Ann Gen Psychiatry. 2007; 6: 25.

The authors acknowledge the written consent for publication obtained from the patient featured in the manuscript.

Ann Gen Psychiatry. 2007; 6: 25.

BACKGROUNDGinkgo biloba (ginkgo) is a herbal remedy used by over 2% of the adult population in the United States. Several review articles have suggested that ginkgo may increase the risk of bleeding.

Ann Gen Psychiatry. 2007; 6: 25.

OBJECTIVETo report a case of bleeding associated with using ginkgo, to systematically review the literature for similar case reports, and to evaluate whether using ginkgo is causally related to bleeding.

Ann Gen Psychiatry. 2007; 6: 25.

REVIEW METHODSPublished case reports of bleeding events in persons using ginkgo were selected. Two reviewers independently abstracted a standard set of information to assess whether ginkgo caused the bleeding event.

Ann Gen Psychiatry. 2007; 6: 25.

Case ReportA 73-year-old white man presented complaining of recent episodes of spontaneous bleeding. He had noticed 3 nose bleeds in the prior month, ecchymosis on his hands and arms after very minor trauma, frequent hemorrhoidal bleeding, and bleeding from his ear after striking it with a blunt comb. The patient was a healthy, retired pediatrician. He had a remote history of bleeding after using aspirin, occasional, mild hemorrhoidal bleeding, and only 1 or 2 previous nosebleeds. The patient had no excessive bleeding after previous surgical procedures. He was taking no prescription medications, but did take vitamins A, C, D, E, and folic acid for preventive care and had used ginkgo, 75 mg per day, for the previous 6 months as an aid to his “failing memory.” The ginkgo supplement, which was produced by a large U.S. manufacturer and distributed by a large supermarket chain, was standardized to 27% ginkgo flavone glycosides and 10% terpene lactones. He had never been diagnosed with cognitive dysfunction, but felt that ginkgo improved both his memory and clarity of thought.

PLoS Med. 2006 October; 3(10): e420.

Nuclear factor erythroid-2 related factor 2 (NRF2) is a redox-sensitive transcription factor that positively regulates the expression of genes encoding antioxidants, xenobiotic detoxification enzymes, and drug efflux pumps, and confers cytoprotection against oxidative stress and xenobiotics in normal cells. Kelch-like ECH-associated protein 1 (KEAP1) negatively regulates NRF2 activity by targeting it to proteasomal degradation. Increased expression of cellular antioxidants and xenobiotic detoxification enzymes has been implicated in resistance of tumor cells against chemotherapeutic drugs.

PLoS Med. 2006 October; 3(10): e420.

Here we report a systematic analysis of the KEAP1 genomic locus in lung cancer patients and cell lines that revealed deletion, insertion, and missense mutations in functionally important domains of KEAP1 and a very high percentage of loss of heterozygosity at 19p13.2, suggesting that biallelic inactivation of KEAP1 in lung cancer is a common event. Sequencing of KEAP1 in 12 cell lines and 54 non-small-cell lung cancer (NSCLC) samples revealed somatic mutations in KEAP1 in a total of six cell lines and ten tumors at a frequency of 50% and 19%, respectively. All the mutations were within highly conserved amino acid residues located in the Kelch or intervening region domain of the KEAP1 protein, suggesting that these mutations would likely abolish KEAP1 repressor activity. Evaluation of loss of heterozygosity at 19p13.2 revealed allelic losses in 61% of the NSCLC cell lines and 41% of the tumor samples. Decreased KEAP1 activity in cancer cells induced greater nuclear accumulation of NRF2, causing enhanced transcriptional induction of antioxidants, xenobiotic metabolism enzymes, and drug efflux pumps.

PLoS Med. 2006 October; 3(10): e420.

This is the first study to our knowledge to demonstrate that biallelic inactivation of KEAP1 is a frequent genetic alteration in NSCLC. Loss of KEAP1 function leading to constitutive activation of NRF2-mediated gene expression in cancer suggests that tumor cells manipulate the NRF2 pathway for their survival against chemotherapeutic agents.

PLoS Med. 2006 October; 3(10): e420.

Lung cancer is the most common cause of cancer-related death worldwide. More than 150,000 people in the US alone die every year from this disease, which can be split into two basic types—small cell lung cancer and non-small-cell lung cancer (NSCLC). Four out of five lung cancers are NSCLCs, but both types are mainly caused by smoking. Exposure to chemicals in smoke produces changes (or mutations) in the genetic material of the cells lining the lungs that cause the cells to grow uncontrollably and to move around the body. In more than half the people who develop NSCLC, the cancer has spread out of the lungs before it is diagnosed, and therefore can't be removed surgically. Stage IV NSCLC, as this is known, is usually treated with chemotherapy—toxic chemicals that kill the fast-growing cancer cells. However, only 2% of people with stage IV NSCLC are still alive two years after their diagnosis, mainly because their cancer cells become resistant to chemotherapy. They do this by making proteins that destroy cancer drugs (detoxification enzymes) or that pump them out of cells (efflux pumps) and by making antioxidants, chemicals that protect cells against the oxidative damage caused by many chemotherapy agents.

PLoS Med. 2006 October; 3(10): e420.

Lung carcinomas are the leading cause of cancer deaths in the United States and worldwide in both men and women. Non-small-cell lung cancer (NSCLC) accounts for approximately 85% of lung cancer cases, with 65% of these patients presenting with unresectable stage III and IV disease [1].

PLoS Med. 2006 October; 3(10): e420.

Nuclear factor erythroid-2 related factor 2 (NRF2), a cap 'n' collar basic leucine zipper transcription factor, regulates a transcriptional program that maintains cellular redox homeostasis and protects cells from oxidative insult, including from chemotherapeutic agents [7–9]. NRF2 activates transcription of its target genes through binding specifically to the antioxidant response element (ARE) found in those gene promoters. The NRF2-regulated transcriptional program includes a broad spectrum of genes, including ones encoding antioxidants (e.g., γ-glutamyl cysteine synthetase modifier subunit [GCLm], γ-glutamyl cysteine synthetase catalytic subunit [GCLc], heme oxygenase-1, superoxide dismutase, glutathione reductase [GSR], glutathione peroxidase, thioredoxin, thioredoxin reductase, peroxiredoxins[PRDX], and cysteine/glutamate transporter [SLC7A11]) [7,8], xenobiotic metabolism enzymes (e.g., NADP[H] quinone oxidoreductase 1 [NQO1], GSTs, and UDP-glucuronosyltransferase) [7,8], and several ATP-dependent drug efflux pumps (e.g., MRP1 and MRP2) [10–12].

PLoS Med. 2006 October; 3(10): e420.

Tumor SamplesTumors for this study were randomly selected from 700 freshly frozen lung cancer tissues from patients who had undergone complete, curative resection of their disease from 1993 to 2001 at Johns Hopkins Hospital, Baltimore, Maryland, United States. Tumors were macrodissected and only those specimens with greater than 50% neoplastic cells were used. A total of 56 cases of lung tumor, including 40 paired lung tumors and adjacent normal tissues (frozen tissue) and 16 pleural fluid (PF) samples, were chosen in accordance with the Institutional Review Board protocol, and DNA was isolated using DNeasy Kit (Qiagen, Valencia, California, United States). Details of the lung tumor samples are listed in Table S1.

PLoS Med. 2006 October; 3(10): e420.

Western Blot AnalysisTo obtain total protein lysates, cancer cells or tissues were lysed in 50 mM Tris (pH 7.2), 1% Triton X-100 containing Halt Protease Inhibitor cocktail (Pierce, Rockford, Illinois, United States) and centrifuged at 12,000g for 15 min at 4 °C. To obtain nuclear extracts, NE-PER Nuclear Extraction Reagents (Pierce) were used. Protein concentrations were estimated by the BCA method (Pierce). For immunoblot analysis, 30 μg and 100 μg of protein from nuclear extracts and total protein lysates, respectively, were used and resolved on 10% SDS-PAGE gels. Proteins were transferred onto PVDF membranes, and the following antibodies were used for immunoblotting: anti-KEAP1 (gift from M. Velichkova, University of California San Diego, La Jolla, California, United States), anti-NRF2 (H-300; Santa Cruz Biotechnology, Santa Cruz, California, United States), anti–Lamin B1 (Santa Cruz Biotechnology), and anti-GAPDH (Imgenex, Sorrento Valley, California, United States). All primary antibodies were diluted in PBS-T/5% nonfat dry milk and incubated overnight at 4 °C.

PLoS Med. 2006 October; 3(10): e420.

Enzyme AssayEnzyme activities of GST, GSR, and NQO1 were determined in the total protein lysates by following methods previously described [8]. Total glutathione (oxidized and reduced) was determined using a modified Tietze method [25] by measuring reduction of 5,5′-dithiobis-2-nitrobenzoic acid in a GSR-coupled assay.

PLoS Med. 2006 October; 3(10): e420.

ImmunohistochemistryFormalin-fixed tissues were treated with anti-NRF2 antibody (H-300, Santa Cruz Biotechnology) at a dilution of 1:250 for 1 h and developed using horseradish peroxidase (Dako, Glostrup, Denmark). Non-immune rabbit IgG (Jackson ImmunoResearch Laboratories, West Grove, Pennsylvania, United States) was used as a negative control. To demonstrate the specificity of antibody staining, we preincubated the anti-NRF2 antibody with NRF2 and luciferase in vitro transcribed and translated protein, respectively, for 30 min and then carried out immunohistochemical staining.