Ovarian cancer is the fifth leading cause of cancer death among women in the United States and Western Europe and has the highest mortality rate of all gynecologic cancers. It is estimated that 21,650 new cases of ovarian cancer will be diagnosed in 2008 and 15,520 women will die of the disease (American Cancer Society, Cancer Facts and Figures 2008). Approximately 75% of cases of epithelial ovarian carcinoma are diagnosed at advanced stage (III/IV) with disseminated intraperitoneal metastases,1 such that most patients succumb to the disease within 5 years. The standard treatment protocol used to treat patients with advanced-stage ovarian cancer includes primary cytoreductive surgery followed by platinum and taxane chemotherapy. Despite the fact that 70% of patients will have a complete clinical response to this initial therapy, most patients will eventually experience platinum-resistant recurrent disease and are treated with salvage agents such as topotecan,2-4 doxorubicin,4 gemcitabine,5 paclitaxel,6 and/or docetaxel7 19% to 35%. Unfortunately, response rates to such salvage agents2-7 are generally less than 20%. An absence of more active therapies is currently a major impediment to improving outcome for patients with advanced-stage ovarian cancer.
Gedunin is a natural product extracted from the neem tree (Azadirachta indica) and has been used in ayurvedic and homoeopathic medicine in India and other parts of Asia for centuries as treatment of a wide range of ailments because it is believed to have anti-inflammatory, antipyretic, analgesic, antiulcer, and antimalarial properties.8 Gedunin, a limonoid, is a heavily oxygenated bitter-tasting modified triterpene. Recently, gedunin has been shown to have antiproliferative properties against prostate9 and colon10 cancer cell lines, possibly via a heat shock protein 90 kd (HSP90) pathway modulation.9 We sought to explore the effect of gedunin on ovarian cancer cell proliferation and cisplatin response. Further, in an effort to elucidate the molecular underpinnings of gedunin action, we analyzed in vitro sensitivity data with genome-wide expression data to identify genes and biological pathways associated with gedunin response.
MATERIALS AND METHODS
We evaluated the in vitro antiproliferative effect of gedunin on 3 ovarian cancer cell lines (SKOV3, OVCAR4, and OVCAR8), alone and in the presence of cisplatin. Further, we analyzed in vitro gedunin sensitivity data integrated with genome-wide expression data from 54 human cancer cell lines in an effort to identify genes and molecular pathways that underlie the mechanism of gedunin action.
All tissue culture reagents were obtained from Sigma-Aldrich, Co (St Louis, Mo). Cisplatin was purchased from Sigma-Aldrich and gedunin was from Gaia Chemical, Corp (Gaylordsville, Conn). The ovarian cancer cell lines SKOV3, OVCAR4, and OVCAR8 were obtained from the National Cancer Institute (Frederick, Md) and cultured as recommended by the supplier. Briefly, these cell lines were grown in RPMI-1640 supplemented with 10% fetal bovine serum, 1% sodium pyruvate, and 1% nonessential amino acids using 75-cm2 Costar flasks (Fisher Scientific, Pittsburgh, PA). Cells were incubated in 5% carbon dioxide-95% air and 100% humidity at 37°C.
Cell Proliferation Assays
To evaluate the effect of gedunin on ovarian cancer cells, we used a luminescence cell viability assay. Cells were harvested by trypsinization, and their numbers were quantified using Trypan blue (4%). In a microplate (CulturPlate-96; PerkinElmer Life Sciences, Boston, Mass), a cell suspension (100 μL) containing 5000 cells was dispensed. The plate was incubated at 37°C with a 5% carbon dioxide-95% air and 100% humidity atmosphere for 24 hours. After incubation, the medium was replaced with a medium containing the study drug (25 μM gedunin alone, 25 μM cisplatin alone, or 25 μM cisplatin + 25 μM gedunin) and incubated at 37°C to specified time points. The IC50 value of cisplatin has previously been reported in this range.11 At the desired time points, a volume of CellTiter-Glo Reagent (Promega, Corp, Madison, Wis) equal to the volume of the cell culture medium (100 μL) was added. The content was mixed for 2 minutes on an orbital shaker to induce cell lysis. The plate was incubated at room temperature for 10 minutes to stabilize the luminescent signal, and luminescence was measured using a VICTOR2 multichannel counter (Wallac, PerkinElmer Life Sciences). Luminescence was measured for 1 second. Effect of treatment is reported as a percentage relative to the effect of vehicle alone, which is normalized to 100% for each condition.
Cell Proliferation Analysis
Data are represented as mean ± SEM for each condition. One-way analysis of variance was used to analyze the data, and a posttest Bonferroni was used to compare all bars at the confidence interval of 95%. This analysis was performed using GraphPad Prism software (San Diego, Calif).
Bioinformatic Analysis of Genes Associated With Gedunin Response
To identify genes associated with in vitro gedunin responsiveness, we analyzed GI50 and Affymetrix U133 GeneChip expression data available for the NCI-60 cancer cell lines. Affymetrix U133 GeneChip data for 54 cell lines were downloaded from the National Cancer Institute (NCI) CellMiner Web site (http://discover.nci.nih.gov/cellminer/loadDownload.do). The gedunin GI50 values for 54 cell lines were obtained from NCI's cancer screening data page (http://dtp.nci.nih.gov/dtpstandard/cancerscreeningdata/index.jsp). Because GI50 data for only 54 cell lines were available, we used GeneChip data for each of these 54 cell lines. Background correction, quantile normalization, and summarization were performed on the data by robust multichip average method provided by Affy (R package).12 The resulting intensity values were analyzed using Significance Analysis of Microarrays (SAM) software.13,14 Using both Affymetrix gene expression data and gedunin GI50 in vitro chemosensitivity data, linear regression analysis was performed to identify messenger RNAs associated with response to gedunin (false discovery rate [FDR], <20%).
In an effort to provide a relevant biologic context to the genes identified to be associated with in vitro gedunin responsiveness, an analysis of biological pathway relationships was performed using the commercially available software Ingenuity Pathway Analysis (IPA; Ingenuity Systems, Redwood City, Calif). This Web-based, literature-curated application correlates gene expression array data to relevant biological pathways, such that one can identify the networks, molecular mechanisms, and biological processes most relevant to developed data. The IPA software uses genes provided for analysis by the user as "seeds," from which it generates networks, which are scored based on the number of network eligible study genes they contain compared with the total number of genes in that network. The higher the score, the lower the probability, that the observed number of network-eligible genes would be observed by random chance.
Effect of Gedunin on Ovarian Cancer Cell Lines
Relative to control (vehicle only, normalized to 100%), the viability of SKOV3 cells treated with gedunin (25 μM) alone at 24, 48, and 72 hours was 69%, 62%, and 52% versus 52%, 29%, and 17% for cells treated with cisplatin (25 μM) alone. In contrast, the viability of SKOV3 cells treated with both gedunin (25 μM) and cisplatin (25 μM) was 47%, 35%, and 28% (Figs. 1A and B). In parallel, the viability of OVCAR4 cells treated with gedunin (25 μM) alone at 24, 48, and 72 hours was 80%, 44%, and 24% versus 37%, 10%, and 3% for cells treated with cisplatin (25 μM) alone and 19%, 8%, and 3% for OVCAR4 cells treated with both gedunin (25 μM) and cisplatin (25 μM; Figs. 1C and D). The viability of OVCAR8 cells treated with gedunin (25 μM) alone at 24, 48, and 72 hours was 69%, 17%, and 5% versus 103%, 52%, and 32% for cells treated with cisplatin (25 μM) alone and 66%, 10%, 2% for OVCAR8 cells treated with both gedunin (25 μM) and cisplatin (25 μM; Figs. 1E and F). Of note, the effect of a reduced dose of cisplatin (12.5 μM) combined with a reduced dose of gedunin (12.5 μM) was equal the effect of treatment with the higher (25 μM) dose of cisplatin alone (data not shown).
Genes Associated With In Vitro Gedunin Response
To elucidate the molecular underpinnings of gedunin action, we analyzed NCI cell line in vitro sensitivity data with genome-wide expression data to identify genes associated with gedunin response. Using linear regression analysis, we identified 52 genes associated with gedunin responsiveness (q value <20%; Table 1). Three genes demonstrated increasing expression with increasing gedunin sensitivity, and 49 genes demonstrated decreasing expression with increasing gedunin sensitivity. Of the 52 genes associated with gedunin responsiveness, 20 are involved in cell cycle control, lipid metabolism, and molecular transport; 14 genes are involved in cellular growth and connective tissue development and function; and 7 genes are involved in cellular growth, cancer, and cell cycle functions.
Pathway Analysis of Genes Associated With In Vitro Gedunin Response
To provide biologic context to our findings, IPA (Ingenuity Systems) was performed on the 52 genes identified. On the basis of IPA's algorithm, which uses a right-tailed Fisher exact test for overrepresented functional/pathways, the 52 genes associated with in vitro gedunin response included canonical pathways for aryl hydrocarbon receptor signaling (P = 0.001), phosphatidylinositol 3-kinase (PI3K)/AKT signaling (P = 0.002), nitric oxide signaling (P = 0.002), neuregulin signaling (P = 0.003), and extracellular signal-regulated kinase/mitogen-activated protein kinase signaling (P = 0.004). The top-scored canonical pathway (based on the number of the 52 study genes contained compared with the total number of genes in that network/pathway) is shown in Figure 2.
We have demonstrated the growth-inhibitory properties of a natural substance called gedunin on ovarian cancer cell lines. Further, we have demonstrated that this substance may also influence ovarian cancer cell cisplatin sensitivity. Using existing cell line expression data coupled with measures of gedunin in vitro sensitivity and pathway analysis, we have identified genes and molecular pathways that may underlie gedunin response.
The bioactivity of many natural substances has increased focus on such agents as potential therapeutics. As an example, curcumin, a component of turmeric, which has been used in Indian cuisine for centuries, was recently shown to suppress inflammation and angiogenesis in ovarian cancer murine models by inhibition of nuclear factor κB.15 One of the best-recognized natural products in oncologic practice today is paclitaxel, used widely for treatment of patients with breast, lung, and ovarian cancers. The drug, which is believed to produce its antineoplastic effect in part by stabilization of microtubules, was first extracted from the bark of the Pacific yew tree (Taxus brevifolia). Other natural products that have demonstrated antiproliferative activity include camptothecin, a derivative of the Camptotheca acuminata tree, analogs of which include topotecan, used to treat ovarian, colon, and other cancers, and combretastatin, extracted from the bark of African willow tree Combretum caffrum and used to treat leukemia, colon, and lung cancers.16 Recently, gedunin has been shown to have antiproliferative properties against prostate9 and colon10 cancer cell lines, possibly via an HSP90 pathway modulation, which activates an androgen receptor-mediated pathway.9
In the current study, we have demonstrated that gedunin has an antiproliferative activity against ovarian cancer cell lines. Further, our analysis suggests that the biologic basis to gedunin responsiveness may involve activation of one or more pathways, including aryl hydrocarbon receptor signaling, PI3K/AKT signaling, nitric oxide signaling, neuregulin signaling, and/or ERK/MAPK signaling pathways. The role of PI3K/AKT in ovarian carcinogenesis has been reported by several groups.17-19 The PI3K/AKT signaling pathway regulates vascular endothelial growth factor expression through hypoxia-inducible factor 1 in ovarian cancer cells,20,21 and an inhibition of PI13 catalytic subunit (p110α) using small interfering RNA decreases ovarian cancer cell invasion, migration, and proliferation through AKT signaling.19
As we have demonstrated, the antiproliferative effect of gedunin may be driven by modulation of several genes that are involved in various pathways related to cell death/survival and apoptosis, including CHK2 checkpoint (apoptosis, DNA damage response, G2 phase, G1 phase, survival, and proliferation), protein phosphatase 2 (cell death and survival), eukaryotic translation initiation factor 4E (growth, proliferation, apoptosis, and cell death), heterogeneous nuclear ribonucleoprotein A1 (apoptosis, growth, and stress response), HSP90 (growth, binding, apoptosis, cell viability, and proliferation), phosphoprotein enriched in astrocytes 15 (apoptosis, cell death, proliferation, and survival), and tumor necrosis factor receptor-associated protein 1 (apoptosis and growth). Recently, inhibition of HSP90 by 17-allylamino-17-demethoxygeldanamycin was shown to induce antiproliferative activity in ovarian cancer cell lines and was associated with down-regulation of a group of MYC-regulated proteins.22
The high mortality associated with advanced-stage epithelial ovarian cancer mandates efforts to identify more active therapeutic agents. The evaluation of natural substances, particularly those that have been used safely and successfully as traditional or herbal remedies in the past, may offer promise in this regard. We have provided evidence to suggest that gedunin, an extract of the neem tree, has activity against ovarian cancer cells and that this effect may be driven by pathways that have previously been shown to be important therapeutic targets in human cancers.
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