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- Original Article -

Gossypol inhibits proliferation of endometrioma cells in culture

Shawky Z. A. Badawy¹, Abdul-Kader Souid², Violeta Cuenca¹, Nicholas Montalto¹, Frances Shue¹

Department of Obstetrics and Gynecology, and Department of Pediatrics, SUNY Upstate Medical University, Syracuse, New York 13210, USA

Abstract

Aim: To evaluate the anti-proliferative activity and mitochondrial toxicity of gossypol in endometrioma cells maintained in short-term cultures. Methods: (A) Three endometrioma cell lines from patients were treated with 25 or 50 nmol/L gossypol for up to 12 days. The effect of gossypol on the cell growth was recorded. (B) A phosphorescence oxygen analyzer was used to determine the effects of gossypol on mitochondrial oxygen consumption of six endometrioma cell lines from patients. (C) Cellular gossypol accumulations in three endometrioma cell lines from patients were measured by high-pressure liquid chromatography. Results: Proliferation of the endometrioma cells was inhibited by 25 and 50 nmol/L gossypol. Respiration of the endometrioma cells was inhibited by 10 μmol/L gossypol. Cellular gossypol was detected in the endometrioma cell lines that were treated for 24 h with 10 and 0.3 μmol/L gossypol. Conclusion: Gossypol invokes a potent toxicity on cultured endometrioma cells. (Asian J Androl 2007 May; 9: 388_393)

Keywords: gossypol; endometrioma; mitochondria; oxygen consumption; cellular respiration

Correspondence to: Prof. Shawky Z. A. Badawy, Department of Obstetrics and Gynecology, SUNY Upstate Medical University, 750 E. Adams Street, Syracuse, New York 13210, USA.

Tel: +1-315-470-7905 Fax: +1-315-470-2838
E-mail: badawys@upstate.edu
Received 2005-10-25 Accepted 2006-02-27

DOI: 10.1111/j.1745-7262.2007.00168.x

1 Introduction

Gossypol [(2,2'-binaphthalene)-8,8'-dicarboxyaldeyde-1,1'6,6',7,7'-hexahydoxy5,5'-di-isopropyl-3,3'-dimethyl] is a yellow pigment found in the cottonseeds of the plant genus Gossypium [1]. This polyphenolic binaphthalene aldehyde has potent contraceptive and anticancer activities, including those against multi-drug resistant tumors [2_6]. Its mechanism of action however remains under investigation.

Gossypol was introduced (in ~1960) as an anti-fertility agent for men [4, 7]. This effect was found to be dose dependent [8, 9]. Animal and human studies showed gossypol reduced sperm counts and increased dead-sperm numbers. These toxic insults were mostly attributed to a direct effect of gossypol on the seminiferous tubules of the testes [10]. The spermatids and spermatocytes were especially sensitive, showing increased drug accumulation and ultrastructural changes in the nuclear membranes, endoplasmic reticulum and mitochondria. In vitro studies on the mitochondria showed gossypol specifically inhibited respiration [11].

Gossypol was also used in China (since ~1970) for treatment of women with uterine fibroids, uterine bleeding and endometriosis [12]. The treatment in these cases was effective in reducing the size of fibroids, leading to amenorrhea and improving the symptoms of endometriosis.

Gossypol primarily impairs mitochondrial functions [6]. The drug uncouples oxidative phosphorylation [13, 14] and initiates apoptosis. The latter process involves release of cytochrome c from the mitochondrial intermembrane space and activation of caspases that inhibit cellular respiration [15, 16].

The present study evaluates the effects of gossypol on endometrioma cells in culture. The results show toxic effects of gossypol on endometrioma cells.

2   Materials and methods

2.1 Endometrioma cells

Six endometrioma cell lines (BP1295, KF681, VG5800, SH391, SH3134, and MCL10275) were developed from 6 patients. The tissues were collected during operative laparoscopy or laparotomy for endometriosis with associated endometrioma. Each patient signed a consent document for the study, which was approved by the Institutional Review Board for the protection of human subjects.

A 1_2 cm piece of each tissue was excised and immediately sent to the research laboratory. The remaining specimen was sent to surgical pathology for histologic studies. The latter tests confirmed the diagnosis of endometrioma in the 6 patients studied [17].

2.2 Endometrioma cell culture

Epithelial and stroma cells were isolated from the tissue using 0.1 mg collagenase (LS00414; Worthington Biomedical Corp., NJ, USA) per mL DPBS (Mediatech, Inc., Westwood, MA, USA). Tissues were digested in a 37ºC water-bath with periodic agitation for 15 min. The cells were then washed with media (M199 with Earle's salts and L-glutamine; Mediatech Inc., Westwood, MA, USA) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. Cells were centrifuged for 5 min at 800 × g. The cell pellet was re-suspended in 10 mL of the above media. The cells were counted and plated at 1_2 × 10⁶ cells per mL on T-25 cm flasks (3038; Falcon Products, Morristown, TN, USA) that were coated with 10 μg/mL fibronectin. The cultures were incubated at 37ºC in an incubator containing 5% CO₂. The media were changed every three days. The cells were grown in culture until confluence, and then passed to another culture. The cells were frozen in the above media with 8% dimethylsulfoxide (Sigma-Aldrich Inc., St. Louis, MO, USA) at 2_3 × 10⁶ cells/mL. Aliquots (1.5 mL each) of the cell suspensions were placed in 2-mL cryo-vials and stored in liquid nitrogen [17].

2.3 Cell proliferation

Thawed frozen stroma cell lines were plated at 3 × 10⁴ viable cells per well on fibronectin-coated (FLPP, Sigma-Aldrich Inc., St. Louis, MO, USA) 6-well plates (3046; Falcon Products, Morristown, TN, USA). On day 3, the media (described above) were exchanged with 25 or 50 nmol/L gossypol along with a control (media without gossypol) that was run simultaneously. The cells were harvested and counted on days 3, 6, 9 and 12 using a hemocytometer. Viability was done using trypan blue exclusion stain.

Gossypol solution was freshly prepared in cold 95% ethanol and kept on ice. The concentration of gossypol was determined by ultraviolet absorbance at 385 nm, using a molar extinction coefficient of 18 000 [18].

2.4 Respiration

Cellular respiration was measured at 25ºC in sealed vials containing ~1_5 × 10⁶ cells per condition [18, 19]. The cells were suspended in 0.5 mL Pd phosphor solution (RPMI medium containing 10 mmol/L glucose, 2 μmol/L Pd phosphor [Porphyrin Products Inc., Logan, UT, USA) and 3% fat-free albumin [final pH, ~7.4]). The solution was freshly made and stirred at 25ºC for ~30 min prior to use. Mixing was with the aid of a ceramic stir bar. Rate of respiration was the negative of the slope of [O₂] vs. t (zero-order rate, in μmol/L O₂ per min per 10⁶ cells). The drift (Pd phosphor solution without cells) was 0.28 ± 0.05 μmol/L O₂ per min.

2.5 Instrument

[O₂] in solution was determined as function of t using the Pd phosphor. The phosphorescence decay of the probe was characterized by a single exponential; the decay time ( t, disappearance of the phosphorescence signal) was inversely proportional to O₂ concentration [18, 19]. Samples were exposed to ten light flashes per sec from a pulsed light-emitting diode array with peak output at 625 nm (OTL630A-5-10-66-E, Opto Technology Inc., Wheeling, Il, USA). Emitted phosphorescent light was detected by a Hamamatsu photomultiplier tube (#928; Stratford, CT, USA) after passing through a wide-band interference filter centered at 800 nm (Omega Optical Inc., Brattleboro, VT, USA). The amplified phosphorescence decay was digitized at 1 MHz by a 20 MHz A/D converter (Computer Board Inc., Middleboro, MA, USA). Two hundred fifty samples were collected from each decay curve and the data from 10 consecutive decay curves were averaged for calculating. [O₂] was calculated using: t⁰/t = 1 + tk_q [O₂]; where t, is the lifetime in the presence of oxygen; t⁰, the lifetime in the absence of oxygen; and k_q, the second-order O₂ quenching constant for the Pd phosphor in the presence of fat-free albumin (k_q, ~4.1× 10⁸ M^-1 s^-1).

2.6 Cellular gossypol accumulation

Cells (in M199 media plus 10% FBS) were incubated with gossypol for 24 h at 37ºC in an incubator containing 5% CO₂. The cell pellets were then rinsed with drug-free media and collected by centrifugation (1 000 × g for 2 min). The cell pellets were suspended in 200 μL of acetonitrile/HCl/ascorbic acid solution. The mixtures were vigorously mixed and place in boiling water for 2 min. The supernatants were then separated on HPLC and gossypol peak was detected by absorbance at 254 nm [20].

The analysis was performed on Beckman HPLC system (Beckman Coulter Inc.,Fullerton, CA, USA). The solvent was acetonitrile : H₂O : acetic acid (7:2:1) [20]. The column (4.6 × 250 mm Beckman ultrasphere IP) was operated isocratically at 1 mL/min. Standards (10 μmol/L gossypol in the acetonitrile/HCl/ascorbic acid solution) were included with each analytical run. The standard curves were linear (r > 0.99) over 5_30 nmoles.

3  Results

3.1 Gossypol inhibited the growth of endometrioma cells

The proliferations of 3 endometrioma cell lines (BP1295, KF681 and VG5800) were studied. Gossypol inhibited the growth of all 3 tumor cells. However, the sensitivity of the cells to the drug varied. The 1st tumor showed mild growth inhibition at 25 nmol/L and complete inhibition at 50 nmol/L (Figure 1A). The 2nd and 3rd tumors were similar, showing milder inhibition at either 25 or 50 nmol/L (Figure 1B and C).

3.2 Gossypol impaired endometrioma cell respiration

The respirations of 6 endometrioma cell lines (BP1295, KF681, VG5800, SH391, SH134 and MCL10275) were studied. In 3 tumors (the same cell lines in Figure 1), the cells were treated with 50 or 300 nmol/L gossypol for 24 h. Respiration was then measured at the end of the treatment period. Representative runs for the cell line VG5800 are shown in Figure 2. Respiration was unaffected by this gossypol treatment (Table 1).

We next investigated the respiration of another 3 cell lines (SH391, SH134 and MCL10275) during continuous exposure to 10 μmol/L gossypol. Respiration was decreased by ~40_75% in the presence of 10 μmol/L gossypol (Table 2).

3.3 Cellular gossypol accumulation

Cellular gossypol was detected in the 2 endometrioma cell lines, KF681 and VG5800 that were treated for 24 h with 10 and 0.3 μmol/L gossypol, respectively (Table 3).

4  Discussion

The contraceptive effect of gossypol was discovered by the observation that the use of uncooked cotton seed oil was responsible for the high rate of infertility in various villages in China [21]. These reports appeared in the Chinese literature as early as 1933. Since then, studies have showed marked suppression of sperm production by gossypol.

Gossypol was also used for treatment of endometriosis, uterine fibroids, adrenal tumors and gliomas [5, 22]. The drug was successful (in ~90% of the cases) in reducing the sizes of endometrial cysts and fibroids [12]; but ~80% of the treated patients suffered from amenorrhea. There was no drug effect on gonadotropin or ovarian steroid hormones, suggesting the amenorrhea was related to a direct effect of gossypol on the endometrium [23]. Some investigators demonstrated reduction in estrogen and progesterone receptors in the endometrium of women treated with gossypol before they underwent hysterectomy [24].

The present study demonstrates an inhibitory effect of gossypol on the proliferation of three endometrioma cell lines in culture. The effect of gossypol on these endometrioma cells is thus similar to that on sperm [25].

The results also suggest that gossypol may have potent cytotoxic activity against endometriomas. The drug inhibits proliferation of endometriosis and interferes with cellular metabolism and mitochondrial functions. Further studies are necessary to address whether gossypol is applicable to the treatment of endometriosis and fibroids.

Acknowledgement

The authors thank Dr She-Pu Xue of the Chinese Academy of Medical Sciences for providing Gossypol as a gift for conducting this study. This work was supported by Paige's Butterfly Run.

References

1 Withers WA, Carruth FE. Gossypol_a toxic substance in cottonseed meal. A preliminary note. Science 1915; 41: 324.

2 Poso H, Wichmann K, Janne J, Luukkainen T. Gossypol, a powerful inhibitor of human spermatozoal metabolism. Lancet 1980;1: 885_6.

3 Tuszynski GP, Cossu G. Differential cytotoxic effect of gossypol on human melanoma, colon cancer, and other tissue culture cell lines. Cancer Res 1984; 44: 768_71.

4 Wu D. An overview of the clinical pharmacology and therapeutic potential of gossypol as a male contraceptive agent and in gynecological disease. Drugs 1989; 38: 333_41.

5 Bushunow P, Reidenberg MM, Wasenko J, Winfield J, Lorenzo B, Lemke S, et al. Gossypol treatment of recurrent adult malignant gliomas. J Neurooncol. 1999; 43: 79_86.

6 Benz CC, Keniry MA, Ford JM, Townsend AJ, Cox FW, Palayoor S, et al. Biochemical correlates of the antitumor and antimitochondria properties of gossypol enantiomers. Mol Pharmacol 1990; 37: 840_7.

7 Qian SZ, Wang ZG. Gossypol: a potential antifertility agent for males. Ann Rev Pharmacol Toxicol 1984; 24: 329_60.

8 Wang YE, Luo YG, Tang XC. Studies on the antifertility actions of cottonseed meal and gossypol. Document of First National Conference on Male Antifertility Agents, Wuhan (September 1972). Acta Pharm Sin 1979; 14: 662_9.

9 Xue SP, Zang SD, Su SY, Wu YW, Lin Y, Zhou ZH, et al. Antifertility effect of gossypol on the germinal epithelium of the rat testis. A cytological, autoradiographical and ultrastructural observation. Document of Second National Conference on Male Antifertility Agents, Qingdao (1973). Sci Siu 1979; 9:915_28.

10 Xue SP. Studies on the antifertility effect of gossypol, a new contraceptive for males. Symposium on Recent Advances in Fertility Regulation. 2_5 September, Beijing, China. 1980;122_46.

11 Dai RX, Pang SN, Liu ZL. Studies on the antifertility effect of gossypol. II. A morphological analysis of the antifertility effect of gossypol. Document of Fourth National Conference on Male Antifertility Agents, Suzhou (1975). Acta Biol Exp Siu 1978; 11: 27_36.

12 Han ML, Wang YF, Tang MY, Ge QS, Zhou LF, Zhu PD, et al. Gossypol in the treatment of endometriosis and uterine myoma. Contrib Gynecol Obstet 1987; 268_70.

13 Abou-Donia MB, Dieckert JW. Gossypol: uncoupling of respiratory chain and oxidative phosphorylation. Life Science 1974; 14: 1955_63.

14 Cuellar A, Ramirez J. Further studies on the mechanism of action of gossypol on mitochondrial membrane. Internat J Biochem 1974; 25: 1149_55.

15 Petit PX, Zamzami N, Vayssiere JL, Mignotte B, Kroemer G, Castedo M. Implication of mitochondria in apoptosis. Mol Cell Biochem 1997; 174: 185_8.

16 Li P, Nijhawan D, Budihardjo I, Srinivasula SM, Ahmad M, Alnemri ES, et al. Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell 1997; 91: 479_89.

17 Badawy SZ, Holland J, Landis S, Frankel L, Cuenca V, Khan S. Role of estradiol, progesterone and transforming factor on human endometrioma cell culture. J Reproductive Immunol 1996; 36: 58_63.

18 Souid AK, Tacka KA Galvan KA, Penefsky HS. Immediate effects of anticancer drugs on mitochondrial oxygen consumption. Biochem Pharmacol 2003; 66: 977_98.

19 Lo LW, Koch CJ, Wilson DF. Calibration of oxygen- dependent quenching of the phosphorescence of Pd-meso-tetra (4-carboxyphenyl) porphine: a phosphor with general application for measuring oxygen concentration in biological systems. Anal Biochem 1996; 236: 153_60.

20 Marcelle GB, Ahmed MS, Pezzuto JM, Cordell GA, Waller DP, Soejarto DD, et al. Analysis of gossypol and gossypol-acetic acid by high performance liquid chromatography. J Pharm Sci 1984; 3: 396_8.

21 Hubei Pronvical Hancuan "Disease of Burning Sensation", Treatment and Prevention Group. Collected material about disease of burning sensation. Document of Conference on Disease of Burning Sensation, Hubei 1970.

22 Flack MR, Pyle RG, Mullen NM, Lorenzo B, Wu YW, Knazek RA, et al. Oral gossypol in the treatment of metastatic adrenal cancer. J Clin Endocrinol Metab 1993; 76: 1019_24.

23. Cheng KF, Wu WY, Tang MY, Chu PT. Endometrial changes after administration of gossypol for menorrhagia. Am J Obstet Gynecol 1980; 138: 1227_9.

24. Liu YX, Jia XC, Zhou SW, Zhou M. Effect of gossypol on the steroid receptors of the endometrium. Chinese J Obstet Gynecol 1981; 16: 129_31.

25. Xue SP, Liang DC, Fei RR, Chen XM, Ye SJ, Liu Y, et al. Subcellular site of antisprematogenic effect of Gossypol and its possible molecular mechanism of action. Sci Sin (B) 1988;26: 614_33.