Eddie Reed

Messenger RNA levels of XPAC and ERCC1 in ovarian cancer tissue correlate with response to platinum-based chemotherapy.

Nucleotide excision repair is a DNA repair pathway that is highly conserved in nature, with analogous repair systems described in Escherichia coli, yeast, and mammalian cells. The rate-limiting step, DNA damage recognition and excision, is effected by the protein products of the genes ERCC1 and XPAC. We therefore assessed mRNA levels of ERCC1 and XPAC in malignant ovarian cancer tissues from 28 patients that were harvested before the administration of platinum-based chemotherapy. Cancer tissues from patients whose tumors were clinically resistant to therapy (n = 13) showed greater levels of total ERCC1 mRNA (P = 0.059), full length transcript of ERCC1 mRNA (P = 0.026), and XPAC mRNA (P = 0.011), as compared with tumor tissues from those individuals clinically sensitive to therapy (n = 15). In 19 of these tissues, the percentage of alternative splicing of ERCC1 mRNA was assessed. ERCC1 splicing was highly variable, with no difference observed between responders and nonresponders. The alternatively spliced species constituted 2-58% of the total ERCC1 mRNA in responders (median = 18%) and 4-71% in nonresponders (median = 13%). These data suggest greater activity of the DNA excision repair genes ERCC1 and XPAC in ovarian cancer tissues of patients clinically resistant to platinum compounds. These data also indicate highly variable splicing of ERCC1 mRNA in ovarian cancer tissues in vivo, whether or not such tissues are sensitive to platinum-based therapy.

Acquired cisplatin resistance in human ovarian cancer cells is associated with enhanced repair of cisplatin-DNA lesions and reduced drug accumulation.

Studies were undertaken to investigate acquired resistance to cisplatin in human ovarian cancer cells. The cell lines A2780 and A2780/CP70 were studied to assess their respective characteristics of drug accumulation and efflux, cytosolic inactivation of drug, and DNA repair. All experiments were performed using 1-h drug exposures. The A2780/CP70 cell line was 13-fold more resistant to cisplatin than A2780 cells. When studied at their respective IC50 doses, drug accumulation rates were similar for the two cell lines. However, the resistant cell line was twofold more efficient at effluxing drug, which was associated with reduced total drug accumulation for equivalent micromolar drug exposures. At equivalent levels of total cellular drug accumulation, the two cell lines formed the same levels of cisplatin-DNA damage, suggesting that cytosolic inactivation of drug does not contribute to the differential in resistance between these cell lines. Resistant cells were also twofold more efficient at repairing cisplatin-DNA lesions in cellular DNA and in transfected plasmid DNA. We conclude that in these paired cell lines, alterations in drug uptake/efflux and in DNA repair are the major contributing factors to acquired resistance to cisplatin.

Apigenin inhibits VEGF and HIF-1 expression via PI3K/AKT/p70S6K1 and HDM2/p53 pathways

Apigenin is a nontoxic dietary flavonoid that has been shown to possess anti‐tumor properties and therefore poses special interest for the development of a novel chemopreventive and/or chemotherapeutic agent for cancer. Ovarian cancer is one of the most common causes of cancer death among women. Here we demonstrate that apigenin inhibits expression of vascular endothelial growth factor (VEGF) in human ovarian cancer cells. VEGF plays an important role in tumor angiogenesis and growth. We found that apigenin inhibited VEGF expression at the transcriptional level through expression of hypoxia‐inducible factor 1α (HIF‐1α). Apigenin inhibited expression of HIF‐1α and VEGF via the PI3K/AKT/p70S6K1 and HDM2/ p53 pathways. Apigenin inhibited tube formation in vitro by endothelial cells. These findings reveal a novel role of apigenin in inhibiting HIF‐1 and VEGF expression that is important for tumor angiogenesis and growth, identifying new signaling molecules that mediate this regulation.—Fang, J., Xia, C., Cao, Z., Zheng, J. Z., Reed, E., Jiang, B.‐H. Apigenin inhibits VEGF and HIF‐1 expression via PI3K/AKT/p70S6K1 and HDM2/ p53 pathways. FASEB J. 19, 342–353 (2005)

Antitumor effect of β-elemene in non-small-cell lung cancer cells is mediated via induction of cell cycle arrest and apoptotic cell death

Abstract.β-Elemene is a novel anticancer drug, which was extracted from the ginger plant. However, the mechanism of action of β-elemene in non-small-cell lung cancer (NSCLC) remains unknown. Here we show that β-elemene had differential inhibitory effects on cell growth between NSCLC cell lines and lung fibroblast and bronchial epithelial cell lines. In addition, β-elemene was found to arrest NSCLC cells at G2-M phase, the arrest being accompanied by decreases in the levels of cyclin B1 and phospho-Cdc2 (Thr-161) and increases in the levels of p27kip1 and phospho-Cdc2 (Tyr-15). Moreover, β-elemene reduced the expression of Cdc25C, which dephosphorylates/activates Cdc2, but enhanced the expression of the checkpoint kinase, Chk2, which phosphorylates/ inactivates Cdc25C. These findings suggest that the effect of β-elemene on G2-M arrest in NSCLC cells is mediated partly by a Chk2-dependent mechanism. We also demonstrate that β-elemene triggered apoptosis in NSCLC cells. Our results clearly show that β-elemene induced caspase-3, −7 and −9 activities, decreased Bcl-2 expression, caused cytochrome c release and increased the levels of cleaved caspase-9 and poly(ADP-ribose) polymerase in NSCLC cells. These data indicate that the effect of β-elemene on lung cancer cell death may be through a mitochondrial release of the cytochrome c-mediated apoptotic pathway.

Phase I study of taxol and granulocyte colony-stimulating factor in patients with refractory ovarian cancer.

PURPOSE To increase the taxol dose beyond the current standard dose intensity of 175 mg/m2 per 21 days in patients with refractory ovarian cancer. PATIENTS AND METHODS Fifteen patients who had platinum-refractory or recurrent advanced-stage ovarian cancer were treated with taxol in a phase I trial and were given granulocyte-colony stimulating factor (G-CSF). Taxol was administered at doses of 170, 200, 250, and 300 mg/m2 every 3 weeks. G-CSF was given as a daily subcutaneous injection that started 24 hours after the completion of the taxol infusion. RESULTS Four patients required either taxol dose reduction or delay. The dose-limiting toxicity (DLT) was peripheral neuropathy, and it occurred at 300 mg/m2. This toxicity was manifested clinically as a stocking-and-glove sensory disturbance that primarily affected proprioception, and was associated with objective changes on nerve conduction studies in affected individuals. Mucositis was rarely observed. Substantial myelosuppression was observed, but was not dose-limiting. Five of 14 assessable patients experienced an objective response to therapy, with another five individuals who experienced a 30% to 45% reduction in tumor mass. CONCLUSION Taxol can be safely administered in doses up to 250 mg/m2 with G-CSF support, which may make it possible to study taxol dose intensification.

Phase I Clinical Trial of Oral COL-3, a Matrix Metalloproteinase Inhibitor, in Patients With Refractory Metastatic Cancer

PURPOSE This phase I clinical trial was designed to determine the maximum-tolerated dose and dose-limiting toxicities of the matrix metalloproteinase (MMP) inhibitor COL-3 in patients with refractory solid tumors. PATIENTS AND METHODS Thirty-five patients with different cancer types were enrolled. COL-3 doses were escalated from 36 mg/m2/d in successive cohorts of at least three patients. Circulating levels of MMP-2, MMP-9, vascular endothelial growth factor, and basic fibroblast growth factor were assessed during treatment. Pharmacokinetic parameters were assessed for single and multiple doses of drug. RESULTS Cutaneous phototoxicity was dose-limiting at 98 mg/m2/d. With the use of prophylactic sunblock, COL-3 was well tolerated at 70 mg/m2/d. The dose of 36 mg/m2/d was well tolerated without the use of sunblock. Other toxicities that did not seem to be related to dose or pharmacokinetics included anemia, anorexia, constipation, dizziness, elevated liver function test results, fever, headache, heartburn, nausea, vomiting, peripheral and central neurotoxicities, fatigue, and three cases of drug-induced lupus. Disease stabilization for periods of 26+ months, 8 months, and 6 months were seen in hemangioendothelioma, Sertoli-Leydig cell tumor, and fibrosarcoma, respectively. There was a potentially statistically significant relationship between changes in plasma MMP-2 levels and cumulative doses of drug when progressive disease patients were compared with those with stable disease or toxicity (P = .042). CONCLUSION COL-3 induced disease stabilization in several patients who had a nonepithelial type of malignancy. Phototoxicity was dose-limiting. We recommend the dose of 36 mg/m2/d for phase II trials.

A phase II study of 5-aza-2'deoxycytidine (decitabine) in hormone independent metastatic (D2) prostate cancer.

Aims and Background Decitabine (5-aza-2′-deoxycytidine) is an S-phase-specific pyrimidine analog with hypomethylation properties. In laboratory models of prostate cancer (PC-3 and DU-145), decitabine induces cellular differentiation and enhanced expression of genes involved in tumor suppression, immunogenicity, and programmed cell death. Methods We conducted a phase II study of decitabine in 14 men with progressive, metastatic prostate cancer recurrent after total androgen blockade and flutamide withdrawal. Decitabine was administered at a dose of 75 mg/m2/dose IV as a 1 hour infusion every 8 hours for three doses. Cycles of therapy were repeated every 5 to 8 weeks to allow for resolution of toxicity. Results Two of 12 patients evaluable for response had stable disease with a time to progression of more than 10 weeks. This activity was seen in 2 of 3 African-American patients. Toxicity was similar to previously reported experience. No significant changes in urinary concentrations of the angiogenic factor bFGF, a potential biomarker of tumor activity, were identified over time in 7 unselected patients with progressive disease. Conclusions We conclude that decitabine is a well tolerated regimen with modest clinical activity against hormone-independent prostate cancer. Further investigations in patients of African-American origin may be warranted.

Antiproliferative effect of β-elemene in chemoresistant ovarian carcinoma cells is mediated through arrest of the cell cycle at the G2-M phase

Abstract.Elemene is a natural antitumor plant drug. However, the effect of elemene on cell growth in ovarian cancer is unknown. In this study, we show that β-elemene inhibited the proliferation of cisplatin-resistant human ovarian cancer cells and their parental cells, but had only a marginal effect in human ovary cells, indicating differential inhibitory effects on cell growth between ovarian cancer cells and normal ovary cells. We also demonstrated for the first time that β-elemene markedly enhanced cisplatin-induced growth inhibition in resistant cells compared to sensitive cells. In addition, cell cycle analysis revealed a synergistic effect of β-elemene and cisplatin on the induction of cell cycle G2-M arrest in our resistant ovarian carcinoma cells. Furthermore, we showed that treatment of these cells with both drugs downregulated cyclin B1 and Cdc2 expression, but elevated the levels of p53, p21waf1/cip1, p27kip1 and Gadd45. Finally, the combination of β-elemene and cisplatin was found to increase the phosphorylation of Cdc2 and Cdc25C, which leads to a reduction in Cdc2-cyclin B1 activity. These novel findings suggest that β-elemene sensitizes chemoresistant ovarian carcinoma cells to cisplatin-induced growth suppression partly through modulating the cell cycle G2 checkpoint and inducing cell cycle G2-M arrest, which lead to blockade of cell cycle progression.

ERCC1 and Clinical Resistance to Platinum-Based Therapy

Praz and colleagues report in this issue on the relationship between the occurrence of a polymorphism of ERCC1 in tumor tissues of patients with colorectal cancer and the rate of disease response to the combination of oxaliplatin and 5-fluorouracil ([1][1]). This polymorphism is associated with

Cisplatin induction of ERCC-1 mRNA expression in A2780/CP70 human ovarian cancer cells.

ERCC-1 is a critical gene within the nucleotide excision repair pathway, and cells without a functionalERCC-1 do not perform cisplatin-DNA adduct repair. We therefore investigated the cisplatin effect on ERCC-1mRNA expression in vitro. In response to a 1-h cisplatin exposure, A2780/CP70 human ovarian cancer cells showed a 6-fold increase in steady-state level of ERCC-1 mRNA. This rise was attributable to increased transcription as measured by nuclear run-on assays and a 60% increase in ERCC-1mRNA half-life. The increase in ERCC-1 mRNA was preceded by a 4–5-fold rise in mRNA expressions ofc-fos and c-jun, a 14-fold increase in c-Jun protein phosphorylation, and an increase in in vitronuclear extract binding activity to the AP-1-like site ofERCC-1. These data suggest that the induction ofERCC-1 expression in A2780/CP70 cells exposed to cisplatin results from two major factors: (a) an increase in the expression of transactivating factors that bind the AP-1-like site in the 5′-flanking region of ERCC-1 and (b) an increase in the level of c-Jun phosphorylation that enhances its transactivation property.