Cheryl Leece

Clinical pharmacology studies of Oltipraz — A potential chemopreventive agent

SummaryPharmacological studies on Oltipraz [4-methyl-5(pyrazinyl-2)-1-2-dithiole-3-thione)] were conducted with normal healthy subjects using various doses and schedules. Administration of single doses (1, 2 and 3 mg/kg) resulted in detectable drug levels in the serum (mean peak serum concentrations 16, 61 and 205 ng, respectively) and urine. The t1/2 was short (4.4, 4.1 and 5.3 hours respectively) and no steady state was achieved after multiple daily doses for 12 days. Introduction of a loading dose during the first day produced a steady state when 1.5 and 2.0 mg/kg/day were used. Daily administration of Oltipraz sustained the steady state with insignificant variations. Consumption of a high fat diet increased the serum and urine concentrations of Oltipraz (30–60%) compared to the low fat diet. Two subjects experienced flatulence during the administration of the drug. One subject developed numbness and pain in the thumbs with occurrence of small purplishblack spots resembling those observed in subacute endocarditis. These changes disappeared 10 days after discontinuation of the drug. No changes in peripheral blood counts, biochemical profile or thyroid function tests were observed after four weeks of Oltipraz. Further studies with a larger number of healthy subjects are needed for clarification of the safety and biological efficacy of small doses of Oltipraz during chronic administration.

Maternal Catecholamine Levels in Midpregnancy and Risk of Preterm Delivery

Associations between stress hormones and preterm delivery have not been fully explored. In this study, pregnant women enrolled from 52 clinics in 5 Michigan communities (1998-2004) provided urine samples for 3 days (waking and bedtime) during midpregnancy. Urinary catecholamine levels (epinephrine, norepinephrine, and dopamine) were measured in a subcohort (247 preterm and 760 term deliveries), and a 3-day median value was calculated. Polytomous logistic regression models assessed relations between catecholamine quartiles (of the median) and a 4-level outcome variable (i.e., term (referent) and 3 preterm delivery subtypes: spontaneous; premature rupture of membranes; and medically indicated). Final models incorporated other relevant covariates (e.g., creatinine, demographic, behavior). The risk of spontaneous preterm delivery was increased in the highest versus lowest quartile of norepinephrine and dopamine: norepinephrine, waking (adjusted odds ratio (AOR) = 3.7, 95% confidence interval (CI): 1.8, 7.9) and bedtime (AOR = 2.5, 95% CI: 1.3, 4.9); dopamine, waking (AOR = 2.6, 95% CI: 1.4, 5.1) and bedtime (AOR = 2.3, 95% CI: 1.2, 4.6). Adjusted odds ratios were further strengthened after removing women whose placentas showed evidence of acute infection or vascular pathology. High catecholamine levels in maternal urine may be indicative of excess stressors and/or predisposition to elevated sympathetic activation that contributes to increased risk of spontaneous preterm delivery.

Endothelin-1 Enriched Tumor Phenotype Predicts Breast Cancer Recurrence

Introduction. Breast cancer recurrence can develop years after primary treatment. Crosstalk between breast cancer cells and their stromal microenvironment may influence tumor progression. Our primary study aim was to determine whether endothelin-1 (ET-1) expression in tumor and stroma predicts breast cancer relapse. The secondary aim was to determine ET-1/endothelin receptor A (ETAR) role on signaling pathways and apoptosis in breast cancer. Experimental Design. Patients with histologically documented stages I–III invasive breast cancer were included in the study. ET-1 expression by immunohistochemistry (IHC) in tumor cells and stroma was analyzed. Association between ET-1 expression and clinical outcome was assessed using multivariate Cox proportional hazard model. Kaplan-Meier curves were used to estimate disease-free survival (DFS). In addition, the effect of ET-1/ETAR on signaling pathways and apoptosis was evaluated in MCF-7 and MDA-MB-231 breast cancer cells. Results. With a median followup of 7 years, ET-1 non-enriched tumor phenotype had a significant association with favorable disease-free survival (HR = 0.16; 95% CI 0.03–0.77; P value <0.02). ER negativity, advanced stage of disease and ET-1-enriched tumor phenotype were all associated with a higher risk for recurrence. Experimental study demonstrated that ET-1 stimulation promoted Akt activation in MCF-7 and MDA-MB-231 cells. Furthermore, silencing of ETAR induced apoptosis in both hormone receptor negative and hormone receptor positive breast cancer cells. Conclusions. We found ET-1 expression in tumor and stroma to be an independent prognostic marker for breast cancer recurrence. Prospective studies are warranted to examine whether ET-1 expression in tumor/stroma could assist in stratifying patients with hormone receptor positive breast cancer for adjuvant therapy.

Inhibition of MLK3 Decreases Proliferation and Increases Antiproliferative Activity of Epidermal Growth Factor Receptor (EGFR) Inhibitor in Pancreatic Cancer Cell Lines

Pancreatic adenocarcinoma is associated with advanced presentation and poor survival. Currently approved therapies have minimal effect on patient survival. Pancreatic adenocarcinomas have a high incidence of activated K-RAS, which may confer resistance to epidermal growth factor receptor (EGFR) inhibitors. Mixed lineage kinase-3 (MLK3) is a MAP3K that activates multiple MAPK pathways. The role of MLK3 in the pathophysiology and resistance to therapy of pancreatic adenocarcinoma has not been investigated. MLK3 is over expressed in pancreatic cancer cell lines compared to an immortalized pancreatic epithelial cell line. The requirement of MLK3 for cell proliferation and survival of pancreatic cancer cell lines, PANC-1 and MiaPaCa-2, was investigated using RNA interference (siRNA) and MLK inhibitor, K252a, alone or in conjunction with the EGFR inhibitor, Compound 56. Ablation of expression of MLK3 via siRNA-mediated gene silencing and pharmacological inhibition of MLK3 by K252a each decreased cell viability in both pancreatic cancer cell lines, with a concurrent decrease in the activation of ERK, JNK and AKT. Concomitant inhibition of EGFR and MLK3 induced apoptosis, as evidenced by increased cleavage of PARP and caspase-3. These results suggest that MLK3 plays an important role in survival and proliferation of pancreatic cancer cell lines and that inhibition of MLK3 may enhance the therapeutic efficacy of EGFR inhibitors in the treatment of pancreatic cancer.

P2-03-06: Endothelin-1/Endothelin A Receptor Signalling in Breast Cancer.

Background: Endothelin-1 (ET-1) and endothelin A receptor (ETAR) are implicated in breast cancer growth and progression. ET-1 is secreted by both tumor cells and stroma (macrophages, endothelial cells). The purpose of this study is to evaluate ET-1/ETAR role on cell proliferation and its effects on EGFR signaling pathway. Materials and Methods: Two breast cancer cell lines: MCF-7 and MDA-MB-231 were stimulated with ET-1. Proliferation of breast cancer cells was analyzed using MTT assay. Protein expression (EGFR, pEGFR, AKT, pAKT, ERK, pERK) was evaluated by Western blot. siRNA knockdown of endothelin A receptor (ETAR) was performed in MCF-7 and MDA-MB-231. Results: ET-1 stimulated proliferation in both cell lines. Interestingly, at higher ET-1 concentrations cell proliferation was more pronounced in MCF-7 when compared with MDA-MB-231. Stimulation with ET-1 activated EGFR and downstream signaling proteins (pAKT and pERK) in both cell lines. Incubation of breast cancer cells with ET-1 for 48 hours had different effect on total EGFR level: 3 fold increase of total EGFR was seen in MCF-7, while no change was detected in MDA-MB-231. siRNA against ETAR decreased ET-1 induced cell proliferation and reduced total EGFR level in MCF-7. Conclusions: These observations suggest that ET-1/ETAR plays an important role in survival and proliferation of breast cancer cells. However, those effects are diverse in different subtypes of breast cancer. Our results may represent an improved selective targeted treatment strategy, especially for estrogen receptor positive breast cancer. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-03-06.

Abstract 1657: HSP90 inhibition down regulates EGFR and its effector signaling proteins in pancreatic cancer cell lines

Rationale: Currently approved therapies, including EGFR inhibitors, have minimal effect on survival in pancreatic cancer (PC). HSP90 is a molecular chaperone that specifically enables activity of numerous protein kinases, including EGFR and other signaling proteins. We have reported that simultaneous inhibition of EGFR and HSP90 may be additive in inhibiting cell viability in PC cell lines. Here we report possible molecular mechanisms underlying HSP90 and EGFR inhibition. Methods: MiaPaCa-2 and PANC-1 were treated with EGFR inhibitor, compound 56 (CMP56) (5 − 35 μmol), and HSP90 inhibitor geldanamycin (250 nmol − 1μmol). Growth inhibition was evaluated using the MTT assay, and apoptosis was evaluated by caspase-3 and PARP cleavage. The effect on relevant cell signaling proteins was assessed by immunoblotting with specific antibodies directed against EGFR and effectors such as AKT, STAT-3 and JNK. Results: Growth inhibition was significantly (p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1657.