Prunella stica inhibits the proliferation but not the prostaglandin ...

Life Sciences 79 (2006) 436 – 441 www.elsevier.com/locate/lifescie

Prunella stica inhibits the proliferation but not the prostaglandin production of Ishikawa cells Jaou-Chen Huang a,⁎, Cheng-Huai Ruan a , Kathy Tang c , Ke-He Ruan b,⁎ a

Department of Obstetrics and Gynecology, University of Texas Health Science Center, 6431 Fannin Street, Houston, TX 77030, USA b Department of Internal Medicine, University of Texas Health Science Center, 6431 Fannin Street, Houston, TX 77030, USA c TR Acupuncture and Herb Clinic, Houston, TX 77005, USA Received 27 September 2005; accepted 16 January 2006

Abstract Chinese herbs have been used to relieve dysmenorrhea associated with endometriosis. Active components in the herbs and their mechanisms of action remain unknown. Prunella stica, a Chinese herb commonly used to treat dysmenorrhea, was chosen for the present studies. Its effects were investigated on Ishikawa cells, an epithelial cell line derived from human endometrium. Cell proliferation and inhibition of interleukin 1β (IL-1β) induced prostaglandin (PG) production were examined. To learn more about the active components, 120 fractions were collected from the crude extract and each fraction was tested individually. To further characterize the active components, aliquots of fractions with activity were subject to mass spectrometry analysis. Crude extract of P. stica inhibited the proliferation of Ishikawa cells but not the IL-1β induced PG production. Active components of P. stica clustered around fractions 64 and 92; they increased cell doubling time from 18.6 to 26.2 and 29.4h, respectively. Mass spectrometry analysis showed fractions 64 and 92 consisted of three components whose molecular weights were 337, 348 and 430 Daltons. The therapeutic effects of P. stica reside, in part, in inhibiting the proliferation of the epithelial cells derived from human endometrium. The active components are small molecules. © 2006 Elsevier Inc. All rights reserved. Keywords: Endometriosis; Endometrial cancer; Herbs; Cyclooxygenase; Traditional medicine

Introduction Endometriosis is the presence of endometrial tissue outside the uterus. It is a benign yet progressive disease. It affects 15% of reproductive age women and as many as 40% of infertile women. One of the symptoms of endometriosis is cyclic pelvic pain (Speroff et al., 1999). Progestins, gonadotropin-releasing hormone (GnRH) agonist, oral contraceptives and danazol have been used for pain relief (Mahutte and Arici, 2003). These hormonal therapies are effective, but side effects such as mood swings, weight gain, hot flashes and bone loss limit the acceptance by patients and/or the duration of therapy (Mahutte and Arici, 2003). ⁎ Corresponding authors. Huang is to be contacted at 6431 Fannin Street, MSB 3.604, Houston, TX 77030, USA Tel.: +1 713 500 6382; fax: +1 713 500 0586. Ruan, 6431 Fannin Street, MSB 5.278, Houston, TX 77030, USA. Tel.: +1 713 500 6769; fax: +1 713 500 6810. E-mail addresses: [email protected] (J.-C. Huang), [email protected] (K.-H. Ruan). 0024-3205/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2006.01.018

Implants of endometriosis elicit an inflammatory response in the pelvis. As a result, more macrophages and T-lymphocytes are found in peritoneal fluid from women with endometriosis. So are elevated cytokines, chemokines and growth factors. All these soluble factors induce the expression of cyclooxygenase-2 (COX-2), a rate-limiting enzyme in PG biosynthesis, and increase PG production. Symptoms of endometriosis such as pain reflect the sequel of such inflammatory reactions (Lebovic et al., 2001). Dysmenorrhea associated with endometriosis is reportedly caused by increased PG production from the implants of endometriosis (Kauppila et al., 1979). The severity of dysmenorrhea was reported to be directly related to the amount of PGs produced by the implants (Koike et al., 1992). Therefore, nonsteroidal anti-inflammatory drugs such as Motrin®, which blocks COX, have been used to relieve dysmenorrhea associated endometriosis (Hurst and Rock, 1989). Endometriosis is thought to be a genetic disorder of polygenetic/multifactorial inheritance (Simpson et al., 2003). Recent studies suggest that intrinsic molecular aberrations in

J.-C. Huang et al. / Life Sciences 79 (2006) 436–441

Materials and methods

counting. The number of cells in 500μl aliquots was determined three times; the total cell number in the original suspension was calculated based on the mean of three determinations. The variations among the determinations were less than 5%. The experiments were carried out as follows. The Ishikawa cells were seeded in 6-well plates in 1 ml media containing 50 μl of PBS, the crude extract or one of the fractions from the crude extract. Seventy-two hours later, the cell number in the control and experimental wells was determined as described above. Calculation of doubling time To express the effects of P. stica on cell proliferation in a universally applicable form, the doubling time (Rew and Wilson, 2000) was calculated based on the initial and the final cell counts and the time elapsed using the following formula: Doubling time (hour) = (Δt × log 2) / log(Nt / N0), where Δt was

a Cell number (X 1000)

implants of endometriosis contribute significantly to the progress of the disease (Bruner et al., 1997; Khorram et al., 1993; Noble et al., 1996; Osteen et al., 1996; Sharpe-Timms et al., 1995; Zeitoun et al., 1999, 1998). One such molecular aberration is increased local production of PGE2, which enhanced aromatase activity and increased local estrogen production. The increased estrogen production creates a feed forward loop to promote the growth of endometriosis (Bulun et al., 2002). Herbs have been used to alleviate dysmenorrhea in different parts of the world including China (Bensky et al., 1986), Japan (Kotani et al., 1997; Tanaka, 2003) and South Africa (Lindsey et al., 1999). Unlike hormonal therapy, herbs are generally better tolerated by patients, because they have fewer side effects. However, herbs are traditionally used as crude extract and, often times, in combination. Therefore, the active components and the mechanisms of action are difficult to ascertain. We hypothesized that herbs relieve endometriosis associated dysmenorrhea by inhibiting the growth of endometriosis and/or decreasing PG production. In this report, we tested Prunella stica, one of the Chinese herbs commonly used to relieve dysmenorrhea. To minimize variations associated with cells obtained from different patients, we used the Ishikawa cells, a well-defined human endometrial epithelial cell line (Gravanis and Gurpide, 1986). We found that P. stica inhibited cell growth but not the PG production. Furthermore, we found that the active components in P. stica consisted of three species of small molecules.

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Unless otherwise specified, all chemicals were purchased from Sigma Co. (St. Louis, MO, USA).

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b Preparation and fractionation of crude extract Five grams of lyophilized extract of P. stica (TR Acupuncture and Herb Clinic, Houston, TX, USA) were reconstituted in 1.5 ml water and centrifuged at 13,000 ×g for 3 min. The supernatant was filtered, sterilized and used directly as described later or fractionated as follows. Pooled crude extract (5ml) was passed through a gel filtration column (Superdex-30, 16 × 600 mm, Amersham Biosciences, Piscataway, NJ, USA) and eluted with water under a pressure of 100lb/in.2. One hundred twenty fractions were collected, lyophilized and stored at − 20 °C until use. Cell proliferation studies The Ishikawa cells were cultured in 6-well plates, each well containing 1ml DMEM/F-12 medium supplemented with 10% fetal calf serum, penicillin (10Units/ml), streptomycin (10μg/ ml), and HEPES (20mM). The proliferation of cells was studied based on the changes of cell number, determined by a cell counter (model Z1, Beckman Coulter Inc. Fullerton, CA, USA). Attached cells were trypsinized with phosphate buffered saline (PBS) containing trypsin (0.5%) and EDTA (10mM) and resuspended in 1ml culture media. The cell suspension was mixed with 9.0ml buffer (Isoton®, Beckman Coulter Inc.) immediately before

Fig. 1. a)The crude extract of Prunella stica inhibited the proliferation of Ishikawa cells. The Ishikawa cells (100,000 cells per well) were cultured in media containing crude extract (with a final concentration of 0.1mg/ml) of Prunella stica or phosphate buffered saline (control). Seventy-two hours later, the cell number was determined by a cell counter. Results from four independent observations (mean ± SD) are depicted. b). Dose response curve of Prunella stica on the effects of Ishikawa cell proliferation. The Ishikawa cells (100,000 cells per well) were cultured in media containing different concentrations of Prunella stica extract (solid squares) or bovine serum albumin (open squares, control). Seventy-two hours later, the cell numbers were determined by a cell counter. Results from three independent observations are averaged and plotted.

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the time elapsed in hours and Nt and N0 were the cell numbers at time t and time 0, respectively.

PGE2 production after incubating with NS398, a specific COX-2 inhibitor, during the last 30 min of IL-1β incubation. The effects of P. stica on the COX-2 activity were evaluated in a similar way. After the incubation with arachidonic acid, the media was collected and stored at −20°C. The concentration of PGE2 was determined in duplicates using an enzyme immunoassay (Cayman Chemical Co., Ann Arbor, MI, USA). The antibody was specific for PGE2; its cross-reactivity to other PGs was negligible. The inter- and intra-assay errors were 15% and 8%, respectively.

Mass spectrometry analysis The mass spectrometry analysis was performed in the Core Protein Chemistry Facility at the Tufts University (Boston, MA, USA) using a MALDI system (Perceptive Biosystems, Albertville, MN, USA). Aliquots (0.5μl) of active fractions were spotted on the plate and mixed with equal volume of the matrix (2, 5-dihydroxybenzoic Acid). The sample was air-dried and read.

Statistical analysis Effects of P. stica on COX-2 activity The Ishikawa cells were cultured as described above. When 70% confluence was reached, the cells were maintained in media with reduced serum (0.5%) for 18h to induce a state of quiescence. The induction of COX-2 expression was achieved by incubating cells for six hours in a fresh, reduced-serum media supplemented with IL-1β (10 ng/ml). Some wells were randomly selected to receive PBS; they served as negative controls. Preliminary studies indicated that PGE2 was one of the major PGs produced by the Ishikawa cells and that P. stica did not affect PGE2 production in quiescent Ishikawa cells. Therefore, the COX activities were estimated as follows. Total COX activity in cells which had been previously incubated with IL1β for 6h was estimated based on the PGE2 production after incubating with 20μM arachidonic acid for 30min. The COX-2 activity in these cells was estimated based on a reduction in

Cell Number

a

The differences in PGE2 concentrations were evaluated by one-way analysis of variance followed by Bonferroni's posttests when appropriate. A p b 0.05 was considered statistically significant. GraphPad InStat version 3.00 (GraphPad Software, San Diego CA, USA) was used. Results P. stica inhibited cell proliferation The crude extract of P. stica inhibited the proliferation of Ishikawa cells by ∼93% (Fig. 1a). Over the 72-h period, control cells increased from ∼100,000 to 1,952,355 ± 235,367 and the experimental cells to 132,975 ± 28,952 cells (mean ± SD, based on four independent observations). A dose response curve for

Control cells 1,711,840

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Fraction Number Fig. 2. Some fractions of the crude extract of Prunella stica inhibited the proliferation of the Ishikawa cells. (a) Ishikawa cells (117,840 per well) were cultured in media containing fractions of crude extract (with a final concentration of 0.05mg/ml) from Prunella stica or phosphate buffered saline (control) for 72h. Control cells increased to 1,711,840 (represented by the line indicated by the arrow). Fractions 64, 92 and their neighboring fractions inhibited cell proliferation. (b) The decreased proliferation translated into an increased doubling time: from 18.6h (control, represented by the line indicated by the arrow) to 26.2h (fraction 64) and 29.4 (fraction 92) h, respectively.


4000

p < 0.0002 One way ANOVA

117,840 to 1,711,840 but the experimental cells, incubated with fractions 64 and 92, only increased to 812,926 and 642,660, respectively. The difference in the final cell numbers reflected an increased doubling time from 18.6h (control) to 26.2 h (fraction 64) and 29.4 h (fraction 92) (Fig. 2b). To see whether the herbal extract induces apotosis in the cells, the P. stica-treated cells were stained with trypan blue. However, no significant difference in viability was observed in the treated cells when compared to the untreated cells.

PG E2 (pg/well)

* p < 0.05 versus IL-1β 3000

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P. stica did not affect IL-1β induced PG production 0

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IL-1β + Prunella stica

Fig. 3. Prunella stica did not inhibit cyclooxygenase-2 (COX-2) activity. Near confluent Ishikawa cells were serum-starved for 18h before incubating with interleukin 1β (IL-1β, 10ng/ml) to induce the expression of COX-2. Control cells received phosphate buffered saline. Total COX activity was indirectly assessed by the conversion of excess arachidonic acid to prostaglandin E2 (PGE2). The inhibition of COX-2 activity was estimated by a reduction in PGE2 production: the positive control cells were incubated with NS 398 (5 μM), a specific COX-2 inhibitor, during the last 30min of COX-2 induction and throughout the arachidonic acid incubation; the experimental cells were incubated with the crude extract (with a final concentration of 0.05mg/ml) of Prunella stica during the same period. The figure depicts PGE2 production (mean ± SD) based on three independent experiments.

To learn more about fractions 64 and 92 of the crude extract, aliquots from the combined fractions were analyzed by mass spectrometry. The results showed that fractions 64 and 92 contained three species, whose molecular weights were between 300 and 500 Daltons (Fig. 4). Thus, active components of P. stica were water soluble, small molecules. Discussion The use of herbs is common in the East and is becoming popular in the West. Herbs continue to be a rich source of raw material that contains therapeutic potentials. Many chemically

1000

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337.3 348.3

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Active components of P. stica are small molecules

231.4

the effects of P. stica on the cell proliferation is shown in Fig. 1b. To isolate components that have the inhibitory activity, 120 fractions (0.8 ml each) were collected from a 5ml pooled crude extract and tested individually. As expected, while most of the fractions did not affect cell proliferation, a few fractions did. The inhibitory activities clustered around fractions 64 and 92 (Fig. 2a). Over the 72-h period, control cells increased from

The six-hour incubation with IL-1β (10 ng/ml) increased PGE2 production by about 100% (Fig. 3). The increased PGE2 production was the result of COX-2 induction, because it was completely abolished by preincubation with NS398, a specific COX-2 inhibitor. Preincubation with the crude extract of P. stica did not reduce the enhanced PGE2 production by IL-1β, suggesting a lack of effect on COX-2.

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Mass (m/z) Fig. 4. Active components of Prunella stica are small molecules. An aliquot of combined fractions 64 and 92 was analyzed by mass spectrometry. The results showed that the active components consisted of three species: 337, 348, and 430 Daltons. The first peak (231 Dalton) is the vehicle.

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synthesized drugs used today are active components of herbs or plants used in the past. Aspirin, digoxin and taxol (LysengWilliamson and Fenton, 2005) are good examples. This report is a first step to understand the components and the mechanisms of action of P. stica, which is used by the Chinese for dysmenorrhea. In addition to Chinese, people from other cultures also used herbs or plants to treat dysmenorrhea. Recent clinical trials showed Japanese herbal medicine effectively relieves primary dysmenorrhea (Kotani et al., 1997) and secondary dysmenorrhea associated with endometriosis or adenomyosis (Tanaka, 2003). In South Africa, certain plants are still being used by the traditional healers to treat dysmenorrhea (Lindsey et al., 1999). Extract of these plants reportedly inhibited PG synthesis by the myometrium and the endometrium (Lindsey et al., 1999). Our results showed that therapeutic effects of P. stica may derive from a reduction of foreign body induced inflammation in the pelvis. Pain associated with endometriosis is the sequel of inflammation caused by endometriosis implants in the pelvis (Lebovic et al., 2001): pain associated with endometriosis is reportedly caused by PGs produced by the implants (Kauppila et al., 1979) and the severity of pain is directly proportional to the amount of PGs produced (Koike et al., 1992). Reducing endometriosis implants minimizes the inflammation and, thus, alleviates dysmenorrhea. In addition to reducing the pelvic inflammatory reactions, P. stica may reduce the molecular aberrations within the implants that propel the growth of endometriosis. Continuous growth of endometriosis is, in part, caused by elevated intrinsic PGE2, which enhances aromatase expression and increases estrogen production within the implants (Bulun et al., 2002). PGE2 is predominantly produced by an inducible microsomal PGE2 synthase (mPGES) in the cell line. IL-1β is one of the common cytokines used to induce mPGES production, which leads to an increased PGE2 level in the cells. P. stica did not affect basal or IL-1β stimulated PGE2 production, it reduces the number of cells capable of producing or responding to PGE2 and, thereby, dampens the momentum of the positive feedback caused by the molecular aberrations. The in vivo effects of P. stica may involve other mechanisms not examined in this study such as the modulation of immune response from the host. The immune system has an important role in the establishment and the progression of endometriosis. A defective immune surveillance may be involved in the former (Hill, 1992); a dysregulated immune response may be involved in the latter (Lebovic et al., 2001). Indeed, another member of the Prunella genus, Prunella vulgaris, reportedly modulates the immune response in a rat model (Shin et al., 2001). The active components of P. stica may be plant alkaloids, because they are heat-stable, water-soluble and have molecular weights less than 500 Daltons. Positive identification and confirmation of their chemical structures require further purification and characterization. This may require high pressure liquid chromatography, gas chromatography, mass spectrometry, and nuclear magnetic resonance. Nevertheless, mass spectrometry analysis is a first step to understand the active components of P. stica.

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