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Ginkgo biloba and ovarian cancer prevention: Epidemiological and biological evidence
Cancer Letters 251 (2007) 43–52 www.elsevier.com/locate/canlet
Ginkgo biloba and ovarian cancer prevention: Epidemiological and biological evidence q Bin Ye a,*, Margarita Aponte a, Yan Dai b, Lily Li c, Ming-Chih D. Ho c, Allison Vitonis a, Dale Edwards a, Tai-Nang Huang c, Daniel W. Cramer a,* a
Laboratory of Gynecologic Oncology and Epidemiology, Department of Obstetrics and Gynecology and Reproductive Biology, Brigham and Women’s Hospital, Harvard Medical School, Dana-Farber Cancer Center, USA b Cancer Research Center, Department of Medicine, Boston University, USA c Linden Bioscience, Woburn, MA, USA Received 10 August 2006; received in revised form 24 October 2006; accepted 26 October 2006
Abstract There is considerable interest in herbal therapies for cancer prevention but often with little scientific evidence to support their use. In this study, we examined epidemiological data regarding effects of commonly used herbal supplements on risk for ovarian cancer and sought supporting biological evidence. 4.2% of 721 controls compared to 1.6% of 668 cases regularly used Ginkgo biloba for an estimated relative risk (and 95% confidence interval) of 0.41 (0.20, 0.84) (p = 0.01); and the effect was most apparent in women with non-mucinous types of ovarian cancer, RR = 0.33 (0.15, 0.74) (p = 0.007). In vitro experiments with normal and ovarian cancer cells showed that Ginkgo extract and its components, quercetin and ginkgolide A and B, have significant anti-proliferative effects (40%) in serous ovarian cancer cells, but little effect in mucinous (RMUG-L) cells. For the ginkgolides, the inhibitory effect appeared to be cell cycle blockage at G0/G1 to S phase. This combined epidemiological and biological data provide supportive evidence for further studies of the chemopreventive or therapeutic effects of Ginkgo and ginkgolides on ovarian cancer. Published by Elsevier Ireland Ltd. Keywords: Ginkgo; Ovarian cancer; Epidemiology; Cell proliferation; Cell biology
Abbreviations: DMSO, dimethyl sulphoxide; EGb761, extract of Ginkgo biloba; MEM, minimum essential medium; FCS, fetal calf serum; LC–MS/MS, liquid chromatography linked tandem mass spectrometry; TFA, trifluoroacetic acid; RR, relative risk; OC, oral contraceptive. q This research is supported by the following Grants: R01CA054419-13, -1P50-CA105009-02, and R21 CA111949-01 from the National Cancer Institute. * Corresponding authors. Tel.: +1 617 732 6976 (B. Ye), +1 617 732 4895 (D.W. Cramer). E-mail addresses: [email protected] (B. Ye), dcramer@part ners.org (D.W. Cramer). 0304-3835/$ - see front matter Published by Elsevier Ireland Ltd. doi:10.1016/j.canlet.2006.10.025
1. Introduction Approximately 38.2 million adults used herbal supplements in the United States during 2002 with greater usage in women than in men (21.0% vs. 16%) [1]. The most commonly used herbal supplements by women include Echinacea, Ginkgo biloba, Ginseng, St. John’s Wort and glucosamine/chondroitin. Use of G. biloba, has been suggested for prevention of Alzheimer’s disease and peripheral
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arterial disease [2–4]; but more recently its potential for treating or preventing cancer has been considered [5]. Extracts of Ginkgo leaves contain both flavonoid and terpenoids constituents, which have anti-oxidant and anti-lipoperoxidative properties considered important in cancer chemoprevention [1]. Anticlastogenic effects on radiation-exposed chromosomes have also been described [6,7]. Cell line studies suggest that Ginkgo may reduce the growth of human breast cancer cells perhaps through its action on the peripheral-type benzodiazepine pathway important in steroid hormone regulation [8] and has significant anti-proliferative and cytotoxic effects on human hepatocellular carcinoma (HCC) cells [9]. In vivo experiments suggest that Ginkgo may promote apoptosis of cancer cells by caspase-3 activation in oral cavity cancer cells in rats [10], G. biloba may also prevent benzo (a)pyrene-induced forestomach carcinogenesis in mice [11]. To date, however, there have been no studies of the effect of Ginkgo on ovarian cancer, the leading cause of mortality from gynecologic cancers in the United States and a cancer for which a chemopreventive approach would be highly desirable. In this study, we examined both epidemiological and biological evidence regarding the potential effects of G. biloba, and its key components on ovarian cancer.
197 (in MA) could not be re-contacted because subjects returned an ‘‘opt out’’ postcard required by the hospital IRB, and 47 no longer had a working telephone. Of the remaining 1281 who were contacted, 152 were ineligible because they had no ovaries or were not the correct age, 59 were incapacitated or did not speak English, and 349 declined leaving 721 who were interviewed and included in this report. After written informed consent, an in-person interview dealing with demographic, medical and family history was conducted. We asked about herbal remedies used continuously for six or more months. Subjects were asked about exposures prior to a reference date defined 1 year prior to their date of diagnosis for ovarian cancer cases and 1 year prior to the date of interview for controls. Subjects also completed a self-administered dietary questionnaire. Heparinized blood specimens were collected from subjects agreeing to provide one, separated into red cell, buffy coat, and plasma components, and stored at 80 centigrade. 2.2. Chemical reagents Dimethyl sulphoxide (DMSO), quercetin, kaempferol, ginkgolide A, and B (>90% HPLC grade) were purchased from Sigma. Standard G. biloba extract powder, with active ingredients of 24% Ginkgoflavon-glycosides, and 6% terpene lactones, was purchased from Spectrum Chemical MFG Corp. (New Brunswick, NJ). Cell culture medium of MCDB-105 and medium 199 were purchased from Sigma–Aldrich (St. Louis, MO) and F12 from Invitrogen (Carlsbad, CA).
2. Materials and methods 2.3. Cell culture 2.1. Epidemiological study This report is based on the second phase of a population-based case-control study of ovarian cancer conducted between 7/1998 and 7/2003 and involving eastern Massachusetts (MA) and all New Hampshire (NH), approved by the Brigham and Women’s Hospital and Dartmouth Medical Center’s Institutional Review. We identified 1267 cases from tumor boards and Statewide Registries and excluded 119 cases who died, 110 who moved from the study area, 1 who had no telephone, 23 who did not speak English, and 46 found to have a non-ovarian primary upon review. Of the remaining 968, physicians denied permission to contact 106 and 171 declined to participate leaving 691 cases interviewed. Of these, 668 had an epithelial ovarian cancer (including borderline malignancies) and are included in this report. Controls were identified through town books in MA and Drivers’ License lists in NH and sampled to match the age and residence of previously accumulated cases. Invitations to participate were sent to 1843 potential controls. Of these 318 had moved and could not be located or had died,
Immortalized normal human ovarian epithelial cell (HOSE-E6E7) and serous type ovarian cancer cell lines (OVCA429, OVCA433, OVCA420) previously established in this laboratory [12,13] were cultured in sterile 75-cm2 cell culture flasks in MCDB 105 and medium 199 supplemented with 10% fetal bovine serum (Gemini Bioproducts Woodland, CA) and 1% antibiotic (200 mM L-glutamine, 10,000 U penicillin and 10 mg streptomycin per milliliter). Two mucinous ovarian cancer cell lines (RMUG-S and RMUG-L) were purchased from The Japanese Collection of Research Bioresource (Tokyo, Japan). RMUG-L cells were cultured in MCDB105/ M199 medium as above, and RMUG-S cells were cultured in F-12 medium with 10% fetal bovine serum and 1% antibiotic as above. Cells were maintained at 37 C under 5% CO2 and 95% air in a high humidity chamber. Monolayer cells at 60–80% confluence were enzymatically removed using trypsin/EDTA and plated in 96-well flat-bottomed plates at concentration of 5 · 103 per well for HOSE-E6E7 cells, 1 · 103 per well for OVCA429 and RMUG-L, respectively. Treatments
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were added 24 h after plating. Ten millimolar stock solutions of G. biloba, quercetin, kaempferol, ginkgolide A, ginkgolide B, were prepared in DMSO and cells were treated with 10, 50 and 100 lM concentrations of each component. Concentration of standard G. biloba treatment was calibrated according to the amount of total active lactones estimated to be 4–6% with average molecular weight about 300 for the standard G. biloba mixture. An equal volume of DMSO (less than <1% concentration) was used as control treatment. 2.4. Cell proliferation and cell cycle analysis Cell proliferation and viability were assessed using an MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay [14] (Promega, Madison, WI). Briefly, the tetrazolium salts are cleaved to formazan by cellular enzymes. An expansion in the number of viable cells results in an increase in the overall activity of mitochondrial dehydrogenases in the sample. This augmentation in enzyme activity leads to an increase in the amount of formazan dye formed, which directly correlates with the number of metabolically active cells in the culture. After 72 h of treatment, 15 lL of the MTT dye solution was added to each well and the plates were incubated at 37 C for 4 h in a humidified chamber. After incubation, 100 lL of the solubilization/stop solution was added to each well. One hour after the addition of the solubilization solution, the contents of the wells were mixed and read by the 96-well plate scanning spectrophotometer (lQuant) and quantitative software (KC-junior, Bio-Tek Instruments, Inc.) at an absorbance of 630 nm for quantitative analysis. Data were collected from at least three separate experiments, and at least eight repeats were performed for each individual treatment. The cell proliferation rate was presented as a percentage of the control, which had been treated with an equivalent volume of DMSO (as 100%). For the cell cycle analysis, ovarian cells were plated in 75 cm2 flasks and treated with 100 lM of ginkgolide A and B; the control was treated with an equal amount of DMSO. After 24 and 48 h, cells were harvested by trypsin digestion and followed by PBS washing and spun for 5 min at room temperature at 12,000 rpm. Cells were fixed with 70% ethanol (in PBS buffer) by suspending the cell pellet and incubated at room temperature for 5 min or at 20 C for two days. The fixed cells were washed once with PBS and then treated with RNase A (50 lg/ ml) at 37 C for 30–60 min. After a single washing with PBS to remove RNase A, propidium iodide (25 lg/ml in PBS buffer) was added. Cells were subjected to analysis using a FACS Calibur Flow Cytometer (Becton–Dickson, San Jose, CA). Each experiment was repeated at least three times and the cell cycle profiles and data were analyzed by ModFit LT software (Verity Software House, Inc., Topsham, ME) [15].
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2.5. LC–MS/MS analysis of ginkgolides To prepare the sample for the ginkgolide analysis, a 50 lL aliquot of human serum was diluted with 50 lL of 45% aqueous acetonitrile and then precipitated with 100 lL of 90% acetonitrile. Standard solutions containing 0.001, 0.005, 0.01, 0.1, 1 and 10 lg/ml of both ginkgolide A and ginkgolide B, respectively, were prepared in the presence of equal volume of human serum and aqueous acetonitrile as described above. All treated samples were centrifuged at 13,000 rpm for 3 min. The supernatants were transferred to a 96-well plate for the LC–MS/MS analysis. The LC–MS/MS system consisted of a CTC Pal auto injector (Leap Technologies, Carrboro, NC), a Rheos CPS-LC binary pump (Flux instruments, Basel, Switzerland), and API 4000 triple quadrupole mass spectrometer (Applied Biosystems, Foster City, CA). HPLC separation was performed using a Luna C18-Cartridge column (20 · 2.0 mm, 5 lm, Phenomenex, Torrance, CA). A combination of an isocratic and a linear gradient from 65% mobile phase A (100% H2O, 0.1% formic acid, 0.01% trifluoroacetic acid (TFA)) to 80% mobile phase B (0.1% formic acid, 0.01% TFA in ACN/ H2O (V/V; 90/ 10)) over 1.5 min at a flow rate of 200 lL/min was used to elute ginkgolides in the serum extracts. Total run time including column re-equilibration, was 3 min. Specific parent/daughter ion pairs (ginkgolide A: 409.3/345.2; and ginkgolide B: 425.2/361.3) were monitored under a multiple-reaction monitoring (MRM) mode using an electrospray positive (ES+) ionization source on the API 4000. 2.6. Statistical analysis Logistic regression analysis was used to calculate the exposure odds ratio to estimate the relative risk (RR) for ovarian cancer associated with the use of any or a particular type of herbal remedy. Adjustment variables included age, study center, oral contraceptive (OC) use, parity, and Jewish ethnic background. Additionally, we adjusted individually for categories of consumption of vitamin A, carotene, tomatoes and tomato sauce and juice, and raw carrots based upon previous work showing the importance of these foods and vitamins on ovarian cancer risk in our data [16]. For the data from the cell proliferation assay, linear regression models were applied to analyze mean OD values from the MTT assays across the different concentration treatments, adjusted for individual experiments. We used a partial F test to determine whether mean OD levels varied across the four concentrations (control, 10, 50, 100 lM). If the means were found to be significantly different, we tested whether or not the treatment OD means were significantly different from the control OD mean. All analyses were conducted with SAS statistical software (SAS Institute, Inc., Cary, NC).
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3. Results 3.1. Epidemiological study Overall 80 (11.1%) of the 721 controls and 67 (10%) of the cases reported regular use of any type of herbal or nutritional supplement (p = 0.71) (Table 1). While over 30 separate supplements were reported, only the five listed in Table 1 were used by more than 1% of subjects. Among these five, only one type of herbal supplement was found to be significantly associated with risk for ovarian cancer, and this was Ginkgo. Thirty (4.2%) of controls versus 11 (1.6%) of cases had regularly used Ginkgo for an adjusted RR (and 95% confidence interval of 0.41 (0.20, 0.84) (p = 0.01)). Use of Ginkgo as the only herbal remedy was also associated with a decreased risk for ovarian cancer, adjusted RR = 0.36 (0.14, 0.91) p = 0.03. Additional adjustment of the Ginkgo association by consumption of vitamin A, carotene, tomatoes, tomato sauce and juice, and raw carrots did not alter the association (data not shown). Stratifying cases by whether they had a mucinous versus non-mucinous type of tumor, we found that the
inverse association of G. biloba use and ovarian cancer was confined to women with non-mucinous types of ovarian cancer RR = 0.33 (0.15, 0.74) (p = 0.007) (Table 2). Because of the small number of exposed subjects, examination of the association by recency and duration of use was very limited (Table 3). The majority of both cases and controls who reported Ginkgo use were using it at their reference date and had used it less than two years; and these were the categories for which the association was significant. No information on the exact type of Ginkgo preparation and number of tablets taken daily was available. 3.2. In vitro cellular study A crude extract of G. biloba as well as its pure chemical components including quercetin, kaempferol, ginkgolide A and ginkgolide B, were used for treatment at different concentrations and time points in serous cancer cells (OVCA429), a mucinous type cancer cell line (RMUGL), and HOSE-E6E7 cells (Fig. 1). The standard G. biloba extract at concentrations equivalent to 10, 50 and 100 lM
Table 1 Distributions and comparison of herbal/nutritional supplements in ovarian cancer case-control population Cases N (%)
Controls N (%)
Adjusteda RR (95% CI)
Any use No Yes
601 (90.0) 67 (10.0)
641 (88.9) 80 (11.1)
1.00 0.94 (0.66, 1.33)
0.71
Ginkgo No Yes
657 (98.4) 11 (1.6)
691 (95.8) 30 (4.2)
1.00 0.41 (0.20, 0.84)
0.01
Echinacea No Yes
657 (98.4) 11 (1.6)
709 (98.3) 12 (1.7)
1.00 0.94 (0.40, 2.19)
0.88
Ginseng No Yes
663 (99.2) 5 (0.8)
713 (98.9) 8 (1.1)
1.00 0.77 (0.25, 2.43)
0.66
St. Johns Wort No Yes
653 (97.8) 15 (2.2)
708 (98.2) 13 (1.8)
1.00 1.26 (0.59, 2.70)
0.55
Glucosamine/chondroitin No Yes
661 (99.0) 7 (1.0)
712 (98.8) 9 (1.2)
1.00 0.90 (0.32, 2.52)
0.84
Mutually exclusive categories No use Ginkgo alone Echinacea alone Ginseng alone St. Johns Wort alone Glucosamine alone Other alone Combination
601 (90.0) 6 (0.9) 10 (1.5) 1 (0.2) 13 (2.0) 7 (1.0) 25 (3.7) 5 (0.8)
641 (88.9) 19 (2.6) 10 (1.4) 4 (0.6) 6 (0.8) 7 (1.0) 22 (3.0) 12 (1.7)
1.00 0.36 0.95 0.35 2.17 1.24 1.31 0.49
0.03 0.92 0.34 0.12 0.70 0.37 0.19
Herbal/nutritional supplement at least weekly for 6 months or longer
a
Adjusted for age, study center, OC use, parity, and Jewish ethnic background.
(0.14, (0.38, (0.04, (0.81, (0.42, (0.72, (0.17,
0.91) 2.36) 3.14) 5.80) 3.69) 2.39) 1.44)
p-value
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Table 2 Ginkgo biloba supplement decreases the risk for non-mucinous ovarian cancers Disease status
Any Ginkgo use N (%)
No Ginkgo use N (%)
Adjusteda RR (95% CI)
p-value
Controls Cases Mucinous Non-mucinous
30 11 3 8
691 657 70 587
1.00 0.41 (0.20, 0.84) 1.17 (0.34, 4.05) 0.33 (0.15, 0.74)
0.01 0.81 0.007
a
(4.2) (1.6) (4.1) (1.3)
(95.8) (98.4) (95.9) (98.7)
Adjusted for age, study center, OC use, parity, and Jewish ethnic background.
Table 3 Recency and duration of Ginkgo use among cases with non-mucinous ovarian cancer and controls Non-mucinous cases N (%)
Controls N (%)
Adjusteda RR (95% CI)
p-value
Ginkgo use at reference age Never used No longer using Currently using
587 (98.7) 2 (0.3) 6 (1.0)
691 (95.8) 9 (1.2) 21 (2.9)
1.00 0.25 (0.05, 1.21) 0.37 (0.15, 0.93)
0.09 0.03
Duration of use No use 62 years >2 years
587 (98.7) 6 (1.0) 2 (0.3)
691 (95.8) 23 (3.2) 7 (1.0)
1.00 0.34 (0.14, 0.85) 0.31 (0.06, 1.54)
0.02 0.15
Ginkgo use at least weekly for 6 months or longer
Adjusted for age, study center, OC use, parity, and Jewish ethnic background. HOSE-E6E7
OVCA 429
120 100 80 Ginkgo biloba 60 40 20 0
Quercetin
Kaempferol
120 100 80 60 40 20 120 100 80 60 40 20
**
120 100 80 Ginkgolide A 60 40 20 120 100 80 Ginkgolide B 60 40 20 0 0
10
50
100
100 80 60 40 20 0 120 100 80 60 40 20 120 100 80 60 40 20 140 120 100 80 60 40 20 120 100 80 60 40 20 0
**
RMUG-L
** **
**
**
120 100 80 60 40 20 0
**
120 100 80 60 40 20
Relative cell proliferation rate (%)
a
120 100 80 60 40 20
**
**
**
**
**
0 10 50 100 Treatment Concentration (μM) T
120 100 80 60 40 20 120 100 80 60 40 20 0 0
10
50
100
Fig. 1. Percent relative cellular proliferation rate (compared to control) after treatment of Ginkgo biloba extract, quercetin, kaempferol, ginkgolide A and B with different concentrations on HOSE-E6E7 cells, serous type ovarian cancer cells (OVCA429), and mucinous type ovarian cancer cells (RMUG-L). Standard error bars are shown in each column. The star symbol (**) indicates the significance (p < 0.001) compared to the controls. Concentration of the standardized mixture Ginkgo biloba was calculated using the 6% active terpene lactones with estimated molecular weight 300.
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of ginkgolides had significant (p < 0.01) inhibitory effects on OVCA429 cells, but much less effect was observed in HOSE and RMUG-L cells. In general, there was little effect observed in quercetin and kaempferol (except for the HOSE-E6E7 cells with 100 lM kaempferol, OVCA429 with 50 and 100 lM quercetin). Ginkgolide A at 50 and 100 lM and ginkgolide B at 10, 50 and 100 lM showed a significant (p < 0.0001) effect on OVCA 429 cancer cell proliferation, which was reduced to about 60% compared to the control treatments. But little effect was observed in HOSE-E6E7 cells and mucinous type cancer cells RMUG-L (Fig. 1). Interestingly, the effect of ginkgolide B on OVCA429 proliferation was comparable in its pattern to the effect of standard G. biloba extract. A similar anti-proliferative effect was also observed in other non-mucinous ovarian cancer cell lines such as OVCA420 and OVCA433, with about 30–40% cell proliferation inhibition by ginkgolide A and B treatment.
3.3. Cell cycle analysis Next, we analyzed the effect of ginkgolide on cell cycle and DNA distribution in HOSE-E6E7 cells, serous type OVCA429 and mucinous type RMUG-L cells. After 24 h treatment, neither ginkgolide A nor ginkgolide B (100 lM) showed a significant effect on DNA content of G0/G1, S and G2/M phases in HOSE-E6E7 cells. After 24 and 48 h treatment with ginkgolide B (100 lM) on OVCA429 cells, G0/G1 phase DNA was significantly (p < 0.01) increased from 47.9% to 51.5% and 65.1%, respectively. S-phase DNA contents were decreased to 35.8% and 24% after ginkgolide B treatment compared to the control (43%) (p < 0.01). However, there was a reverse effect observed in mucinous cells (RMUG-L). After 48 h treatment, the G0/G1 phase DNA was decreased from 57% to 44%, and S-phase DNA was increased from 28.7% to 40.9% (Fig. 2). There were no
Fig. 2. Flow cytometry analysis for the effect of ginkgolide on the cell cycle of ovarian cancer cell lines. HOSE-E6E7 cells with ginkgolide A and B for 24 h, serous type ovarian cancer cells (OVCA429) and mucinous type ovarian cancer cells (RMUG-L) were exposed to 100 lM ginkgolide B for 24 and 48 h and equal volume of DMSO as control. The cells were then harvested and stained with propidium iodide and analyzed for perturbation in the cell cycle. Relative distribution of DNA contents (%) of the cell cycle phases was indicated in each cytometric flow chart. Ginkgolide B induced a G0/G1 phase arrest and S phase decreased significantly (p < 0.01) compared to the control were measured in OVCA429 cells, but not in HOSE-E6E7 and RMUG-L cells.
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observed changes in G0/G1, S-phase and M-phase DNA distribution in mucinous RMUG-S cells after treatment. Similar results were observed in OVCA429, after 48 h treatment with ginkgolide A. Cell G0/G1 DNA was increased from 50.7% to 86.1% and S-phase DNA contents was decreased from 36.5% to 10.3%.
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1.0 ng/ml ginkgolide B were detected in the plasma sample of a woman, who had routinely taken G. biloba for 136 months (Fig. 3C). There were no signals detected in the plasma of a woman with no reported use of G. biloba (Fig. 3D).
4. Discussion 3.4. LC–MS/MS quantification of ginkgolides Because we identified that ginkgolides A and B were the most active components of G. biloba extract, we sought at least descriptive evidence that subjects who used G. biloba had detectable ginkgolides in their plasma. Using LC–MS/MS described in Methods, we constructed the standard curves of ginkgolides at 0.001–1 lg per ml concentrations and these were used for calibration (Fig. 3A and B). Up to 8.5 ng/ml of ginkgolide A and
We have presented epidemiological data supporting an association between regular (at least six months continuous) use of G. biloba and a decreased risk for ovarian cancer, and biological data supporting anti-proliferative effects of key Ginkgo components, which might underlie a chemopreventive effect. The epidemiologic association between Ginkgo use and ovarian cancer appeared to be
Fig. 3. Quantitative assay for serum level of ginkgolide A and B by liquid chromatography and mass spectrometry (LC–MS/MS). (A) Standard curves of pure ginkgolide A and B, mixed with serum (0.001–1.0 lg/ml) detected by the LC–MS/MS. (B) Mass spectrometry signals of specific parent/daughter ion pair (ginkgolide A, and B) with their retention time of 0.001 lg/ml of standard ginkgolide A and B present in the MS profiles. (C) Detectable mass spectrum signals of ginkgolide A and B in serum from a Ginkgo user. (D) Not detectable mass spectrometry signals of ginkgolide A and B in serum from the no-Ginkgo users.
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confined to women with non-mucinous ovarian tumors and indicated that regular use was associated with a RR (and 95% CI) of 0.33 (0.15, 0.74) indicating about a 67% decrease in risk for non-mucinous tumors associated with use of Ginkgo. We asked women with ovarian cancer to focus on the use of herbal supplements at least one year prior to their cancer diagnosis, so that the addition or discontinuation of herbal supplements because of cancer symptoms or treatments was not a factor in the association. In addition, we adjusted for key potential confounders including the well-known protective factors of parity and oral contraceptive use as well as age, study center, dietary factors and Jewish ethnic background – a strong correlate of BRCA1 or BRAC2 cancers. Neither do we believe that the finding represents overselection of healthy controls in our study more like to use herbal supplements since the association was specific for Ginkgo and not other commonly used remedies. In addition our finding that between 4% and 5% of women were using Ginkgo matches use reported by national surveys in the US [17]. The ability to examine for a dose response was limited by the small number of exposed subjects and by the fact that we did not ask about number of tablets taken daily. Another limitation is the small number of exposed subjects which introduces imprecision in the magnitude of the effect. Given these limitations, supporting biological data was clearly needed. Although the potential effects of Ginkgo in the prevention of circulatory disturbances have received considerable attention, its role as a cancer chemopreventive or supplement to conventional therapy is receiving more attention [5]. Ginkgo biloba and its components such as quercetin and ginkgolide may affect a number of cancers through many different pathways as illustrated by the following studies: (1) increased antioxidant activity observed against bladder and breast cancer [8,18,19]; (2) inhibition of cell proliferation and induced cytotoxity in human liver cancer cells [9,20]; (3) blockage of the angiogenic response in lung cancer and decrease metastases [21,22]; (4) induction of gene expression patterns associated with cell proliferation and cell cycle in breast cancer cells [8,19]; (5) induced detoxification enzymes such as cytochrome P450 (CYP), glutathione S-transferase and quinone reductase to prevent colon cancer [23]; and (6) induction of anti-clastogenic effects in leukemia [7]. In our review, we were unable to identify any direct biological data, which would specifically link
Ginkgo to ovarian cancer prevention. Thus we performed our own set of experiments that focused on both the standardized crude Ginkgo extract as well as its purified individual components. We found that crude Ginkgo extract, its pure diterpene components of ginkgolide A and B, and its pure flavonoid, quercetin, have a significant inhibitory effect on ovarian cancer cells. In addition, the antiproliferative effect of ginkgolide A and B appeared to be, at least partially due to cell cycle blockage at G0/G1 to S phase checking point, evident in serous type cancer cells, but not on mucinous type cancer cells, i.e., RMUG-L cells (Fig. 2). It is of some interest that Wei et al. reported that ginkgolide B can inhibit smooth muscle cell proliferation in a concentration dependent manner with inhibition related to a G1 to S phase blockage in cell cycle [24]. It is not clear why ginkgolide A and B have no effect on the RMUG-L line of mucinous ovarian cancer cells (or even a reverse effect in the cell cycle data) (Fig. 2). However, this observation is consistent with both epidemiologic and molecular data suggesting mucinous ovarian tumors do appear to differ from non-mucinous types in many ways [25– 28]. Although other components of Ginkgo, including quercetin, which may have independent anti-tumor activity [29–33], may underlie the basis for the effect of the extract on cell proliferation, we have emphasized the ginkgolides, especially ginkgolide B, because of its more pronounced dose-response. Ginkgolides are unique compounds and can only be found in G. biloba extract, therefore we believe that the inhibitory effect of Ginkgo may primarily result from its diterpene components ginkgolide A and B. In fact, many types of natural diterpenes such as paclitaxel [34] and triterpenes such as 2-cyano3,12-dioxooleana-1,9,-dien-28-oic acid (CDDO) [35–37] have anticancer activities. The fact that the antiproliferative effect of Ginkgo extract at about 2 mM (equivalent to 100 lM ginkgolide) was somewhat greater compared to the individual ginkgolides may suggest a synergistic effect of quercetin, kaempferol, ginkgolide A and B. Because our in vitro experimental data indicates the anti-proliferative effects may reside primarily with the ginkgolide components of Ginkgo extract, we thought it is important to demonstrate that this component is actually present in women who used Ginkgo. Using an LC–MS/MS based quantitative assay, up to 8.5 ng/ml of ginkgolide A and 1.0 ng/ ml ginkgolide B were detected in the plasma sample
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of a woman, who had routinely taken G. biloba for 136 months while no signals were detected in the control plasma of a non-user. These concentrations are consistent with averages of 15 ng/ml measured for ginkgolide A and 4 ng/ml for ginkgolide B reported in 10 young healthy volunteers after a single oral dose of 80 mg of G. biloba (EGb 761 formula) [38]. Here, we must note that a concentration of ginkgolide A of about 8.5 ng/ml in blood is equivalent to about 21 nM and would be one magnitude order below the effective concentration (lM) demonstrated in vitro experiments. Of course the peripheral level of a drug may not reflect the concentration at the tissue of interest if there is preferential binding of the drug to receptors in the preventive pathway. We are currently examining the connection between the ginkolides as known antagonists against the platelet activating factor receptor (PAFR) in ovarian cancer and evidence that PAFR is over expressed in other cancers [39]. In conclusion, we have presented epidemiologic and biologic data revealing that G. biloba, perhaps through its ginkgolide A and B components may have chemopreventive properties against ovarian cancer. Further in vitro and in vivo studies, especially those that explore the biologic mechanism underlying this effect are necessary. Given the high case/ fatality ratio for ovarian cancer and absence of any general population screening methods, we believe such studies are a high priority. Acknowledgements We thank Drs. Samuel C. Mok and Ross S. Berkowitz from the OB/GYN department at the Brigham and Women’s Hospital for their kind support in this project. We are thankful to Peter Schow and Kat Folz-Donahue from Dana-Farber Cancer Institute’s core facility for cell cycle analysis and technical assistance. References [1] B.J. Tesch, Herbs commonly used by women: an evidencebased review, Am. J. Obstet. Gynecol. 188 (2003) S44–S55. [2] F.V. DeFeudis, A brief history of EGb 761 and its therapeutic uses, Pharmacopsychiatry 36 (Suppl. 1) (2003) S2–S7. [3] J. Blume, M. Kieser, U. Holscher, Placebo-controlled double-blind study of the effectiveness of Ginkgo biloba special extract EGb 761 in trained patients with intermittent claudication, Vasa 25 (1996) 265–274.
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[4] B. Schneider, Ginkgo biloba extract in peripheral arterial diseases. Meta-analysis of controlled clinical studies, Arzneimittelforschung 42 (1992) 428–436. [5] F.V. DeFeudis, V. Papadopoulos, K. Drieu, Ginkgo biloba extracts and cancer: a research area in its infancy, Fundam. Clin. Pharmacol. 17 (2003) 405–417. [6] A. Alaoui-Youssefi, I. Lamproglou, K. Drieu, I. Emerit, Anticlastogenic effects of Ginkgo biloba extract (EGb 761) and some of its constituents in irradiated rats, Mutat. Res. 445 (1999) 99–104. [7] I. Emerit, N. Oganesian, T. Sarkisian, R. Arutyunyan, A. Pogosian, K. Asrian, A. Levy, L. Cernjavski, Clastogenic factors in the plasma of Chernobyl accident recovery workers: anticlastogenic effect of Ginkgo biloba extract, Radiat. Res. 144 (1995) 198–205. [8] V. Papadopoulos, A. Kapsis, H. Li, H. Amri, M. Hardwick, M. Culty, P.G. Kasprzyk, M. Carlson, J.P. Moreau, K. Drieu, Drug-induced inhibition of the peripheral-type benzodiazepine receptor expression and cell proliferation in human breast cancer cells, Anticancer Res. 20 (2000) 2835– 2847. [9] J.C. Chao, C.C. Chu, Effects of Ginkgo biloba extract on cell proliferation and cytotoxicity in human hepatocellular carcinoma cells, World J. Gastroenterol. 10 (2004) 37–41. [10] K.S. Kim, K.H. Rhee, J.H. Yoon, J.G. Lee, J.H. Lee, J.B. Yoo, Ginkgo biloba extract (EGb 761) induces apoptosis by the activation of caspase-3 in oral cavity cancer cells, Oral Oncol. 41 (2005) 383–389. [11] A.M. Agha, A.A. El-Fattah, H.H. Al-Zuhair, A.C. AlRikabi, Chemopreventive effect of Ginkgo biloba extract against benzo(a)pyrene-induced forestomach carcinogenesis in mice: amelioration of doxorubicin cardiotoxicity, J. Exp. Clin. Cancer Res. 20 (2001) 39–50. [12] S.W. Tsao, S.C. Mok, E.G. Fey, J.A. Fletcher, T.S. Wan, E.C. Chew, M.G. Muto, R.C. Knapp, R.S. Berkowitz, Characterization of human ovarian surface epithelial cells immortalized by human papilloma viral oncogenes (HPVE6E7 ORFs), Exp. Cell Res. 218 (1995) 499–507. [13] R.C. Bast Jr., M. Feeney, H. Lazarus, L.M. Nadler, R.B. Colvin, R.C. Knapp, Reactivity of a monoclonal antibody with human ovarian carcinoma, J. Clin. Invest. 68 (1981) 1331–1337. [14] T. Mosmann, Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays, J. Immunol. Methods 65 (1983) 55–63. [15] C. Plasencia, R. Dayam, Q. Wang, J. Pinski, T.R. Burke Jr., D.I. Quinn, N. Neamati, Discovery and preclinical evaluation of a novel class of small-molecule compounds in hormone-dependent and -independent cancer cell lines, Mol. Cancer Ther. 4 (2005) 1105–1113. [16] D.W. Cramer, H. Kuper, B.L. Harlow, L. Titus-Ernstoff, Carotenoids, antioxidants and ovarian cancer risk in preand postmenopausal women, Int. J. Cancer 94 (2001) 128– 134. [17] J.P. Kelly, D.W. Kaufman, K. Kelley, L. Rosenberg, T.E. Anderson, A.A. Mitchell, Recent trends in use of herbal and other natural products, Arch. Intern. Med. 165 (2005) 281– 286. [18] H. Kobuchi, M.T. Droy-Lefaix, Y. Christen, L. Packer, Ginkgo biloba extract (EGb 761): inhibitory effect on nitric oxide production in the macrophage cell line RAW 264.7, Biochem. Pharmacol. 53 (1997) 897–903.
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[19] K. Gohil, R.K. Moy, S. Farzin, J.J. Maguire, L. Packer, mRNA expression profile of a human cancer cell line in response to Ginkgo biloba extract: induction of antioxidant response and the Golgi system, Free Radic. Res. 33 (2000) 831–849. [20] F. Cheung, Y.L. Siow, K. O, Inhibition by ginkgolides and bilobalide of the production of nitric oxide in macrophages (THP-1) but not in endothelial cells (HUVEC), Biochem. Pharmacol. 61 (2001) 503–510. [21] C.P. Juarez, J.C. Muino, H. Guglielmone, R. Sambuelli, J.R. Echenique, M. Hernandez, J.D. Luna, Experimental retinopathy of prematurity: angiostatic inhibition by nimodipine, ginkgo-biloba, and dipyridamole, and response to different growth factors, Eur. J. Ophthalmol. 10 (2000) 51– 59. [22] M. Monte, L.E. Davel, E.S. de Lustig, Inhibition of lymphocyte-induced angiogenesis by free radical scavengers, Free Radic. Biol. Med. 17 (1994) 259–266. [23] R. Suzuki, H. Kohno, S. Sugie, K. Sasaki, T. Yoshimura, K. Wada, T. Tanaka, Preventive effects of extract of leaves of ginkgo (Ginkgo biloba) and its component bilobalide on azoxymethane-induced colonic aberrant crypt foci in rats, Cancer Lett. 210 (2004) 159–169. [24] E.H. Wei, M.R. Rao, N.D. Ji, X.Y. Chen, Q. Chen, Inhibitory effects of ginkgolide B on proliferation of bovine aortic smooth muscle cells, Yao Xue Xue Bao 37 (2002) 90–93. [25] C. Pisano, S. Greggi, R. Tambaro, S. Losito, F. Iodice, M. Di Maio, E. Ferrari, M. Falanga, R. Formato, V.R. Iaffaioli, S. Pignata, Activity of chemotherapy in mucinous epithelial ovarian cancer: a retrospective study, Anticancer Res. 25 (2005) 3501–3505. [26] D. Pectasides, G. Fountzilas, G. Aravantinos, H.P. Kalofonos, E. Efstathiou, E. Salamalekis, D. Farmakis, D. Skarlos, E. Briasoulis, T. Economopoulos, M.A. Dimopoulos, Advanced stage mucinous epithelial ovarian cancer: the Hellenic Cooperative Oncology Group experience, Gynecol. Oncol. 97 (2005) 436–441. [27] V. Wang, C. Li, M. Lin, W. Welch, D. Bell, Y.F. Wong, R. Berkowitz, S.C. Mok, C.A. Bandera, Ovarian cancer is a heterogeneous disease, Cancer Genet. Cytogenet. 161 (2005) 170–173. [28] F.W. Wamunyokoli, T. Bonome, J.Y. Lee, C.M. Feltmate, W.R. Welch, M. Radonovich, C. Pise-Masison, J. Brady, K. Hao, R.S. Berkowitz, S. Mok, M.J. Birrer, Expression profiling of mucinous tumors of the ovary identifies genes of
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Ginkgo biloba and ovarian cancer prevention: Epidemiological and biological evidence q Bin Ye a,*, Margarita Aponte a, Yan Dai b, Lily Li c, Ming-Chih D. Ho c, Allison Vitonis a, Dale Edwards a, Tai-Nang Huang c, Daniel W. Cramer a,* a
Laboratory of Gynecologic Oncology and Epidemiology, Department of Obstetrics and Gynecology and Reproductive Biology, Brigham and Women’s Hospital, Harvard Medical School, Dana-Farber Cancer Center, USA b Cancer Research Center, Department of Medicine, Boston University, USA c Linden Bioscience, Woburn, MA, USA Received 10 August 2006; received in revised form 24 October 2006; accepted 26 October 2006
Abstract There is considerable interest in herbal therapies for cancer prevention but often with little scientific evidence to support their use. In this study, we examined epidemiological data regarding effects of commonly used herbal supplements on risk for ovarian cancer and sought supporting biological evidence. 4.2% of 721 controls compared to 1.6% of 668 cases regularly used Ginkgo biloba for an estimated relative risk (and 95% confidence interval) of 0.41 (0.20, 0.84) (p = 0.01); and the effect was most apparent in women with non-mucinous types of ovarian cancer, RR = 0.33 (0.15, 0.74) (p = 0.007). In vitro experiments with normal and ovarian cancer cells showed that Ginkgo extract and its components, quercetin and ginkgolide A and B, have significant anti-proliferative effects (40%) in serous ovarian cancer cells, but little effect in mucinous (RMUG-L) cells. For the ginkgolides, the inhibitory effect appeared to be cell cycle blockage at G0/G1 to S phase. This combined epidemiological and biological data provide supportive evidence for further studies of the chemopreventive or therapeutic effects of Ginkgo and ginkgolides on ovarian cancer. Published by Elsevier Ireland Ltd. Keywords: Ginkgo; Ovarian cancer; Epidemiology; Cell proliferation; Cell biology
Abbreviations: DMSO, dimethyl sulphoxide; EGb761, extract of Ginkgo biloba; MEM, minimum essential medium; FCS, fetal calf serum; LC–MS/MS, liquid chromatography linked tandem mass spectrometry; TFA, trifluoroacetic acid; RR, relative risk; OC, oral contraceptive. q This research is supported by the following Grants: R01CA054419-13, -1P50-CA105009-02, and R21 CA111949-01 from the National Cancer Institute. * Corresponding authors. Tel.: +1 617 732 6976 (B. Ye), +1 617 732 4895 (D.W. Cramer). E-mail addresses: [email protected] (B. Ye), dcramer@part ners.org (D.W. Cramer). 0304-3835/$ - see front matter Published by Elsevier Ireland Ltd. doi:10.1016/j.canlet.2006.10.025
1. Introduction Approximately 38.2 million adults used herbal supplements in the United States during 2002 with greater usage in women than in men (21.0% vs. 16%) [1]. The most commonly used herbal supplements by women include Echinacea, Ginkgo biloba, Ginseng, St. John’s Wort and glucosamine/chondroitin. Use of G. biloba, has been suggested for prevention of Alzheimer’s disease and peripheral
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arterial disease [2–4]; but more recently its potential for treating or preventing cancer has been considered [5]. Extracts of Ginkgo leaves contain both flavonoid and terpenoids constituents, which have anti-oxidant and anti-lipoperoxidative properties considered important in cancer chemoprevention [1]. Anticlastogenic effects on radiation-exposed chromosomes have also been described [6,7]. Cell line studies suggest that Ginkgo may reduce the growth of human breast cancer cells perhaps through its action on the peripheral-type benzodiazepine pathway important in steroid hormone regulation [8] and has significant anti-proliferative and cytotoxic effects on human hepatocellular carcinoma (HCC) cells [9]. In vivo experiments suggest that Ginkgo may promote apoptosis of cancer cells by caspase-3 activation in oral cavity cancer cells in rats [10], G. biloba may also prevent benzo (a)pyrene-induced forestomach carcinogenesis in mice [11]. To date, however, there have been no studies of the effect of Ginkgo on ovarian cancer, the leading cause of mortality from gynecologic cancers in the United States and a cancer for which a chemopreventive approach would be highly desirable. In this study, we examined both epidemiological and biological evidence regarding the potential effects of G. biloba, and its key components on ovarian cancer.
197 (in MA) could not be re-contacted because subjects returned an ‘‘opt out’’ postcard required by the hospital IRB, and 47 no longer had a working telephone. Of the remaining 1281 who were contacted, 152 were ineligible because they had no ovaries or were not the correct age, 59 were incapacitated or did not speak English, and 349 declined leaving 721 who were interviewed and included in this report. After written informed consent, an in-person interview dealing with demographic, medical and family history was conducted. We asked about herbal remedies used continuously for six or more months. Subjects were asked about exposures prior to a reference date defined 1 year prior to their date of diagnosis for ovarian cancer cases and 1 year prior to the date of interview for controls. Subjects also completed a self-administered dietary questionnaire. Heparinized blood specimens were collected from subjects agreeing to provide one, separated into red cell, buffy coat, and plasma components, and stored at 80 centigrade. 2.2. Chemical reagents Dimethyl sulphoxide (DMSO), quercetin, kaempferol, ginkgolide A, and B (>90% HPLC grade) were purchased from Sigma. Standard G. biloba extract powder, with active ingredients of 24% Ginkgoflavon-glycosides, and 6% terpene lactones, was purchased from Spectrum Chemical MFG Corp. (New Brunswick, NJ). Cell culture medium of MCDB-105 and medium 199 were purchased from Sigma–Aldrich (St. Louis, MO) and F12 from Invitrogen (Carlsbad, CA).
2. Materials and methods 2.3. Cell culture 2.1. Epidemiological study This report is based on the second phase of a population-based case-control study of ovarian cancer conducted between 7/1998 and 7/2003 and involving eastern Massachusetts (MA) and all New Hampshire (NH), approved by the Brigham and Women’s Hospital and Dartmouth Medical Center’s Institutional Review. We identified 1267 cases from tumor boards and Statewide Registries and excluded 119 cases who died, 110 who moved from the study area, 1 who had no telephone, 23 who did not speak English, and 46 found to have a non-ovarian primary upon review. Of the remaining 968, physicians denied permission to contact 106 and 171 declined to participate leaving 691 cases interviewed. Of these, 668 had an epithelial ovarian cancer (including borderline malignancies) and are included in this report. Controls were identified through town books in MA and Drivers’ License lists in NH and sampled to match the age and residence of previously accumulated cases. Invitations to participate were sent to 1843 potential controls. Of these 318 had moved and could not be located or had died,
Immortalized normal human ovarian epithelial cell (HOSE-E6E7) and serous type ovarian cancer cell lines (OVCA429, OVCA433, OVCA420) previously established in this laboratory [12,13] were cultured in sterile 75-cm2 cell culture flasks in MCDB 105 and medium 199 supplemented with 10% fetal bovine serum (Gemini Bioproducts Woodland, CA) and 1% antibiotic (200 mM L-glutamine, 10,000 U penicillin and 10 mg streptomycin per milliliter). Two mucinous ovarian cancer cell lines (RMUG-S and RMUG-L) were purchased from The Japanese Collection of Research Bioresource (Tokyo, Japan). RMUG-L cells were cultured in MCDB105/ M199 medium as above, and RMUG-S cells were cultured in F-12 medium with 10% fetal bovine serum and 1% antibiotic as above. Cells were maintained at 37 C under 5% CO2 and 95% air in a high humidity chamber. Monolayer cells at 60–80% confluence were enzymatically removed using trypsin/EDTA and plated in 96-well flat-bottomed plates at concentration of 5 · 103 per well for HOSE-E6E7 cells, 1 · 103 per well for OVCA429 and RMUG-L, respectively. Treatments
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were added 24 h after plating. Ten millimolar stock solutions of G. biloba, quercetin, kaempferol, ginkgolide A, ginkgolide B, were prepared in DMSO and cells were treated with 10, 50 and 100 lM concentrations of each component. Concentration of standard G. biloba treatment was calibrated according to the amount of total active lactones estimated to be 4–6% with average molecular weight about 300 for the standard G. biloba mixture. An equal volume of DMSO (less than <1% concentration) was used as control treatment. 2.4. Cell proliferation and cell cycle analysis Cell proliferation and viability were assessed using an MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay [14] (Promega, Madison, WI). Briefly, the tetrazolium salts are cleaved to formazan by cellular enzymes. An expansion in the number of viable cells results in an increase in the overall activity of mitochondrial dehydrogenases in the sample. This augmentation in enzyme activity leads to an increase in the amount of formazan dye formed, which directly correlates with the number of metabolically active cells in the culture. After 72 h of treatment, 15 lL of the MTT dye solution was added to each well and the plates were incubated at 37 C for 4 h in a humidified chamber. After incubation, 100 lL of the solubilization/stop solution was added to each well. One hour after the addition of the solubilization solution, the contents of the wells were mixed and read by the 96-well plate scanning spectrophotometer (lQuant) and quantitative software (KC-junior, Bio-Tek Instruments, Inc.) at an absorbance of 630 nm for quantitative analysis. Data were collected from at least three separate experiments, and at least eight repeats were performed for each individual treatment. The cell proliferation rate was presented as a percentage of the control, which had been treated with an equivalent volume of DMSO (as 100%). For the cell cycle analysis, ovarian cells were plated in 75 cm2 flasks and treated with 100 lM of ginkgolide A and B; the control was treated with an equal amount of DMSO. After 24 and 48 h, cells were harvested by trypsin digestion and followed by PBS washing and spun for 5 min at room temperature at 12,000 rpm. Cells were fixed with 70% ethanol (in PBS buffer) by suspending the cell pellet and incubated at room temperature for 5 min or at 20 C for two days. The fixed cells were washed once with PBS and then treated with RNase A (50 lg/ ml) at 37 C for 30–60 min. After a single washing with PBS to remove RNase A, propidium iodide (25 lg/ml in PBS buffer) was added. Cells were subjected to analysis using a FACS Calibur Flow Cytometer (Becton–Dickson, San Jose, CA). Each experiment was repeated at least three times and the cell cycle profiles and data were analyzed by ModFit LT software (Verity Software House, Inc., Topsham, ME) [15].
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2.5. LC–MS/MS analysis of ginkgolides To prepare the sample for the ginkgolide analysis, a 50 lL aliquot of human serum was diluted with 50 lL of 45% aqueous acetonitrile and then precipitated with 100 lL of 90% acetonitrile. Standard solutions containing 0.001, 0.005, 0.01, 0.1, 1 and 10 lg/ml of both ginkgolide A and ginkgolide B, respectively, were prepared in the presence of equal volume of human serum and aqueous acetonitrile as described above. All treated samples were centrifuged at 13,000 rpm for 3 min. The supernatants were transferred to a 96-well plate for the LC–MS/MS analysis. The LC–MS/MS system consisted of a CTC Pal auto injector (Leap Technologies, Carrboro, NC), a Rheos CPS-LC binary pump (Flux instruments, Basel, Switzerland), and API 4000 triple quadrupole mass spectrometer (Applied Biosystems, Foster City, CA). HPLC separation was performed using a Luna C18-Cartridge column (20 · 2.0 mm, 5 lm, Phenomenex, Torrance, CA). A combination of an isocratic and a linear gradient from 65% mobile phase A (100% H2O, 0.1% formic acid, 0.01% trifluoroacetic acid (TFA)) to 80% mobile phase B (0.1% formic acid, 0.01% TFA in ACN/ H2O (V/V; 90/ 10)) over 1.5 min at a flow rate of 200 lL/min was used to elute ginkgolides in the serum extracts. Total run time including column re-equilibration, was 3 min. Specific parent/daughter ion pairs (ginkgolide A: 409.3/345.2; and ginkgolide B: 425.2/361.3) were monitored under a multiple-reaction monitoring (MRM) mode using an electrospray positive (ES+) ionization source on the API 4000. 2.6. Statistical analysis Logistic regression analysis was used to calculate the exposure odds ratio to estimate the relative risk (RR) for ovarian cancer associated with the use of any or a particular type of herbal remedy. Adjustment variables included age, study center, oral contraceptive (OC) use, parity, and Jewish ethnic background. Additionally, we adjusted individually for categories of consumption of vitamin A, carotene, tomatoes and tomato sauce and juice, and raw carrots based upon previous work showing the importance of these foods and vitamins on ovarian cancer risk in our data [16]. For the data from the cell proliferation assay, linear regression models were applied to analyze mean OD values from the MTT assays across the different concentration treatments, adjusted for individual experiments. We used a partial F test to determine whether mean OD levels varied across the four concentrations (control, 10, 50, 100 lM). If the means were found to be significantly different, we tested whether or not the treatment OD means were significantly different from the control OD mean. All analyses were conducted with SAS statistical software (SAS Institute, Inc., Cary, NC).
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3. Results 3.1. Epidemiological study Overall 80 (11.1%) of the 721 controls and 67 (10%) of the cases reported regular use of any type of herbal or nutritional supplement (p = 0.71) (Table 1). While over 30 separate supplements were reported, only the five listed in Table 1 were used by more than 1% of subjects. Among these five, only one type of herbal supplement was found to be significantly associated with risk for ovarian cancer, and this was Ginkgo. Thirty (4.2%) of controls versus 11 (1.6%) of cases had regularly used Ginkgo for an adjusted RR (and 95% confidence interval of 0.41 (0.20, 0.84) (p = 0.01)). Use of Ginkgo as the only herbal remedy was also associated with a decreased risk for ovarian cancer, adjusted RR = 0.36 (0.14, 0.91) p = 0.03. Additional adjustment of the Ginkgo association by consumption of vitamin A, carotene, tomatoes, tomato sauce and juice, and raw carrots did not alter the association (data not shown). Stratifying cases by whether they had a mucinous versus non-mucinous type of tumor, we found that the
inverse association of G. biloba use and ovarian cancer was confined to women with non-mucinous types of ovarian cancer RR = 0.33 (0.15, 0.74) (p = 0.007) (Table 2). Because of the small number of exposed subjects, examination of the association by recency and duration of use was very limited (Table 3). The majority of both cases and controls who reported Ginkgo use were using it at their reference date and had used it less than two years; and these were the categories for which the association was significant. No information on the exact type of Ginkgo preparation and number of tablets taken daily was available. 3.2. In vitro cellular study A crude extract of G. biloba as well as its pure chemical components including quercetin, kaempferol, ginkgolide A and ginkgolide B, were used for treatment at different concentrations and time points in serous cancer cells (OVCA429), a mucinous type cancer cell line (RMUGL), and HOSE-E6E7 cells (Fig. 1). The standard G. biloba extract at concentrations equivalent to 10, 50 and 100 lM
Table 1 Distributions and comparison of herbal/nutritional supplements in ovarian cancer case-control population Cases N (%)
Controls N (%)
Adjusteda RR (95% CI)
Any use No Yes
601 (90.0) 67 (10.0)
641 (88.9) 80 (11.1)
1.00 0.94 (0.66, 1.33)
0.71
Ginkgo No Yes
657 (98.4) 11 (1.6)
691 (95.8) 30 (4.2)
1.00 0.41 (0.20, 0.84)
0.01
Echinacea No Yes
657 (98.4) 11 (1.6)
709 (98.3) 12 (1.7)
1.00 0.94 (0.40, 2.19)
0.88
Ginseng No Yes
663 (99.2) 5 (0.8)
713 (98.9) 8 (1.1)
1.00 0.77 (0.25, 2.43)
0.66
St. Johns Wort No Yes
653 (97.8) 15 (2.2)
708 (98.2) 13 (1.8)
1.00 1.26 (0.59, 2.70)
0.55
Glucosamine/chondroitin No Yes
661 (99.0) 7 (1.0)
712 (98.8) 9 (1.2)
1.00 0.90 (0.32, 2.52)
0.84
Mutually exclusive categories No use Ginkgo alone Echinacea alone Ginseng alone St. Johns Wort alone Glucosamine alone Other alone Combination
601 (90.0) 6 (0.9) 10 (1.5) 1 (0.2) 13 (2.0) 7 (1.0) 25 (3.7) 5 (0.8)
641 (88.9) 19 (2.6) 10 (1.4) 4 (0.6) 6 (0.8) 7 (1.0) 22 (3.0) 12 (1.7)
1.00 0.36 0.95 0.35 2.17 1.24 1.31 0.49
0.03 0.92 0.34 0.12 0.70 0.37 0.19
Herbal/nutritional supplement at least weekly for 6 months or longer
a
Adjusted for age, study center, OC use, parity, and Jewish ethnic background.
(0.14, (0.38, (0.04, (0.81, (0.42, (0.72, (0.17,
0.91) 2.36) 3.14) 5.80) 3.69) 2.39) 1.44)
p-value
B. Ye et al. / Cancer Letters 251 (2007) 43–52
47
Table 2 Ginkgo biloba supplement decreases the risk for non-mucinous ovarian cancers Disease status
Any Ginkgo use N (%)
No Ginkgo use N (%)
Adjusteda RR (95% CI)
p-value
Controls Cases Mucinous Non-mucinous
30 11 3 8
691 657 70 587
1.00 0.41 (0.20, 0.84) 1.17 (0.34, 4.05) 0.33 (0.15, 0.74)
0.01 0.81 0.007
a
(4.2) (1.6) (4.1) (1.3)
(95.8) (98.4) (95.9) (98.7)
Adjusted for age, study center, OC use, parity, and Jewish ethnic background.
Table 3 Recency and duration of Ginkgo use among cases with non-mucinous ovarian cancer and controls Non-mucinous cases N (%)
Controls N (%)
Adjusteda RR (95% CI)
p-value
Ginkgo use at reference age Never used No longer using Currently using
587 (98.7) 2 (0.3) 6 (1.0)
691 (95.8) 9 (1.2) 21 (2.9)
1.00 0.25 (0.05, 1.21) 0.37 (0.15, 0.93)
0.09 0.03
Duration of use No use 62 years >2 years
587 (98.7) 6 (1.0) 2 (0.3)
691 (95.8) 23 (3.2) 7 (1.0)
1.00 0.34 (0.14, 0.85) 0.31 (0.06, 1.54)
0.02 0.15
Ginkgo use at least weekly for 6 months or longer
Adjusted for age, study center, OC use, parity, and Jewish ethnic background. HOSE-E6E7
OVCA 429
120 100 80 Ginkgo biloba 60 40 20 0
Quercetin
Kaempferol
120 100 80 60 40 20 120 100 80 60 40 20
**
120 100 80 Ginkgolide A 60 40 20 120 100 80 Ginkgolide B 60 40 20 0 0
10
50
100
100 80 60 40 20 0 120 100 80 60 40 20 120 100 80 60 40 20 140 120 100 80 60 40 20 120 100 80 60 40 20 0
**
RMUG-L
** **
**
**
120 100 80 60 40 20 0
**
120 100 80 60 40 20
Relative cell proliferation rate (%)
a
120 100 80 60 40 20
**
**
**
**
**
0 10 50 100 Treatment Concentration (μM) T
120 100 80 60 40 20 120 100 80 60 40 20 0 0
10
50
100
Fig. 1. Percent relative cellular proliferation rate (compared to control) after treatment of Ginkgo biloba extract, quercetin, kaempferol, ginkgolide A and B with different concentrations on HOSE-E6E7 cells, serous type ovarian cancer cells (OVCA429), and mucinous type ovarian cancer cells (RMUG-L). Standard error bars are shown in each column. The star symbol (**) indicates the significance (p < 0.001) compared to the controls. Concentration of the standardized mixture Ginkgo biloba was calculated using the 6% active terpene lactones with estimated molecular weight 300.
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B. Ye et al. / Cancer Letters 251 (2007) 43–52
of ginkgolides had significant (p < 0.01) inhibitory effects on OVCA429 cells, but much less effect was observed in HOSE and RMUG-L cells. In general, there was little effect observed in quercetin and kaempferol (except for the HOSE-E6E7 cells with 100 lM kaempferol, OVCA429 with 50 and 100 lM quercetin). Ginkgolide A at 50 and 100 lM and ginkgolide B at 10, 50 and 100 lM showed a significant (p < 0.0001) effect on OVCA 429 cancer cell proliferation, which was reduced to about 60% compared to the control treatments. But little effect was observed in HOSE-E6E7 cells and mucinous type cancer cells RMUG-L (Fig. 1). Interestingly, the effect of ginkgolide B on OVCA429 proliferation was comparable in its pattern to the effect of standard G. biloba extract. A similar anti-proliferative effect was also observed in other non-mucinous ovarian cancer cell lines such as OVCA420 and OVCA433, with about 30–40% cell proliferation inhibition by ginkgolide A and B treatment.
3.3. Cell cycle analysis Next, we analyzed the effect of ginkgolide on cell cycle and DNA distribution in HOSE-E6E7 cells, serous type OVCA429 and mucinous type RMUG-L cells. After 24 h treatment, neither ginkgolide A nor ginkgolide B (100 lM) showed a significant effect on DNA content of G0/G1, S and G2/M phases in HOSE-E6E7 cells. After 24 and 48 h treatment with ginkgolide B (100 lM) on OVCA429 cells, G0/G1 phase DNA was significantly (p < 0.01) increased from 47.9% to 51.5% and 65.1%, respectively. S-phase DNA contents were decreased to 35.8% and 24% after ginkgolide B treatment compared to the control (43%) (p < 0.01). However, there was a reverse effect observed in mucinous cells (RMUG-L). After 48 h treatment, the G0/G1 phase DNA was decreased from 57% to 44%, and S-phase DNA was increased from 28.7% to 40.9% (Fig. 2). There were no
Fig. 2. Flow cytometry analysis for the effect of ginkgolide on the cell cycle of ovarian cancer cell lines. HOSE-E6E7 cells with ginkgolide A and B for 24 h, serous type ovarian cancer cells (OVCA429) and mucinous type ovarian cancer cells (RMUG-L) were exposed to 100 lM ginkgolide B for 24 and 48 h and equal volume of DMSO as control. The cells were then harvested and stained with propidium iodide and analyzed for perturbation in the cell cycle. Relative distribution of DNA contents (%) of the cell cycle phases was indicated in each cytometric flow chart. Ginkgolide B induced a G0/G1 phase arrest and S phase decreased significantly (p < 0.01) compared to the control were measured in OVCA429 cells, but not in HOSE-E6E7 and RMUG-L cells.
B. Ye et al. / Cancer Letters 251 (2007) 43–52
observed changes in G0/G1, S-phase and M-phase DNA distribution in mucinous RMUG-S cells after treatment. Similar results were observed in OVCA429, after 48 h treatment with ginkgolide A. Cell G0/G1 DNA was increased from 50.7% to 86.1% and S-phase DNA contents was decreased from 36.5% to 10.3%.
49
1.0 ng/ml ginkgolide B were detected in the plasma sample of a woman, who had routinely taken G. biloba for 136 months (Fig. 3C). There were no signals detected in the plasma of a woman with no reported use of G. biloba (Fig. 3D).
4. Discussion 3.4. LC–MS/MS quantification of ginkgolides Because we identified that ginkgolides A and B were the most active components of G. biloba extract, we sought at least descriptive evidence that subjects who used G. biloba had detectable ginkgolides in their plasma. Using LC–MS/MS described in Methods, we constructed the standard curves of ginkgolides at 0.001–1 lg per ml concentrations and these were used for calibration (Fig. 3A and B). Up to 8.5 ng/ml of ginkgolide A and
We have presented epidemiological data supporting an association between regular (at least six months continuous) use of G. biloba and a decreased risk for ovarian cancer, and biological data supporting anti-proliferative effects of key Ginkgo components, which might underlie a chemopreventive effect. The epidemiologic association between Ginkgo use and ovarian cancer appeared to be
Fig. 3. Quantitative assay for serum level of ginkgolide A and B by liquid chromatography and mass spectrometry (LC–MS/MS). (A) Standard curves of pure ginkgolide A and B, mixed with serum (0.001–1.0 lg/ml) detected by the LC–MS/MS. (B) Mass spectrometry signals of specific parent/daughter ion pair (ginkgolide A, and B) with their retention time of 0.001 lg/ml of standard ginkgolide A and B present in the MS profiles. (C) Detectable mass spectrum signals of ginkgolide A and B in serum from a Ginkgo user. (D) Not detectable mass spectrometry signals of ginkgolide A and B in serum from the no-Ginkgo users.
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B. Ye et al. / Cancer Letters 251 (2007) 43–52
confined to women with non-mucinous ovarian tumors and indicated that regular use was associated with a RR (and 95% CI) of 0.33 (0.15, 0.74) indicating about a 67% decrease in risk for non-mucinous tumors associated with use of Ginkgo. We asked women with ovarian cancer to focus on the use of herbal supplements at least one year prior to their cancer diagnosis, so that the addition or discontinuation of herbal supplements because of cancer symptoms or treatments was not a factor in the association. In addition, we adjusted for key potential confounders including the well-known protective factors of parity and oral contraceptive use as well as age, study center, dietary factors and Jewish ethnic background – a strong correlate of BRCA1 or BRAC2 cancers. Neither do we believe that the finding represents overselection of healthy controls in our study more like to use herbal supplements since the association was specific for Ginkgo and not other commonly used remedies. In addition our finding that between 4% and 5% of women were using Ginkgo matches use reported by national surveys in the US [17]. The ability to examine for a dose response was limited by the small number of exposed subjects and by the fact that we did not ask about number of tablets taken daily. Another limitation is the small number of exposed subjects which introduces imprecision in the magnitude of the effect. Given these limitations, supporting biological data was clearly needed. Although the potential effects of Ginkgo in the prevention of circulatory disturbances have received considerable attention, its role as a cancer chemopreventive or supplement to conventional therapy is receiving more attention [5]. Ginkgo biloba and its components such as quercetin and ginkgolide may affect a number of cancers through many different pathways as illustrated by the following studies: (1) increased antioxidant activity observed against bladder and breast cancer [8,18,19]; (2) inhibition of cell proliferation and induced cytotoxity in human liver cancer cells [9,20]; (3) blockage of the angiogenic response in lung cancer and decrease metastases [21,22]; (4) induction of gene expression patterns associated with cell proliferation and cell cycle in breast cancer cells [8,19]; (5) induced detoxification enzymes such as cytochrome P450 (CYP), glutathione S-transferase and quinone reductase to prevent colon cancer [23]; and (6) induction of anti-clastogenic effects in leukemia [7]. In our review, we were unable to identify any direct biological data, which would specifically link
Ginkgo to ovarian cancer prevention. Thus we performed our own set of experiments that focused on both the standardized crude Ginkgo extract as well as its purified individual components. We found that crude Ginkgo extract, its pure diterpene components of ginkgolide A and B, and its pure flavonoid, quercetin, have a significant inhibitory effect on ovarian cancer cells. In addition, the antiproliferative effect of ginkgolide A and B appeared to be, at least partially due to cell cycle blockage at G0/G1 to S phase checking point, evident in serous type cancer cells, but not on mucinous type cancer cells, i.e., RMUG-L cells (Fig. 2). It is of some interest that Wei et al. reported that ginkgolide B can inhibit smooth muscle cell proliferation in a concentration dependent manner with inhibition related to a G1 to S phase blockage in cell cycle [24]. It is not clear why ginkgolide A and B have no effect on the RMUG-L line of mucinous ovarian cancer cells (or even a reverse effect in the cell cycle data) (Fig. 2). However, this observation is consistent with both epidemiologic and molecular data suggesting mucinous ovarian tumors do appear to differ from non-mucinous types in many ways [25– 28]. Although other components of Ginkgo, including quercetin, which may have independent anti-tumor activity [29–33], may underlie the basis for the effect of the extract on cell proliferation, we have emphasized the ginkgolides, especially ginkgolide B, because of its more pronounced dose-response. Ginkgolides are unique compounds and can only be found in G. biloba extract, therefore we believe that the inhibitory effect of Ginkgo may primarily result from its diterpene components ginkgolide A and B. In fact, many types of natural diterpenes such as paclitaxel [34] and triterpenes such as 2-cyano3,12-dioxooleana-1,9,-dien-28-oic acid (CDDO) [35–37] have anticancer activities. The fact that the antiproliferative effect of Ginkgo extract at about 2 mM (equivalent to 100 lM ginkgolide) was somewhat greater compared to the individual ginkgolides may suggest a synergistic effect of quercetin, kaempferol, ginkgolide A and B. Because our in vitro experimental data indicates the anti-proliferative effects may reside primarily with the ginkgolide components of Ginkgo extract, we thought it is important to demonstrate that this component is actually present in women who used Ginkgo. Using an LC–MS/MS based quantitative assay, up to 8.5 ng/ml of ginkgolide A and 1.0 ng/ ml ginkgolide B were detected in the plasma sample
B. Ye et al. / Cancer Letters 251 (2007) 43–52
of a woman, who had routinely taken G. biloba for 136 months while no signals were detected in the control plasma of a non-user. These concentrations are consistent with averages of 15 ng/ml measured for ginkgolide A and 4 ng/ml for ginkgolide B reported in 10 young healthy volunteers after a single oral dose of 80 mg of G. biloba (EGb 761 formula) [38]. Here, we must note that a concentration of ginkgolide A of about 8.5 ng/ml in blood is equivalent to about 21 nM and would be one magnitude order below the effective concentration (lM) demonstrated in vitro experiments. Of course the peripheral level of a drug may not reflect the concentration at the tissue of interest if there is preferential binding of the drug to receptors in the preventive pathway. We are currently examining the connection between the ginkolides as known antagonists against the platelet activating factor receptor (PAFR) in ovarian cancer and evidence that PAFR is over expressed in other cancers [39]. In conclusion, we have presented epidemiologic and biologic data revealing that G. biloba, perhaps through its ginkgolide A and B components may have chemopreventive properties against ovarian cancer. Further in vitro and in vivo studies, especially those that explore the biologic mechanism underlying this effect are necessary. Given the high case/ fatality ratio for ovarian cancer and absence of any general population screening methods, we believe such studies are a high priority. Acknowledgements We thank Drs. Samuel C. Mok and Ross S. Berkowitz from the OB/GYN department at the Brigham and Women’s Hospital for their kind support in this project. We are thankful to Peter Schow and Kat Folz-Donahue from Dana-Farber Cancer Institute’s core facility for cell cycle analysis and technical assistance. References [1] B.J. Tesch, Herbs commonly used by women: an evidencebased review, Am. J. Obstet. Gynecol. 188 (2003) S44–S55. [2] F.V. DeFeudis, A brief history of EGb 761 and its therapeutic uses, Pharmacopsychiatry 36 (Suppl. 1) (2003) S2–S7. [3] J. Blume, M. Kieser, U. Holscher, Placebo-controlled double-blind study of the effectiveness of Ginkgo biloba special extract EGb 761 in trained patients with intermittent claudication, Vasa 25 (1996) 265–274.
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