| IntroductionIn the year2011, approximately8,010women succumbed to endometrial cancer and nearly47,130patients were newly diagnosed with this cancer. In about70%of the women with a diagnosis of endometrial cancer, the disease is found localized to the corpus and five year survival is as high as85%. Advanced and recurrent endometrial cancer patients, enrolled in several gynecologic oncology group (GOG) trials for agents including platinum, taxanes and anthracyclines, rarely have complete responses to therapy. Combination regimens show higher response rates, but the progression free period with these therapies is relatively low (5-7months) with higher morbidity and continued lack of cure.These statistics highlight the need for the development of novel and effective chemopreventive and chemotherapeutic agents for endometrial cancer.Naturally occurring dietary components provide an important source of bioactive compounds that can serve as both chemopreventive as well as chemotherapeutic agents against endometrial and other types of cancers. Our lab is currently investigating the anti-cancer properties of compounds present in the rhizomes of ginger (Zingiber officinale). These studies are supported by previous investigations demonstrating that dry ginger powder or solvent extracts of ginger roots induce cell cycle arrest and apoptosis in skin, breast, prostate, colon, and ovarian cancer cells. Topical application of the ethanolic extract of ginger decreased the incidence, size, and the number of DMBA/TPA induced tumors in SENCAR mice.The majority of the previous studies have concluded that the bioactive components of the dry powder and solvent extract of ginger rhizomes responsible for the anti-cancer activities are the phenolic compounds4-,6-,8-and10-gingerols, paradol, and shogaol, a product formed after drying or heating of the roots. These phenolic compounds, and especially the gingerols exhibit anti-proliferative and anti-angiogenic properties as demonstrated by in vitro and in vivo studies in various cancer models. Human colorectal cancer cells when treated with6-gingerol, inhibited cell proliferation by inducing G1cell cycle arrest and apoptosis. Gingerols exhibit these anti-cancer effects via multiple mechanisms, which include protein degradation as well as β-catenin, PKC delta, and GSK3beta pathways. Studies in the ovarian cancer model have demonstrated that6-shogaol inhibits the secretion of VEGF by the cancer cells.6-gingerol induces apoptosis in the prostate cancer cell line LnCaP by increasing the expression of p53and Bax and simultaneously decreasing the experssion of Bcl-2.In addition to the powdered ginger and the solvent extraction, bioactive compounds can also be isolated by steam distillation of this rhizomes. To the best of our knowledge, only limited studies have been conducted to demonstrate the anti-cancer properties of the steam distilled extracts of ginger. Chemical analysis of the steam distilled extract of ginger indicates that the previously identified bioactive phenolic compounds are present at very low concentration in the steam distilled extracts of ginger. In the current study we demonstrate that the steam distilled extracts of ginger are potent mediators of apoptosis in endometrial cancer cells. Our studies suggest that one of the major bioactive components of the steam distilled extract of ginger is citral (a mixture of two terpenoid isomers, neral and geranial). We demonstrate that treatment of the endometrial cancer cells with the steam distilled extract of ginger results in significant increase in intracellular calcium, decrease in the mitochondrial membrane potential, increase in the expression of caspase3, phosphorylation of P53, and a significant decrease in the expression of Bcl-2. The observations outlined in our studies demonstrate that the steam distilled extract of ginger and its bioactive components have the potential to be developed as chemopreventive and chemotherapeutic agents for endometrial cancer.The purpose of our study is to discover the bioactive components in the steam distilled extract of ginger and to study the underlying mechanisms of its anti-cancer property.MethodsSteam distillation of ginger rhizomes. Ginger rhizomes were obtained from local vendors, cleaned with distilled water and cut into0.5cm pieces. Approximately250-300g of the cut ginger pieces were transferred to the1000ml round bottom flask of the Clevenger steam distillation apparatus. The ginger roots were submerged in500ml of deionized water (18MOhm-cm) and steam distillation was carried out for4-6hours by heating the flask. The oil separating in the Clevenger apparatus was lighter than water and was separated by periodically draining the liquid accumulating in the separation tube of the unit. The oil was immediately aliquoted in microfuge tubes and frozen until used in assays. The density of the oil was calculated to be0.87g/ml and this measurement was used to calculate the concentration of the extract used to conduct the biological assays.Cell proliferation assays. Effect of steam distilled ginger extracts, citral, and6-gingerol on the proliferation of the cancer cell lines was determined by the3-(4.5-dimethythiazol-2-yl)-2.5-diphenyl tetrazolium bromide (MTT) uptake method [32,33]. Briefly, the cancer cells were plated in96-well plate at a density of5000cells/well in their respective medium. The cells were then treated with various concentrations of ginger extract (0.025μg,0.25μg,2.5μg,6.25μg and12.50μg/ml) and incubated at370C in a5%CO2environment for24h,48h and72h. After the designated time period,20μl3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide was added to each well and the plates were incubated at370C for additional3h. The formazan crystals formed in the wells were dissolved in100μl DMSO. The absorbance was measured at570nm using a Spectra MAX190(Molecular Devices, Sunnyvale, CA).Combined treatment of cancer cells with SDGE and radiation or chemotherapy. MTT assays were conducted to determine if SDGE enhanced the anti-proliferation effect of radiation or chemotherapy in the endometrial cancer cells. Ishikawa or ECC-1cells were plated in multiple96well plates (5X103cells/well) on day1of the experiment. After allowing the cells to stabilize, media or SDGE were added to the wells containing the endometrial cancer cells on Day2. Cells in some of the wells were also treated with cisplatin (5μM) while others were irradiated with a single dose of4Gy using a Cesium-137radiator. Following these treatments, the cells were cultured for72h at37℃in5%CO2environment. Effect of the treatment on proliferation of the endometrial cancer cells was determined by conducting the MTT assays as described above.Gas Chromatography-Mass Spectrometry of SDGE. Separation and identification of compounds in SDGE samples used a Shimadzu GC-17A gas chromatograph equipped with a QP-5000quadrupole mass analyzer (Shimadzu Scientific Instruments, Columbia, MD). Prior to analysis,20μL of freshly defrosted SDGE was dissolved in1000μL of pentane.1μl of this dissolved extract was injected manually to the gas chromatograph using a1:50inlet split ratio and helium as the carrier gas at a flow rate of1.4-ml/min. The gas chromatograph contained a nonpolar RTX-5MS column (30m length,0.25mm ID,0.25μm film thickness; Restek, Bellefonte, PA.) Column temperature was initially70℃followed by a ramp at4℃/min to180℃. Electron ionization detection was in full-scan, positive ion mode over a mass-to-charge ratio (m/z) range of41to300. Compounds were tentatively identified by searching a NIST library and by comparison of arithmetic retention indices to values reported by Adams. Measurement of Apoptosis by flow cytometry. Apoptosis was measured using the FITC-Annexin V Apoptosis Detection kit (BD Pharmingen, San Diego, CA). Briefly,2×106cells were treated with0.25μg/ml ginger extract with or without100μM Pifithrin-a. After incubation at370C for0-16h, the cells were washed twice with cold PBS and resuspended in1×binding buffer,(10mM HEPES/NaOH, pH7.4,140mM NaCl,2.5mM CaC12) at a concentration of1×106cells/ml. Then1×105cells in100μl binding buffer, were transferred to5ml tubes and stained with5μl of FITC-Annexin V and5μl propidium iodide (PI). The cells were gently vortexed and incubated at room temperature for15min. After washing the cells with lx binding buffer to remove the excess FITC-Annexin V and PI, the cells were analyzed on a FACSCalibur flow cytometer. The data were analyzed using FlowJo software.Cell cycle assay. The endometrial cancer cells were treated with SDGE (250ng/ml or2.5μg/ml) for24,48. and72h. Following treatment, the cells were harvested, washed with PBS and fixed in75%ethanol, washed with PBS, and stained with propidium iodide. Flow cytometry was then performed to analyze the samples for both apoptosis and cell cycle status as described earlier [35].Western blot analysis. After treatment of the cells with the steam distilled extracts of ginger, the cancer cells were washed with ice cold phosphate buffered saline (PBS) and lysed with RIPA buffer (Pierce, Rockford, IL) containing a protease inhibitor cocktail (Thermo Scientific, Rockford. IL). The total amount of protein in the lysate was determined by using the BCA assay (Pierce). Cell lysates were loaded at25μg/well onto a7.5or12%resolving polyacrylamide gel and separated by electrophoresis, after which, the proteins were transferred to PVDF membranes. The membranes were blocked with5%milk in Tris buffered saline and probed with the appropriate primary antibodies. Horseradish peroxidase conjugated secondary antibodies and SuperSignal West Dura Extended Duration Substrate (Thermo Scientific, Rockford, IL) were used for detection of the proteins on the blots. The films were scanned using FLUORCHEM890and Image J software was used to quantify the intensities of the bands.Mitochondrial membrane potential assay. The endometrial cancer cells were grown in T25tissue culture flasks. Exponentially growing cells were treated with0.025μg/ml or0.25μg/ml of ginger extract for24hrs. The cells were then washed and harvested,1×106cells were added to each flow tube from untreated,0.025μg/ml and0.25μg/ml ginger extract treated cells. The cells were treated with40nM DiOC6at37℃for30min. The cells were then washed, resuspended in400μl of PBS containing2%FBS and analyzed by FACSCALIBUR flowcytometer to assess the mitochondrial membrane potential.The data were analyzed using FlowJo software.Calcium flux measurements. The Ishikawa cells in the log phase of growth were harvested using trypsin. The cells (1.2×107) were washed three times and suspended in1ml of0.5%bovine serum albumin (BSA) containing Hanks buffered saline that did not contain any divalent cations. The cells were loaded with Indo1-AM (2μM) in the presence of4mM probenecid for30min at37℃in5%CO2environment. The cells were then washed and resuspended in Dulbecco’s phosphate buffered saline containing0.5%BSA and1mM CaCl2to a final concentration of2X106cells/ml. The cells were filtered through a35micron membrane filter prior to flow cytometry on LSR-Ⅱ cytometer. Cells were initially analyzed for3min to determine the baseline intracellular calcium concentration. SDGE (0.025.0.25, or2.5μg/ml) or Ionomycin (1μM used as a positive control) were subsequently added to the cells and the change in the Indo-1fluorescence was determined by continuously streaming the cells through the flow cytometer for approximately7mins. The data obtained were analyzed using FlowJo software.Statistical analysis. Statistical analysis was done using the GraphPad Prizm software. The threshold for statistical significance is a probability of0.05. The data was analyzed using unpaired T-test.ResultNovel strategies are necessary to improve chemotherapy response in advanced and recurrent endometrial cancer. Here, we demonstrate that terpenoids present in the Steam Distilled Extract of Ginger (SDGE) are potent inhibitors of proliferation of endometrial cancer cells. At concentrations as low as0.25mg/ml. SDGE, isolated from six different batches of ginger rhizomes, consistently showed40%inhibition in the proliferation of the endometrial cancer cell lines Ishikawa and ECC-1. SDGE also enhanced the anti-proliferative effect of radiation and cisplatin. Decreased proliferation of Ishikawa and ECC-1cells was a direct result of SDGE-induced apoptosis as demonstrated by annexin V staining and expression of cleaved caspase3. GC/MS analysis identified a total of22different terpenoid compounds in SDGE. with the isomers neral and geranial constituting30-40%. Citral, a mixture of neral and geranial inhibited the proliferation of Ishikawa and ECC-1cells at an IC5010μM (2.3μg/ml). Phenolic compounds such as gingerol, shogaol were not detected in SDGE and6-gingerol was a weaker inhibitor of the proliferation of the endometrial cancer cells. SDGE was more effective in inducing cancer cell death than citral, suggesting that other terpenes present in SDGE were also contributing to endometrial cancer cell death. SDGE treatment resulted in a rapid and strong increase in intracellular calcium and a20-40%decrease in the mitochondrial membrane potential. Ser-15of p53was phosphorylated after15min treatment of the cancer cells with SDGE. This increase in p53was associated with90%decrease in Bcl2whereas no effect was observed on Bax. Inhibitor of p53, pifithrin-α, attenuated the anti-cancer effects of SDGE. Our studies demonstrate that terpenoids from SDGE activate p53, resulting in decreased expression of the anti-apoptotic Bcl-2. The studies indicate that terpenoids from ginger rhizomes should be investigated as therapeutic agents for endometrial cancer. |
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