Necrotic cell death of ovarian adenocarcinoma caused by seminal plasma

Gynecologic Oncology 93 (2004) 671 – 679 www.elsevier.com/locate/ygyno

Necrotic cell death of ovarian adenocarcinoma caused by seminal plasma Lae Ok Park, a Joo Hee Yoon, a Jae Dong Kim, a Ju Tae Seo, b and Seog Nyeon Bae a,* a

Division of Gynecologic Oncology, Department of Obstetrics and Gynecology, Kangnam St. Mary’s Hospital, The Catholic University of Korea, College of Medicine, Seoul, South Korea b Division of Male Infertility, Department of Urology of Samsung Cheil Hospital, Sungkyunkwan University School of Medicine, Seoul, South Korea Received 30 October 2003 Available online 15 April 2004

Abstract Objective. From the knowledge of risk factors of epithelial ovarian cancer, we deduced a hypothesis that human seminal plasma (HSP) has a preventive role in the development of epithelial ovarian cancer. To examine whether HSP directly influences the growth of ovarian cancer, we have investigated the in vitro and in vivo effect of HSP on ovarian adenocarcinoma cell lines (SK-OV-3 and OVCAR-3) in comparison with its effects on normal ovarian surface epithelial cells (NOSE). Methods. Cell viability was determined by MTT assay. Cytotoxic effect was evaluated by flow cytometry analysis, by DNA laddering, and by morphological analysis. In vivo therapeutic effect of HSP was evaluated by the subcutaneous inoculation of SK-OV-3 cells in nude mice (BALB-c) model. Results. HSP at a final concentration of 1:50 induced a time- and dose-dependent inhibition of SK-OV-3 and OVCAR-3 growth, whereas NOSE was not affected. Flow cytometric analysis, DNA laddering, and morphological analysis indicated that HSP induced necrosis, rather than apoptosis, of both ovarian carcinoma cell lines. In in vivo experiment that used the nude mice (Balb-C) with tumor inoculation of SKOV-3 cells, HSP induced necrosis of tumor with no detectable toxic effects on the major organs. Conclusion. These results show that HSP inhibits the growth and induces the necrosis of epithelial ovarian cancer cells and suggests that one or more components of HSP may provide a scientific basis for preventing epithelial ovarian cancer. D 2004 Elsevier Inc. All rights reserved. Keywords: Necrosis; Ovarian adenocarcinoma; Human seminal plasma; Xenograft

Introduction Epithelial ovarian cancer is not only the most common type of ovarian cancer but also the leading cause of death from gynecological malignancy. The highest fatality-tocase ratio is due to most of patients diagnosed at advanced stages because of its insidious progression [1]. Many studies have uniformly indicated that there is a close relationship between the frequency of ovulation and the development of epithelial ovarian cancer. The incidence of epithelial ovarian cancer is increased in women * Corresponding author. Division of Gynecologic Oncology, Department of Obstetrics and Gynecology, Kangnam St. Mary’s Hospital, The Catholic University of Korea, College of Medicine, 137-040 Seocho-gu Banpo-dong 505, Seoul, South Korea. Fax: +82-2-595-1549. E-mail address: [email protected] (S.N. Bae). 0090-8258/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ygyno.2004.02.030

with late age at first birth, early menarche and late menopause, and in women who have never married [2 –6]. Epithelial ovarian cancers are thought to arise from the single layer of ovarian surface epithelium or lines in inclusion cysts. After ovulation, these epithelial cells proliferate to repair defects in the ovarian surface [7]. During the course of repairing process, transformed epithelial cells were vulnerable to spontaneous mutation. Epithelial ovarian cancer arises from the transformed epithelial cells by activation of protooncogenes and inactivation of tumor suppressor genes. Factors that suppress ovulation reduce the risk of epithelial ovarian cancer. It is generally accepted that oral contraceptives have a protective effect against the development of epithelial ovarian cancer [8,9]. But it could not explain the highest incidence rate of epithelial ovarian cancer in postmenopausal women.

672

L.O. Park et al. / Gynecologic Oncology 93 (2004) 671–679

Although the pathogenesis of epithelial ovarian cancer is well known, there is little information about the natural defense mechanism for the development of epithelial ovarian cancer. Recently, a case-control study revealed that a reduced exposure to human semen factors increased the risk of breast cancer, and an experimental study showed a significant efficacy of the semen factors in prevention of mammary lesions and in the preventive effects on ovarian and thyroid cancer [10,11]. There is growing agreement that human seminal plasma (HSP) has diverse biological activities such as immunosuppressive activity [12 –14], bacteriostatic activity [17,18], and antitumoral effect [19 – 21]. From knowledge of risk factors of epithelial ovarian cancer and above-cited reports, we deduced a hypothesis that a reduced exposure to HSP during the period of ovulation is one of etiological risk factors in the development of epithelial ovarian cancer and HSP has a preventive effect in the development of epithelial ovarian cancer. To examine whether HSP directly influences the growth of ovarian cancer, we have been investigating the in vivo and in vitro effects of sperm-free HSP on epithelial ovarian cancer cells (SK-OV-3, OVCAR-3) and, as a control, we also tested its effects in NOSE.

Ag/ml AO and 100 Ag/ml ethidium bromide from stocks prepared in phosphate-buffered saline (PBS), pH 7.2. Primary normal ovarian epithelial cell culture The conditions for growing NOSE in vitro were modifications of the method as described previously [22]. Briefly, normal ovarian tissue was obtained from the operating room from consenting donors and placed in DMEM containing 10% FBS, 100 Ag/ml penicillin, and 100 Ag/ml streptomycin. Epithelial cells were microdissected or scraped from the ovarian surface. The epithelial explants were placed in culture medium and were allowed to attach and proliferate. Once the epithelial cells reached confluency, the explants were removed and the cells were subcultured. Ovarian cancer cell lines and culture

Materials and methods

NIH:OVCAR-3 (human ovarian adenocarcinoma) and SK-OV-3 (human ovarian adenocarcinoma, ascites) cell lines from American Type Culture Collection (Rockville, MD) were cultured in DMEM supplemented with 10% v/v heat-inactivated FBS, 100 Ag/ml of streptomycin, 100 Ag/ml of penicillin, and 100 Ag/ml of L-glutamine. Cells were maintained in a humidified atmosphere of 5% carbon dioxide and 95% air at 37jC.

Semen donors and preparation

Cell viability assay

Semen was obtained from healthy married donors without a history of sexually transmitted disease and any signs and symptoms of genital infection including human immunodeficiency virus and human papilloma virus. Semen sample was collected by masturbation into sterile plastic container, allowed to liquefy for 20 min at room temperature, and then centrifuged at 13,000  g for 10 min. The cell-free supernatants (HSP) were removed to a new tube. The HSP was sterilized by passage through a 0.22-Am Millipore (Millipore Corp, Bedford, MA), filtered and added immediately to cell cultures at the appropriate concentration. All analyses were done with pooled samples.

To investigate the potential effect of HSP on cell growth and survival, cells were treated with different doses and were exposed to different times. Cell viability was measured by MTT assay as described previously [23]. In brief, cells were seeded into 96-well plates at a density of 3  103 cells/ well for 24 h. And then cells were cultured in media containing various concentrations (1:25, 50, 100, 200, and 400 dilution) of HSP for 24 h. Cells were also cultured in media containing HSP at a final concentration of 1:50 for the different time (6, 12, 24, and 48 h). Following exposure to HSP, 20 Al MTT solution (2 mg/ml in PBS) was added to each well, and the plates were incubated for 4 h at 37jC. After the medium was aspirated from each well as completely as possible, 200 Al of DMSO was added to each well to dissolve the formazan crystals. The plates were shaken at room temperature for 5 min and read immediately at 545 nm with a reference wavelength of 620 nm using a Bio-Rad model 3550 microplate reader (Richmond, CA). Wells containing only DMEM-FBS and MTT were used as controls. All experiments were performed on three separate cultures and data were analyzed statistically using one-way analysis of variance of unpaired Student’s t test.

Reagents Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from Life Technologies, Inc., USA. Streptomycin, Penicillin, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), RNase A, proteinase K, dimethyl sulfoxide (DMSO), acridine orange (AO), fluorescein diacetate (FDA) and propidium iodide (PI) were purchased from Sigma Co. (St. Louis, MO). L-glutamine was purchased form Gibco Life Technologies (Grand Island, NY). A stock solution of FDA (1 mg/ ml) was prepared in acetone. A stock solution of PI (1 mg/ ml) was prepared in distilled water. A cocktail of AO and ethidium bromide (AO/EtBr) was prepared by adding 100

Flow cytometric detection of DNA fragmentation A PI staining technique was used to assess the status of cellular DNA as described previously [24]. Cells were

L.O. Park et al. / Gynecologic Oncology 93 (2004) 671–679

incubated in culture media with HSP at a final concentration of 1:50 for 24 h. At the end of the incubation, all floating and adherent cells were collected and fixed in 70% ice-cold ethanol until analysis. After suspending in 500 Al of PBS, cells were digested with 20 Ag/ml RNase A at 37jC for 30 min and then chilled on ice for 10 min. At this point, the PI solution (50 Ag/ml) was added to the cells in RNase solution and the incubation was continued for 1 h at room temperature in dark. After that, the cells were analyzed by flow cytometry using Becton Dickinson FACS system. DNA histograms were collected with a Becton Dickinson flow cytometer (Becton Dickinson, USA). Propidium fluorescence was excited with a 15-mW air-cooled argon ion laser and collected with a 617-long pass optical filter. DNA modeling was performed with ModFit (Verity Software House, Topsham, ME).

673

processed and analyzed in the FACSCalibur by a CELLQuest program. Fluorescence microscopy: AO/ethidium bromide staining Morphologic changes in cells were evaluated by staining with AO and ethidium bromide as described previously [28]. Briefly, cells were cultured in HSP at a final concentration of 1:50 for 24 h and then washed and resuspended in PBS at 1  106 cells/ml. About 1 Al of AO/EtBr was added to 25 Al of cell suspension, mixed gently, and was allowed to sit at room temperature for 2 min. A 10 Al of cell suspension was placed on a glass slide, covered with a coverslip, and examined under 40 magnification with a

DNA laddering analysis The DNA was extracted as described previously [25] with some modification. After cells (2  106) were treated with HSP at a final concentration of 1:50 for 12 and 24 h, the detached cells and remaining adherent cells were pooled together for DNA extraction. SK-OV-3 cells were incubated in the medium with Taxol (5 Ag/ml) for 24 h as a control. The cell pellets were suspended in lysis buffer consisting of 0.5% sodium dodecyl sulfate, 2 mM EDTA, 20 mg/ml proteinase K, and 50 mM Tris – HCl buffer, pH 8.0. The cell lysates were incubated at 55jC for 3 h and the DNA was extracted with an equal volume of phenol/chloroform/ isoamyl alcohol (25:24:1). The aqueous phase was collected and DNA was precipitated with 0.1 volume ammonium acetate and 2.5 volumes cold absolute ethanol, and stored overnight at 20jC. The DNA was dissolved in TE (0.5 M EDTA in 1 M Tris – HCl buffer, pH 8.0) and incubated with RNase A (10 mg/ml) at 37jC for 1 h. The samples were electrophoresed for 3 h at 60 V in 1.5% agarose gel containing 0.5 Ag/ml ethidium bromide and visualized by UV transillumination. Exclusion of PI combined with hydrolysis of FDA To investigate the mechanism of cell death, this assay was used to assess the status of plasma membrane integrity as described previously [26,27]. Cells (1  105 cells/well) were cultured in 6-well plates for 24 h. HSP was added at a final concentration of 1:50 in each well for 6, 12, and 24 h. Cells were washed twice with PBS. About 2 Al of FDA stock solution added in each well was incubated for 15 min at 37jC and then 20 Al of PI stock solution was added and incubated for 5 min at room temperature. After that, the cells were collected and applied to a FACSCalibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA). The excitation wavelength was 488 nm and emitted light was collected via a 530/20 band-pass filter. Data were

Fig. 1. Cytotoxicity of culture medium containing human seminal plasma on the normal ovarian surface epithelial cell (NOSE) and ovarian adenocarcinoma cells (SK-OV-3 and OVCAR-3). (A) The cells were cultured with various concentration of human seminal plasma for 24 h. (B) The cells were incubated at a final concentration of 1:50 dilution over a period of 48 h. At each time period, MTT assay was conducted for measurement of cell viability. Results are expressed as the mean percentage of control. Control cells received an equal volume of vehicle (DMSO). The value of control being 100%. Data are shown as the mean F SD of three independent experiments preformed in triplicate.

674

L.O. Park et al. / Gynecologic Oncology 93 (2004) 671–679

laser-scanning confocal imaging system (MRC1024, BioRad Co., Ltd. Richmond, CA).

Results Cell viability assay

Tumor induction and treatment with HSP Animal studies were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Antitumoral effect of HSP was evaluated by the subcutaneous inoculation of SK-OV-3 cells in nude mice (BALB-c, Charles River Inc., Japan) model. Nude mice were maintained in a specific, pathogen-free environment. Aliquot of SK-OV-3 cells (3  106) in 200 Al of PBS were injected on dorsal subcutaneous site of female nude mice, 4– 5 weeks of age. After the tumor reached 1 cm in diameter on inoculation site, mice received treatment with a 200 Al of 1:50 diluted HSP or a same dose of sterile PBS through intratumoral injection. Twenty-four hours later, five nude mice were sacrificed and the tumors, lungs, hearts, livers, kidneys were excised and fixed in 10% neutral formalin. The specimens were evaluated histologically using hematoxylin and eosin (H&E)-stained sections.

Human seminal plasma has a dose- and a time-dependent toxicity to ovarian adenocarcinoma cells. Exposing the ovarian cancer cells to various concentration of HSP (diluted with medium as 1: 25, 50, 100, 200, and 400) for 24 h, HSP at a final concentration of 1:50 led to a marked inhibition of cell viability as determined by the MTT assay, whereas NOSE had a little effect on the cell viability at the same concentrations (Fig. 1A). Viability of NOSE was 89% at a 1:50 diluted HSP and 93.8% at a 1:400 dilution, whereas SK-OV-3 and OVCAR-3 cell viability was 29%, 25% at a 1:50 dilution and 82%, 80% at a 1:400 dilution, respectively. Therefore, in this experiment, we used HSP at a final concentration of 1:50. Exposure of the OVCAR-3 and SK-OV-3 to HSP at a final concentration of 1:50 over a period of 48 h led to a marked inhibition of cell viability according to an exposure time, whereas viability of NOSE was not affected (Fig. 1B). Viability of SK-OV-3, OVCAR3, and NOSE was 25%, 22%, and 91% at 48 h, respectively.

Fig. 2. Analysis of DNA content and cell cycle status after exposure to human seminal plasma. NOSE, SK-OV-3, and OVCAR-3 were incubated for 24 h in the presence of HSP at a final concentration of 1:50 or in the absence of HSP. At the end of incubation, cells were analyzed for DNA content and cell cycle status by PI staining and subsequent FACS analysis. Events (counts) were plotted against intensity of DNA-staining (FL2, PI) after 24 h of incubation.

L.O. Park et al. / Gynecologic Oncology 93 (2004) 671–679

675

Flow cytometric detection of DNA fragmentation

Fig. 3. DNA fragmentation in HSP-treated NOSE, SK-OV-3, and OVCAR3. Fragmented DNA was isolated from cells treated with HSP at a final concentration of 1:50 for 24 h, and electrophoresed on a 1.5% agarose gel. Then, DNA was visualized with ethidium bromide staining. Lane M, marker (100 bp DNA ladder; Invitrogen Co. Carlsbad, CA).

The dead cells can be separated from normal one by their lower DNA content (Fig. 2). Flow cytometry analyses of cell cycle distribution in NOSE showed that HSP did not have an effect on the growth and division of these cells. Ovarian adenocarcinoma cells exposed to HSP displayed degradation and fragmentation of their DNA after 24 h. Exposure of SK-OV-3 and OVCAR-3 cells to HSP h led not only to G1 arrest, but also to cell death as indicated by an enrichment of cells in G0 –G1 phase (sub-G1 fraction) of the cell cycle. Compared to HSP-untreatment, SK-OV-3 exposed to HSP did show a strong fragmentation after 24 h (0.51% versus 8.01%). Significant fragmentation of G0/1 phase was observed in OVCAR-3, where 26.55% of the nuclei were stained in the sub G0/1 area (HSP-untreatment, 1.73%). These results showed that HSP at a final concentration of 1:50 induced ovarian adenocarcinoma cell death effectively.

Fig. 4. Stainability of untreated (control) and human seminal plasma (HSP) treated cell with PI and FDA. Exponentially growing cells (NOSE, SK-OV-3, and OVCAR-3) were treated with human seminal plasma (HSP) at a final concentration of 1:50 for 6, 12, and 24 h. A short incubation with PI and FDA results in fluorescent labeling in green of live and early apoptotic cells, which still can exclude PI, whereas necrotic and isolated nuclei stained red (PI). Cell fluorescence was analyzed using a FACScan flow cytometer. Loss of esterase activity and an increase in PI stainability are typical of necrotic (N) cells.

676

L.O. Park et al. / Gynecologic Oncology 93 (2004) 671–679

To differentiate a mechanism of cell death, we used a DNA laddering analysis. Internucleosomal DNA fragmentation is a very specific event in apoptosis. Agarose gel electrophoresis was used to examine the production of apoptotic DNA fragments. No obvious internucleosomal ladder of DNA fragments was detected after treatment with HSP at a final concentration of 1:50 for 24 h (Fig. 3). In contrast, SK-OV-3 cells displayed the characteristic DNA fragmentation after treatment with Taxol (1.5 Ag/ml) for 24 h. This result showed that these cells are unable to undergo apoptosis in response to DNA damage by HSP.

predominantly green with FDA, whereas necrotic cells that cannot exclude PI have more intense red fluorescence and proportionally lower green fluorescence. NOSE stained strongly with FDA, in proportion to their DNA content, and have low PI fluorescence. FDA stainability and accessibility to PI of NOSE was not affected by HSP at a final concentration of 1:50. On the other hand, ovarian adenocarcinoma cells (SK-OV-3 and OVCAR-3) that undergo necrosis (6 h treatment with HSP at a final concentration of 1:50) showed diminished FDA fluorescence and more intense PI staining. With time (after 6 h), necrotic cells have unchanged DNA content and would stain more intensely with PI than with FDA (Fig. 4).

Exclusion of PI combined with hydrolysis of FDA

Fluorescence microscopy: AO/ethidium bromide staining

The PI fluoresces red when it binds to DNA or doublestranded RNA. Because the PI is charged and excluded from cells that have their plasma membrane integrity preserved, only those cells that have a ruptured membrane are stained. FDA is uncharged and easily penetrates live cells. The charged product of hydrolysis of FDA, the green fluorochrome fluorescein, becomes entrapped only in the live cells, but escapes from necrotic cells. In this assay, the cells with undamaged plasma membrane exclude PI and stain

AO is cell permeable and fluoresces green in viable cells with intact membranes. In addition, ethidium bromide is excluded from viable cells and fluoresces orange in the presence of DNA. After incubation, cells changed morphologically, viable cells appeared as green fluorescence but necrotic cells had orange fluorescence. Necrotic cells were present in both SK-OV-3 and OVCAR-3 after incubation in HSP at the final concentration of 1:50 when compared to untreated cells (Fig. 5).

DNA laddering analysis

Fig. 5. Morphological changes in cells treated with HSP using AO/ethidium bromide fluorescence microscopy. NOSE, SK-OV-3, and OVCAR-3 cells treated with HSP at a final concentration of 1:50 (HSP+) for 24 h. Necrotic bodies were detected by AO/ethidium bromide staining of nuclei in ovarian adenocarcinoma cell lines (200 magnification). On the other hand, there were no necrotic bodies without HSP (HSP ) for 24 h. Viable cells: green fluorescence, necrotic cells: orange fluorescence.

L.O. Park et al. / Gynecologic Oncology 93 (2004) 671–679

Fig. 6. Representative histology of a subcutaneous tumor formed by SKOV-3 cells. (A) There was a tumor of ovarian cancer cells in the dermis and subcutaneous tissue of non-treated mouse. The tumor showed high cellularity of cancer cells without definite evidence of necrosis. (B) After treatment with a 1:50 diluted HSP, there was a formation of tumor mass in the dermis and subcutaneous tissue. It showed central extensive necrosis and surrounding viable ovarian cancer cells.

Tumor induction and treatment of the animals with HSP Antitumoral effect of HSP was assessed in the BALB-c nude mice model. Xenografted tumors were identified macroscopically and were confirmed by histochemical analysis with H&E staining. As shown in Fig. 6, the treated tumors with 1:50 diluted HSP showed severe necrosis. Neither gross nor microscopic metastases were detected in the liver, lungs, hearts, or kidneys. The histological examination showed that there was no severe damage to the vital organs.

Discussion Although the pathogenesis of epithelial ovarian cancer is well known, unfortunately, there is little information about the natural defense mechanism for the development of epithelial ovarian cancer. Recently, a case-control study revealed that a reduced exposure to human semen factors increased the risk of breast cancer [10] and an experimental study showed a significant efficacy of the semen factors in prevention of mammary lesions and in the preventive effects on thyroid cancer [11]. The HSP contains fluids originating from the prostate glands that include acid phosphatase, citrate, zinc, and magnesium, and from seminal vesicle include fructose and prostaglandins. Secretory products specific for the epididy-

677

mis are lactoferrin, L( )-carnitine, glycerophosphocholine, and the enzyme neutral a, 1 – 4 glucosidase or a-glucosidase [15,16,29]. The immunoglobulin, transferrin, and albumin are highest in ejaculation. Although precise roles for all these substrates have not yet been determined, it is well known that lactoferrin and transferrin are iron-binding protein with bacteriostatic property [17,18]. The immunoglobulins are probably a part of the antibody system of seminal plasma. Immunosuppressive activity of HSP is partly accounted for by its high levels of prostaglandins [13]. There are numerous reports that show that BS-RNase has special biological properties that include specific antitumor, immunosuppressive, and antispermatogenic activities. BS-RNase has an ability to induce time- and dosedependent apoptosis in human lymphocytes and human tumor cells. BS-RNase selectively kills human multidrugresistant neuroblastoma cells via induction of apoptosis and was non-toxic to normal fibroblasts and epithelial cells [20,21]. We treated HSP to eliminate proteins or fatty acids by boiling, ethanol, and/or charcoal treatment. But the results of these treatments were not different with the results of untreated HSP (data are not shown). Furthermore, there was no different activity between different semen samples. The speed of centrifugation did not influence the effect of HSP on these cell lines. Untergasser et al. [30] reported that a low-molecular weight fraction of HSP activates adenylyl cyclase and induces caspase 3-dependent apoptosis in prostatic epithelial cells by decreasing mitochondrial potential and Bcl-2/Bax ratio. We also examined with fractionated HSP according to the molecular weight but the results were not different. Although we do not know which components of the HSP were primarily responsible for necrosis, we thought that more than one component of HSP may play concurrently to induce cytotoxic effects on these carcinoma cells and at least it was not induced solely by a specific protein or prostaglandin component. HSP contains very high concentration of zinc. Much of zinc in HSP is chelated by citrate. Zinc induced apoptosis by decreasing mitochondrial transmembrane potential and Bcl2 protein levels in proliferating prostate epithelial cells [31]. Lately, Lan et al. [32] reported that citric acid could induce human gingival fibroblast death. But its cytotoxic effect is associated with its acidity and it could be prevented by adjusting the pH value of the culture medium to pH 7.5. Because the pH of seminal plasma is 7.2 – 8.0, citric acid alone does not seem to play a cytotoxicity on epithelial ovarian cancer cell in this experiment. We tried to investigate the impact of zinc-citrate on numerous cancer cells. In this report, we showed that HSP induced necrosis of epithelial ovarian cell in vitro and in vivo. This observation is significant because exposure to HSP was associated with natural extermination of malignant transformed ovarian epithelial cells. Considering this experiment, and the risk factors for epithelial ovarian cancer, suggests that advent of maltransformed epithelial cells in the process of disruption

678

L.O. Park et al. / Gynecologic Oncology 93 (2004) 671–679

and healing of epithelial surface by ovulation could be eradicated by some components of seminal plasma. After ejaculation of semen in vaginal canal, one or more cytotoxic components of HSP will be reached to the mutated ovarian epithelial cells directly through reproductive canal or indirectly by absorption from reproductive canal. Sperm entered the peritoneal cavity by their mobile activity but HSP is hardly spread into peritoneal cavity through canal of uterus and fallopian tube. Considering the fact that the tubal ligation had no adverse effects on the prevention of breast cancer [10], most of the cytotoxic components of HSP may be absorbed from vaginal mucosa. Therefore, tubal ligation does not influence the absorption of cytotoxic components of HSP from reproductive canal and scarcely adverse effects on the prevention of ovarian carcinoma. It may be important to keep the HSP in the vagina for a long time after ejaculation for the prevention of breast and epithelial ovarian cancer. Unfortunately, HSP is less likely to use therapeutic purpose because this study showed that HSP induced necrosis of epithelial ovarian cancer. Necrosis is not desirable because cell death through necrosis caused inflammation in the surrounding tissue. Nonetheless, this investigation has provided the first experimental evidence to support that HSP has a preventive and therapeutic effect on epithelial ovarian cancer. Much work remains to be done to determine that HSP could eliminate the transformed ovarian epithelial cells and consequently has a preventive effect on the development of epithelial ovarian cancer. We need a case-control study and several ecological surveys to test the hypothesis that a reduced exposure to HSP during the reproductive lives is an etiological risk factor in the development of ovarian carcinoma. Further in vivo investigation on ovarian cancer in the peritoneal cavity is needed.

[4]

[5]

[6]

[7] [8] [9]

[10]

[11]

[12] [13]

[14]

[15]

[16] [17] [18]

Acknowledgments Cell cycle analyses and confocal imaging analysis were performed at the flow cytometry core facility of the Catholic University School of Medicine of Seoul, Catholic Research Institute of Medical Science. We thank Young Chun Lee and core facility personnel for their professional service. We acknowledge the Catholic Research Foundation for Gynecologic Cancer in Korea Grant for support of this work.

[19]

[20]

[21]

[22]

References [1] Ozols RF. Current status of chemotherapy for ovarian cancer. Semin Oncol 1995;22:61 – 6. [2] Franceschi S, La Vecchia C, Booth M, Tzonou A, Negri E, Parazzini F, et al. Pooled analysis of 3 European case-control studies of ovarian cancer: II. Age at menarche and at menopause. Int J Cancer 1991; 49:57 – 60. [3] Taylor SR, Carroll BE, Lloyd JW. Mortality among women in 3

[23]

[24]

[25]

Catholic religious orders with special reference to cancer. Cancer 1959;12:1207 – 15. Fraumeni Jr HF, Lloyd JW, Smith EM, Wagoner JK. Cancer mortality among nuns: role of marital status in etiology of neoplastic disease in women. J Natl Cancer Inst 1969;42:455 – 68. Weiss NS, Young JL, Roth GJ. Marital status and incidence of ovarian cancer: The U.S. Third National Cancer Survey, 1969 – 71. J Natl Cancer Inst 1977;58:913 – 5. Negri E, Franceschi S, Tzonou A, Booth M, La Vecchia C, Parazzini F, et al. Pooled analysis of 3 European case-control studies: I. Reproductive factors and risk of epithelial ovarian cancer. Int J Cancer 1991;49:50 – 6. Fathalla MF. Incessant ovulation: a factor in ovarian neoplasia? Lancet 1971;2:163. Stanford JL. Oral contraception and neoplasia of the ovary. Contraception 1991;43:543 – 56. Franceschi S, Parazzini F, Negri E, Booth M, La Vecchia C, Beral V, et al. Pooled analysis of 3 European case-control studies of epithelial ovarian cancer: III. Oral contraceptive use. Int J Cancer 1991; 49:61 – 5. Gjorgov AN. Breast cancer and barrier contraception: postulated and corroborated potential for prevention. Folia Med 1998;40(3B Suppl. 3): 17 – 23. Gjorgov AN, Junaid TA, Burns GR, Temmim L, Alex G. Efficacy of preventive prostaglandin treatment of mammary lesions in rats: an experimental trial. Eur J Cancer Prev [London] 1996;5(Suppl. 2): 104 – 5. James K, Hargreave TB. Immunosuppression by seminal plasma and its possible clinical significance. Immunol Today 1984;5:357 – 63. Rees RC, Vallelt P, Clegg A, Potter CW. Suppression of natural and activated human antitumor cytotoxicity by human seminal plasma. Clin Exp Immunol 1986;63:687 – 95. Anderson DJ, Tarter TH. Immunosuppressive effects of mouse seminal plasma components in vivo and in vitro. J Immunol 1982;128: 535 – 9. MacLeod J, Hotchkiss RS. The distribution of spermatozoa and certain chemical constituents in the human ejaculate. J Urol 1942; 48:225 – 9. Amelar RD, Hotchkiss RS. The split ejaculate. Fertil Steril 1965; 16:46 – 60. Masson PL, Heremans JF. Studies on lactoferrin, the iron-biding protein of secretions. Protides Biol Fluids 1966;14:115 – 24. Van Sande M, Van Camp K. Lactoferrin in human prostate tissue. Urol Res 1981;9:241 – 4. Cinatl Jr J, Cinatl J, Kotchetkov R, Vogel JU, Woodcock BG, Matousek J, et al. Bovine seminal ribonuclease selectively kills human multidrug-resistant neuroblastoma cell via induction of apoptosis. Int J Oncol 1999;15:1001 – 9. Cinatl Jr J, Cinatl J, Kotchetkov R, Matousek J, Woodcock BG, Koehl U, et al. Bovine seminal ribonuclease exerts selective cytotoxicity toward neuroblastoma cells both sensitive and resistant to chemotherapeutic drugs. Anticancer Res 2000;20:853 – 9. Liang JY, Liu YY, Zou J, Fanklin RB, Costello LC, Feng P. Inhibitory effect of zinc on human prostatic carcinoma cell growth. Prostate 1999;40:200 – 7. Auersperg N, Siemens CH, Myrdal SE. Human ovarian surface epithelium in primary cultures. In Vitro (Rockville) 1984;20:743 – 55. Hansen MB, Nielsen SE, Berg K. Re-examination and further development of a precise and rapid dye method for measuring cell growth/ cell kill. J Immunol Methods 1989;119:203 – 10. Telford WG, King LE, Fraker PJ. Rapid quantitation of apoptosis in pure and heterogeneous cell population using flow cytometry. J Immunol Methods 1994;172:1 – 16. Leszczynski D, Fagerholm S, Leszczynski K. The effect of the broadband UVA radiation on myeloid leukemia cells: the possible role of protein kinase C in mediation of UVA-induced effects. Photochem Photobiol 1996;64:936 – 42.

L.O. Park et al. / Gynecologic Oncology 93 (2004) 671–679 [26] Horan PK, Kappler JW. Automated fluorescent analysis for cytotoxicity assays. J Immunol Methods 1977;18:309 – 16. [27] Hamori E, Arndt-Jovin DJ, Grimwade BG, Jovin TM. Selection of viable cells with known DNA content. Cytometry 1980;1:132 – 5. [28] Gorman A, McCarthy J, Finucane D, Reville W, Cotter T. Morphological assessment of apoptosis. In: Cotter TG, Martin SJ, editors. Techniques in Apoptosis. A user’s guide. London: Portland Press Ltd; 1996. p. 8 – 10. [29] Cooper TG, Yeung CH, Nashan D, Nieschlag E. Epididymal makers in human infertility. J Androl 1988;9:91 – 101. [30] Untergasser G, Rumpold H, Plas E, Madersvacher S, Berger P. A low-molecular-weight fraction of human seminal plasma activates

679

adenylyl cyclase and induces caspase 3-dependent apoptosis in prostatic epithelial cells by decreasing mitochondrial potential and Bcl-2/ Bax ratio. FASEB J 2001;15:673 – 83. [31] Untergasser G, Rumpold H, Plas E, Witkowski M, Pfister G, Berger P. High levels of zinc ions induce loss of mitochondrial potential and degradation of antiapoptotic Bcl-2 protein in in vitro cultivated human prostate epithelial cells. Biochem Biophys Res Commun 2000;279:607 – 14. [32] Lan WC, Lan WH, Chan CP, Hsieh CC, Chang MC, Jeng JH. The effects of extracellular citric acid acidosis on the viability, cellular adhesion capacity and protein synthesis of cultured human gingival fibroblasts. Aust Dent J 1999;44:123 – 30.