde affects both peptide binding to the plate and orientation of the peptide was confirmed by analysis of several peptide analogs. These studies indicate that peptide binding was mediated through a free amino group leaving the carboxy-terminal portion of the target peptide accessible for antibody binding. It was observed that the length of the peptide also affects the amount of monoclonal antibody that will bind. Under the optimal conditions, results from quantitation of gastrin-releasing peptide in relevant samples agree well with those from previously reported techniques. Thus, we report here a modified microplate assay which may be generally applied for the rapid and sensitive quantitation of peptide hormones.