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An in vitro study on antiproliferative and antiestrogenic effects of Boerhaavia diffusa L. extracts
Journal of Ethnopharmacology 126 (2009) 221–225
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Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jethpharm
An in vitro study on antiproliferative and antiestrogenic effects of Boerhaavia diffusa L. extracts Sreekumar Sreeja, Sreeharshan Sreeja ∗ Cancer Endocrinology, Integrated Cancer Research Programme, Rajiv Gandhi Centre for Biotechnology, Thycaud P.O., Thiruvananthapuram, Kerala 695014, India
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Article history: Received 5 June 2008 Received in revised form 3 August 2009 Accepted 26 August 2009 Available online 31 August 2009 Keywords: Antiestrogen Selective estrogen receptor modulators Boerhaavia diffusa L. Breast cancer Phytoestrogens
a b s t r a c t Ethnopharmacological relevance: Boerhaavia diffusa L. (Nyctinaceae) is a plant of tropical region used in Indian traditional medicine for the treatment of human ailments including abdominal tumor, jaundice, dyspepsia, menstrual disorders, etc. This plant also has antilymphoproliferative, antimetastatic and immunomodulatory effects. Aim of the study: This study aimed to assess the antiproliferative and antiestrogenic properties of methanol extract of Boerhaavia diffusa (BME) in MCF-7 breast cancer cell lines. Materials and methods: The effective concentration range of BME on cell viability was analyzed using MTT assay. Hydroxylapatite assay (HAP) was carried out to confirm the competitive binding of BME to the estrogen receptor (ER). The effect of BME on the expression of a selected estrogen responsive gene pS2 was analyzed by RT-PCR. The ability of BME to alter the cell cycle phases and distributions were studied using FACS analysis. Results: Treatment with varying concentrations of BME (20–320 g/mL) resulted in moderate to very strong growth inhibition in MCF-7 cell lines. BME competed with [3 H]-estradiol for binding to ER with IC50 value of 320 ± 25 g/mL. RT-PCR analysis revealed that BME reduced the mRNA expression of pS2 indicating the antiestrogenic action of BME. BME treatment for 48 h resulted in a remarkable increase in the number of MCF-7 cells in the G0-G1 fraction from 69.1% to 75.8%, with a reciprocal decrease of cells in all other phases indicating cell cycle arrest at G0-G1 phase. Conclusions: The results demonstrate that Boerhaavia diffusa possess antiproliferative and antiestrogenic properties and suggest that it may have therapeutic potential in estrogen dependent breast cancers. © 2009 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Estrogens, mainly 17-estradiol exert numerous pharmacological effects in a large number of targets including the breast, uterus, bone, brain and cardiovascular tissues. Most of their actions are mediated by estrogen receptor (ER) which exists in two different forms ER␣ and ER which reside predominantly in the nucleus of target cells (Cheng et al., 2004). ERs have also been detected in the cellular membrane (Sreeja and Thampan, 2003; Zivadinovic et al., 2005). Phytoestrogens are widely known plant compounds, which bind to ER and affect the physiology of various systems. Mounting evidence points out that phytoestrogens exert estrogenic
Abbreviations: DMEM, Dulbecco’s modified eagle’s medium; E2, estradiol; ER, estrogen receptor; FACS, fluorescence activated cell sorting; HAP, hydroxylapatite; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; RT-PCR, reverse transcriptase polymerase chain reaction; SERM, selective estrogen receptor modulator; BME, Boerhaavia diffusa methanol extract. ∗ Corresponding author. Tel.: +91 471 2529474; fax: +91 471 2348096. E-mail address: [email protected] (S. Sreeja). 0378-8741/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2009.08.041
effects on skeleton, affect favorably the cardiovascular function and menopausal symptoms while demonstrate antiestrogenic action on breast and prostrate tissue (Finkel, 1998; Lissin and Cooke, 2000). This has led to the hypothesis that phytoestrogens may act as selective estrogen receptor modulators (SERMs) and can be considered therapeutically for the prevention of breast and prostate cancer, osteoporosis and cardiovascular diseases (Middleton et al., 2000; Ewies, 2002). Boerhaavia diffusa L. commonly known as ‘Punarnava’ is an abundant creeping weed found all over India (Wahi et al., 1997). The plant has drawn lot of attention due to its uses in Indian traditional medicine. The various parts of the plant are used in the treatment of cancer, jaundice, dyspepsia, inflammation, enlargement of spleen, abdominal pain and as an anti-stress agent (Kirtikar and Basu, 1956; Chakraborti and Handa, 1989; Leslie Taylor, 2005). Root extracts of Boerhaavia diffusa inhibited the growth of several monocytic, lymphoblastoid, fibroblast and erythroleukemic cell lines of mouse and human origin (Mehrotra et al., 2002). Boerhaavia diffusa extracts prevented DMBA-induced skin carcinogenesis in mice (Bharali et al., 2003). Ethanolic extract of the plant is well demonstrated to have anti-mitotic activity in in vitro systems (Obong and Madi,
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1987). Administration of aqueous methanol extract of Boerhaavia diffusa was found to be effective in reducing the metastases formation by B167-10 melanoma cells and Punarnavine, an alkaloid from Boerhaavia diffusa enhanced the immune response against metastatic progression of B16F-10 melanoma cells in mice (Leyon et al., 2005; Manu and Kuttan, 2007). Considering the pronounced antiproliferative and antimetastatic activity of Boerhaavia diffusa, in this study we evaluated the effect of methanol extract of Boerhaavia diffusa (BME) in MCF-7 breast cancer cell lines and validated its antiestrogenic potential to demonstrate its suitability in hormone dependent breast cancer chemoprevention.
protein (ER preparations) was incubated overnight at 4 ◦ C with the varying concentration of BME (range 20–320 g/mL), 20 nM [3 H]-estradiol ± 100-fold molar excess of estradiol (E2) in a final volume of 250 L. 250 L of a 60% HAP suspension in TEM buffer was added and the mixture was incubated at 4 ◦ C for 15 min. The HAP bound receptor–[3 H]-E2 complex was separated by centrifugation at 200 × g for 15 min. After washing twice with Tris buffer (10 mM) the HAP pellet was extracted with 1 mL absolute ethanol. These extracts were added to 4 mL of scintillation cocktail and the radioactivity was measured in a Wallac 1409 liquid scintillation counter. Data was expressed as the ratio of bound [3 H]-E2 in the presence of a competitor to the bound [3 H]-E2 in control × 100.
2. Materials and methods
2.5. Semi-quantitative RT-PCR
2.1. Preparation of extract from Boerhaavia diffusa
MCF-7 cells were seeded onto 100 mm plates and were incubated with phenol red free DMEM supplemented with 10% charcoal treated fetal bovine serum for 48 h before using in the assay. After 48 h, test compounds (BME, E2, ICI 182-780) were added to this medium. After incubating for 24 h total RNA was purified from the cells using Tri-reagent (Sigma chemical Co., St. Louis, MO, USA) according to the manufacturer’s protocol. cDNA was prepared from 5 g of total RNA using AccuScriptTM 1st strand cDNA synthesis kit (Stratagene, USA) as per manufacturer’s instruction. The pS2 cDNA fragments were amplified using the primer pairs (Fotovati et al., 2006), forward—5 -TTTGGAGCAGAGAGGAGGCAATGG-3 , reverse—5 -TGGTATTAGGATAGAAGCACCAGGG-3 (product size 240 bp). The reaction mixture containing 10 mM Tris–HCl (pH 8.5), 50 mM KCl, 2 mM MgCl, 200 M dNTPs, primers (2.5 pM each) and 2.5 units of Taq polymerase were subjected to amplification cycles of 94 ◦ C for 4 min, followed by 30 cycles of 94 ◦ C for 1 min, 59 ◦ C for 30 s, 72 ◦ C for 1 min in a (Eppendorf) thermal cycler. Aliquots (5 L) of each PCR mixture were analyzed by electrophoresis in a 1.2% agarose gel and fragments were visualized by ethidium bromide staining. The intensity of bands was quantified in a Flour-S multi imager (BioRad, Hercules, USA) by using Quantity one densitometry software. The transcripts were normalized with gapdh expression level. The gene expression was shown as ratio of densitometric value of target mRNA to that of gapdh.
Whole plant of Boerhaavia diffusa (Nyctinaceae) were collected from South Kerala during August 2007 and authenticated by Prof. Sheela, Department of Botany, Govt. College Madapally, Calicut University, Kerala. A voucher specimen (accession no. MMCB 004) of the plant was deposited in the herbarium of Cancer Endocrinology, Rajiv Gandhi Centre for Biotechnology, Trivandrum, India. Air-dried powdered plants (10 g) were extracted with methanol in a Soxhlet apparatus for 20 h and then the extract was concentrated using rotary vacuum to get the solid mass. The yield of dried extract obtained from starting crude material was 5% (w/v). The concentrate was dissolved in dimethyl sulphoxide (DMSO, Sigma chemical Co., St. Louis, MO, USA), referred as BME and was used for further experiments. The final concentration of DMSO used was less than 0.1% (v/v) for each treatment. 2.2. Cell and cell culture MCF-7 cell lines obtained from ATCC (Manassas, VA, USA) were cultured in phenol red free Dulbecco’s modified Eagle’s medium, DMEM (Sigma chemical Co., St. Louis, MO, USA) supplemented with 10% heat inactivated fetal bovine serum (Sigma chemical Co., St. Louis, MO, USA), 100 U/mL benzyl penicillin and 100 g/mL streptomycin. The culture was maintained at 37 ◦ C in a humidified atmosphere of 5% CO2 . 2.3. Cell proliferation assay To evaluate the effect of BME on cell proliferation, MTT assay was performed (Haridas et al., 1998). Cells were seeded in 96-well plates at a density of 5 × 103 cells per well and treated with 0, 20, 40, 80, 160 and 320 g/mL of BME. After incubating for 48 h, the drug containing medium was aspirated and then 100 L of MTT reagent (2 mg/mL) was added and incubated for 2 h at 37 ◦ C. The viable cell number is directly proportional to the production of formazan following solubilization with MTT lysis buffer (20% sodium dodecyl sulphate in 50% dimethyl formamide), which can be measured spectrophotometrically at 570 nm. Cell survival was expressed as percentage over the untreated control. Cell survival (CS) was calculated as CS = (Optical density of drug exposed cells/Mean optical density of control cells) × 100. 2.4. Competitive ER binding assay (HAP assay) Hydroxylapatite (HAP) assay was carried out to confirm the competitive binding of BME to ER (Clark et al., 1979). MCF-7 cytosolic extract was used for competition-binding studies. Cytosol was prepared from cells grown for 4 days in estrogen depleted medium. The protein content was measured spectrophotometrically at 570 nm using Bradford reagent. About 40 g of the total
2.6. FACS analysis MCF-7 cells were treated with varying concentrations of BME (20–320 g/mL) for 48 h. The supernatant was then collected and the cells were trypsinised, collected and fixed in 70% cold ethanol (4 ◦ C) overnight. After washing twice with PBS, cells were resuspended in PBS. The cells were stained with propidium iodide (20 g/mL) after treatment with RNase-A (10 g/mL) for 30 min. The DNA content of the cells was then analyzed with a FACS caliber instrument (Becton Dickinson, San Jose, CA, USA). The percentage of cells in different cell cycle phases was calculated using DIVA software. 2.7. Phytochemical screening Qualitative screening of BME was made to investigate the major chemical classes of components present. It was screened for the presence of alkaloids, flavonoids, polyuronides, phenols, reducing compounds, saponins and tannins (Canel, 1998; Karumi et al., 2004). 2.8. Estimation of total flavonoids in BME Aluminum chloride colorimetric method was used for flavonoids determination (Chang et al., 2002). BME (0.5 mL of 0.5 mg/mL) in 80% methanol were separately mixed with 0.1 mL
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3. Results 3.1. Effect of BME on MCF-7 cell proliferation
Fig. 1. Effect of BME on MCF-7 cell viability/proliferation. The cell line was treated with BME at different concentrations of 20, 40, 80, 160, 320 g/mL for 48 h and the cell viability was determined by MTT assay. The cell survival was expressed as % over the untreated control. A dose dependent growth inhibition of MCF-7 cells was observed after treatment with BME. Results are mean values ± S.E. of five replicates. * P ≤ 0.05 when compared to untreated control.
of 10% aluminum chloride, 0.1 mL of 1 M potassium acetate and 4.3 mL of 80% methanol. It was kept at room temperature for 40 min; the absorbance of the reaction mixture was measured at 415 nm with a double beam PerkinElmer UV/Visible spectrophotometer (USA). The calibration curve was prepared by preparing quercetin solutions at concentrations 25–200 g/mL in methanol.
MCF-7 cells were treated with or without BME to study its antiproliferative effects. The effect of BME on growth inhibition was assessed as percentage cell viability where DMSO treated cells was taken as 100% viable. MCF-7 cells were treated with increasing concentrations of BME (20–320 g/mL) for 48 h. As shown in Fig. 1 there was a decrease in viability of cells treated with BME in comparison to the DMSO treated control. The cell viability decreased to 46.8% in 320 g/mL, 48 h BME treated cells. It also inhibited the estradiol induced proliferation in MCF-7 cell lines suggesting its potential antiestrogenic properties. 3.2. ER binding ability of BME In an attempt to ascertain whether BME interacts with ER, competitive binding studies were carried out. MCF-7 cytosolic lysate was used as a source of ER. As shown in Fig. 2 BME (20–320 g/mL) inhibited the binding of [3 H]-E2 to cytosolic ER in a dose dependent manner. The IC50 value of binding of BME, i.e. the concentration of the extract required to reduce the specific radioligand binding by 50% is 320 ± 25 g/mL. 3.3. BME downregulates the ER mediated transcription
2.9. Estimation of total phenols in BME Total phenols were determined by Folin Ciocalteu reagent (Mc Donald et al., 2001). A dilute extract of each plant extract (0.5 mL of 0.5 mg/mL) or gallic acid (standard phenolic compound) was mixed with Folin Ciocalteu reagent (5 mL, 1:10 diluted with distilled water) and aqueous Na2 CO3 (4 mL, 1 M). The mixtures were allowed to stand for 15 min and the total phenols were determined by colorimetry at 765 nm. The standard curve was prepared using 25–200 mg/L solutions of gallic acid in methanol:water (50:50, v/v). Total phenol values are expressed in terms of gallic acid equivalent (mg/g of dry mass), which is a common reference compound.
The effect of BME on pS2 gene expression was studied as a model for endogenous estrogen responsive gene expressed by MCF-7 cells. E2 and ICI 182-780 were used as agonistic control and antagonistic control respectively. MCF-7 cells were incubated with BME (100 g/mL) or estradiol (10 nM) or ICI 182-780 (10 nM) for 24 h and RT-PCR was performed to amplify the pS2 mRNA using gapdh as endogenous control. As shown in Fig. 3A, BME decreased the expression of pS2 gene to levels approximately as great as those produced by ICI 182-780. E2 up-regulated the expression of the
2.10. Statistical analysis The experiments were performed in triplicates. All values were expressed as mean ± S.E. and Tukey’s post hoc test was done to analyze significance of difference between different groups using the statistical analysis software package SPSS (Version 16.0). Values with P ≤ 0.05 were considered as significant.
Fig. 2. ER affinity assay of BME. Binding of 20 nM [3 H]-E2 to cytosolic ER in the presence of varying concentrations (20–320 g/mL) of BME. Specific bound radioligand was calculated by subtracting non-specific bound counts from total bound counts. Data presented as mean ± S.E. from three separate experiments for each data point.
Fig. 3. Effects of BME on pS2 gene expression in MCF-7 cells. MCF-7 cells were incubated with the BME (100 g/mL) or estradiol (10 nM) or ICI 182-780 (10 nM) for 24 h (A). The graph (B) shows the ratio of density of pS2 expression to that of endogenous control gapdh and it represents mean ± S.E. of three replicates. * P ≤ 0.05 when compared to untreated control.
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gene. The graph (Fig. 3B) shows the ratio of density of pS2 to that of endogenous control gapdh and it represents mean ± S.E. of three replicates. * P ≤ 0.05 when compared to untreated control. 3.4. Cell cycle analysis by flow cytometry The effect of BME on cell cycle progression of MCF-7 cells was determined by flow cytometry. MCF-7 cells treated with varying concentrations of BME (20–320 g/mL) showed G0-G1 arrest by increasing the population of G0-G1 phase from 69.1% to 75.8% as compared with that of the untreated control (Fig. 4A–F). 3.5. Phytochemical screening Phytochemical investigation has revealed the presence of alkaloids, flavonoids, phenolics and saponins in BME. The flavonoid contents of the extracts in terms of quercetin equivalent (the standard curve equation: y = 0.0096x + 0.0114, r2 = 0.999) was 18.5 ± 1.18 mg/g of dry extract powder. The total phenols measured by Folin Ciocalteu reagent in terms of gallic acid equivalent (standard curve equation: y = 0.0648x + 0.005, r2 = 0.9921) was 24.28 ± 3 mg/g in the extract powder. 4. Discussion and conclusions
Fig. 4. Inhibition of cell cycle progression in breast cancer cell lines as analyzed by flow cytometry. Flow cytometric analysis (propidium iodide staining) of the cell cycle distribution of MCF-7 cells without the extracts (A) and after treatment with 20 g/mL (B), 40 g/mL (C), 80 g/mL (D), 160 g/mL (E) and 320 g/mL (F) of BME for 48 h.
Estrogen level is a critical factor in the human physiology in that it influence the development, sexual differentiation, fertility, control of the female reproductive tract, organ responsiveness and female diseases due to hormone imbalance. In the classical ER mediated pathway, the ligand binds to the ligand-binding domain of the ER followed by the formation of a liganded receptor homodimeric complex. The complex binds to the distal ERE’s in the 5 -promoter regions of the E2-responsive genes, further interacts with the components of the general transcription factor complex and recruit coactivators or adaptors and finally induces transcription (Korach et al., 1997). Antiestrogenic compounds act on one hand by directly competing with E2 for binding to ER, resulting in a functionally inactive ligand bound complex. On the other hand, they can force the metabolism of E2 or restrain the biosynthesis of E2, leading to the depletion of endogenous E2 binding to the ER in an indirect manner (Porter and Safe, 1999). Inhibition of estrogen is a common therapeutic strategy for estrogen dependent breast tumors in postmenopausal females and tumor prevention in premenopausal women (Papoutsi et al., 2007). In this study the antiestrogenic effect of methanol extract of Boerhaavia diffusa (BME) through direct ER binding was analyzed in ER positive human mammary carcinoma cell line MCF-7. Estrogens are known stimulants of breast cancer growth whereas antiestrogens arrest the growth of cells (Pratt and Pollak, 1993). To assess the antiproliferative effects of the extracts we applied MTT assay which depends on the reduction of tetrazolium salt by the mitochondria of living cells to form a blue formazan product (Denizot and Lang, 1986). The results depend on both number of cells present and on the mitochondrial activity per cell and showed a dose dependent decrease in viability of MCF-7 cells treated with BME in comparison to the control. BME inhibited E2 induced proliferation in MCF-7 cells suggesting its possible antiestrogenic role. In support to this, competitive radioactive binding studies revealed that BME binds to ER in a dose dependent manner with an IC50 value of 320 ± 25 g/mL. These findings emphasize further the involvement of ER regulated genes in controlling cell proliferation. Estrogen induces the expression of various genes such as cfos, c-erb-b2, ER, PR, pS2, etc. pS2 gene transcription is a primary response to E2 in human breast cancer cell line MCF-7 (Brown et al.,
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1984). pS2 is an estrogen regulated gene whose expression can be increased by estrogen and inhibited by antiestrogen (Masiakowski et al., 1982). Since BME down-regulated pS2 gene transcription (Fig. 3A and B), it was confirmed that BME has some potential compounds that could down-regulate the ER mediated transcription and cause antiestrogenic effects in breast tissue. E2 via ER influences the genes controlling cell proliferation, cell cycle progression or apoptosis resulting in uncontrolled proliferation leading to cancers. Many antiestrogenic anticancer agents from plants inhibit cancer cell growth through cell cycle regulation including G0/G1 accumulation (Cariou et al., 2000). As shown in Fig 4A–F, MCF-7 cells treated with BME caused G0-G1 arrest by increasing the population of G0-G1 phase from 69.1% to 75.8% as compared with that of the control. The results indicated that the BME could suppress MCF-7 proliferation via cell cycle blockage by down-regulating the ER mediated pathways. In conclusion, the potential antiestrogenic activity of Boerhaavia diffusa against human breast cancer cells was investigated in this experimental study for the first time. Boerhaavia diffusa extracts exhibited a strong inhibitory effect on the proliferation of human breast cancer cells in vitro and the antiestrogenic effects are mediated by ER. Phytochemical studies have revealed the presence of alkaloids, flavonoids, phenols and saponins in BME. The antiestrogenic activity shown by the extract may be attributed to these diverse compounds. However it warrants further identification of the active components of the extract and thorough analysis of its antiestrogenic and health promoting effects in vivo to demonstrate its suitability in hormone dependent breast cancer chemoprevention. Acknowledgements We express our deep sense of gratitude to Dr. M. Radhakrishna Pillai, Director, Rajiv Gandhi Centre for Biotechnology for providing the facilities and encouragement. We thank the members of our laboratory and summer training students for their wholehearted support and help throughout this work. The financial assistance from Indian Council of Medical Research (ICMR), Govt. of India, as Junior Research Fellowship to Ms. S. Sreeja is gratefully acknowledged. References Bharali, R., Azad, M.R., Tabassum, J., 2003. Chemopreventive action of Boerhaavia diffusa on DMBA-induced skin carcinogenesis in mice. Indian Journal of Physiology and Pharmacology 47, 459–464. Brown, A.M., Jeltsch, J.M., Roberts, M., Chambon, P., 1984. Activation of pS2 gene transcription is a primary response to estrogen in the human breast cancer cell line MCF-7. Proceedings of the National Academy of Sciences of the United States of America 81, 6344–6348. Canel, J.P., 1998. Natural products isolation. Methods in Biotechnology 4, 220–222. Cariou, S., Donovan, J.C., Flanagan, W.M., Milic, A., Bhattacharya, N., Slingerland, J.M., 2000. Down-regulation of p21WAF1/CIP1 or p27Kip1 abrogates antiestrogenmediated cell cycle arrest in human breast cancer cells. Proceedings of the National Academy of Sciences of the United States of America 97, 9042–9046. Chakraborti, K.K., Handa, S.S., 1989. Antihepatotoxic activity of Boerhaavia diffusa. Indian drugs 2713, 161–166. Chang, C., Yang, M., Wen, H., Chern, J., 2002. Estimation of total flavonoids content in propolis by two complementary colorimetric methods. Journal of Food Drug Analysis 10, 178–182.
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Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jethpharm
An in vitro study on antiproliferative and antiestrogenic effects of Boerhaavia diffusa L. extracts Sreekumar Sreeja, Sreeharshan Sreeja ∗ Cancer Endocrinology, Integrated Cancer Research Programme, Rajiv Gandhi Centre for Biotechnology, Thycaud P.O., Thiruvananthapuram, Kerala 695014, India
a r t i c l e
i n f o
Article history: Received 5 June 2008 Received in revised form 3 August 2009 Accepted 26 August 2009 Available online 31 August 2009 Keywords: Antiestrogen Selective estrogen receptor modulators Boerhaavia diffusa L. Breast cancer Phytoestrogens
a b s t r a c t Ethnopharmacological relevance: Boerhaavia diffusa L. (Nyctinaceae) is a plant of tropical region used in Indian traditional medicine for the treatment of human ailments including abdominal tumor, jaundice, dyspepsia, menstrual disorders, etc. This plant also has antilymphoproliferative, antimetastatic and immunomodulatory effects. Aim of the study: This study aimed to assess the antiproliferative and antiestrogenic properties of methanol extract of Boerhaavia diffusa (BME) in MCF-7 breast cancer cell lines. Materials and methods: The effective concentration range of BME on cell viability was analyzed using MTT assay. Hydroxylapatite assay (HAP) was carried out to confirm the competitive binding of BME to the estrogen receptor (ER). The effect of BME on the expression of a selected estrogen responsive gene pS2 was analyzed by RT-PCR. The ability of BME to alter the cell cycle phases and distributions were studied using FACS analysis. Results: Treatment with varying concentrations of BME (20–320 g/mL) resulted in moderate to very strong growth inhibition in MCF-7 cell lines. BME competed with [3 H]-estradiol for binding to ER with IC50 value of 320 ± 25 g/mL. RT-PCR analysis revealed that BME reduced the mRNA expression of pS2 indicating the antiestrogenic action of BME. BME treatment for 48 h resulted in a remarkable increase in the number of MCF-7 cells in the G0-G1 fraction from 69.1% to 75.8%, with a reciprocal decrease of cells in all other phases indicating cell cycle arrest at G0-G1 phase. Conclusions: The results demonstrate that Boerhaavia diffusa possess antiproliferative and antiestrogenic properties and suggest that it may have therapeutic potential in estrogen dependent breast cancers. © 2009 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Estrogens, mainly 17-estradiol exert numerous pharmacological effects in a large number of targets including the breast, uterus, bone, brain and cardiovascular tissues. Most of their actions are mediated by estrogen receptor (ER) which exists in two different forms ER␣ and ER which reside predominantly in the nucleus of target cells (Cheng et al., 2004). ERs have also been detected in the cellular membrane (Sreeja and Thampan, 2003; Zivadinovic et al., 2005). Phytoestrogens are widely known plant compounds, which bind to ER and affect the physiology of various systems. Mounting evidence points out that phytoestrogens exert estrogenic
Abbreviations: DMEM, Dulbecco’s modified eagle’s medium; E2, estradiol; ER, estrogen receptor; FACS, fluorescence activated cell sorting; HAP, hydroxylapatite; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; RT-PCR, reverse transcriptase polymerase chain reaction; SERM, selective estrogen receptor modulator; BME, Boerhaavia diffusa methanol extract. ∗ Corresponding author. Tel.: +91 471 2529474; fax: +91 471 2348096. E-mail address: [email protected] (S. Sreeja). 0378-8741/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2009.08.041
effects on skeleton, affect favorably the cardiovascular function and menopausal symptoms while demonstrate antiestrogenic action on breast and prostrate tissue (Finkel, 1998; Lissin and Cooke, 2000). This has led to the hypothesis that phytoestrogens may act as selective estrogen receptor modulators (SERMs) and can be considered therapeutically for the prevention of breast and prostate cancer, osteoporosis and cardiovascular diseases (Middleton et al., 2000; Ewies, 2002). Boerhaavia diffusa L. commonly known as ‘Punarnava’ is an abundant creeping weed found all over India (Wahi et al., 1997). The plant has drawn lot of attention due to its uses in Indian traditional medicine. The various parts of the plant are used in the treatment of cancer, jaundice, dyspepsia, inflammation, enlargement of spleen, abdominal pain and as an anti-stress agent (Kirtikar and Basu, 1956; Chakraborti and Handa, 1989; Leslie Taylor, 2005). Root extracts of Boerhaavia diffusa inhibited the growth of several monocytic, lymphoblastoid, fibroblast and erythroleukemic cell lines of mouse and human origin (Mehrotra et al., 2002). Boerhaavia diffusa extracts prevented DMBA-induced skin carcinogenesis in mice (Bharali et al., 2003). Ethanolic extract of the plant is well demonstrated to have anti-mitotic activity in in vitro systems (Obong and Madi,
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1987). Administration of aqueous methanol extract of Boerhaavia diffusa was found to be effective in reducing the metastases formation by B167-10 melanoma cells and Punarnavine, an alkaloid from Boerhaavia diffusa enhanced the immune response against metastatic progression of B16F-10 melanoma cells in mice (Leyon et al., 2005; Manu and Kuttan, 2007). Considering the pronounced antiproliferative and antimetastatic activity of Boerhaavia diffusa, in this study we evaluated the effect of methanol extract of Boerhaavia diffusa (BME) in MCF-7 breast cancer cell lines and validated its antiestrogenic potential to demonstrate its suitability in hormone dependent breast cancer chemoprevention.
protein (ER preparations) was incubated overnight at 4 ◦ C with the varying concentration of BME (range 20–320 g/mL), 20 nM [3 H]-estradiol ± 100-fold molar excess of estradiol (E2) in a final volume of 250 L. 250 L of a 60% HAP suspension in TEM buffer was added and the mixture was incubated at 4 ◦ C for 15 min. The HAP bound receptor–[3 H]-E2 complex was separated by centrifugation at 200 × g for 15 min. After washing twice with Tris buffer (10 mM) the HAP pellet was extracted with 1 mL absolute ethanol. These extracts were added to 4 mL of scintillation cocktail and the radioactivity was measured in a Wallac 1409 liquid scintillation counter. Data was expressed as the ratio of bound [3 H]-E2 in the presence of a competitor to the bound [3 H]-E2 in control × 100.
2. Materials and methods
2.5. Semi-quantitative RT-PCR
2.1. Preparation of extract from Boerhaavia diffusa
MCF-7 cells were seeded onto 100 mm plates and were incubated with phenol red free DMEM supplemented with 10% charcoal treated fetal bovine serum for 48 h before using in the assay. After 48 h, test compounds (BME, E2, ICI 182-780) were added to this medium. After incubating for 24 h total RNA was purified from the cells using Tri-reagent (Sigma chemical Co., St. Louis, MO, USA) according to the manufacturer’s protocol. cDNA was prepared from 5 g of total RNA using AccuScriptTM 1st strand cDNA synthesis kit (Stratagene, USA) as per manufacturer’s instruction. The pS2 cDNA fragments were amplified using the primer pairs (Fotovati et al., 2006), forward—5 -TTTGGAGCAGAGAGGAGGCAATGG-3 , reverse—5 -TGGTATTAGGATAGAAGCACCAGGG-3 (product size 240 bp). The reaction mixture containing 10 mM Tris–HCl (pH 8.5), 50 mM KCl, 2 mM MgCl, 200 M dNTPs, primers (2.5 pM each) and 2.5 units of Taq polymerase were subjected to amplification cycles of 94 ◦ C for 4 min, followed by 30 cycles of 94 ◦ C for 1 min, 59 ◦ C for 30 s, 72 ◦ C for 1 min in a (Eppendorf) thermal cycler. Aliquots (5 L) of each PCR mixture were analyzed by electrophoresis in a 1.2% agarose gel and fragments were visualized by ethidium bromide staining. The intensity of bands was quantified in a Flour-S multi imager (BioRad, Hercules, USA) by using Quantity one densitometry software. The transcripts were normalized with gapdh expression level. The gene expression was shown as ratio of densitometric value of target mRNA to that of gapdh.
Whole plant of Boerhaavia diffusa (Nyctinaceae) were collected from South Kerala during August 2007 and authenticated by Prof. Sheela, Department of Botany, Govt. College Madapally, Calicut University, Kerala. A voucher specimen (accession no. MMCB 004) of the plant was deposited in the herbarium of Cancer Endocrinology, Rajiv Gandhi Centre for Biotechnology, Trivandrum, India. Air-dried powdered plants (10 g) were extracted with methanol in a Soxhlet apparatus for 20 h and then the extract was concentrated using rotary vacuum to get the solid mass. The yield of dried extract obtained from starting crude material was 5% (w/v). The concentrate was dissolved in dimethyl sulphoxide (DMSO, Sigma chemical Co., St. Louis, MO, USA), referred as BME and was used for further experiments. The final concentration of DMSO used was less than 0.1% (v/v) for each treatment. 2.2. Cell and cell culture MCF-7 cell lines obtained from ATCC (Manassas, VA, USA) were cultured in phenol red free Dulbecco’s modified Eagle’s medium, DMEM (Sigma chemical Co., St. Louis, MO, USA) supplemented with 10% heat inactivated fetal bovine serum (Sigma chemical Co., St. Louis, MO, USA), 100 U/mL benzyl penicillin and 100 g/mL streptomycin. The culture was maintained at 37 ◦ C in a humidified atmosphere of 5% CO2 . 2.3. Cell proliferation assay To evaluate the effect of BME on cell proliferation, MTT assay was performed (Haridas et al., 1998). Cells were seeded in 96-well plates at a density of 5 × 103 cells per well and treated with 0, 20, 40, 80, 160 and 320 g/mL of BME. After incubating for 48 h, the drug containing medium was aspirated and then 100 L of MTT reagent (2 mg/mL) was added and incubated for 2 h at 37 ◦ C. The viable cell number is directly proportional to the production of formazan following solubilization with MTT lysis buffer (20% sodium dodecyl sulphate in 50% dimethyl formamide), which can be measured spectrophotometrically at 570 nm. Cell survival was expressed as percentage over the untreated control. Cell survival (CS) was calculated as CS = (Optical density of drug exposed cells/Mean optical density of control cells) × 100. 2.4. Competitive ER binding assay (HAP assay) Hydroxylapatite (HAP) assay was carried out to confirm the competitive binding of BME to ER (Clark et al., 1979). MCF-7 cytosolic extract was used for competition-binding studies. Cytosol was prepared from cells grown for 4 days in estrogen depleted medium. The protein content was measured spectrophotometrically at 570 nm using Bradford reagent. About 40 g of the total
2.6. FACS analysis MCF-7 cells were treated with varying concentrations of BME (20–320 g/mL) for 48 h. The supernatant was then collected and the cells were trypsinised, collected and fixed in 70% cold ethanol (4 ◦ C) overnight. After washing twice with PBS, cells were resuspended in PBS. The cells were stained with propidium iodide (20 g/mL) after treatment with RNase-A (10 g/mL) for 30 min. The DNA content of the cells was then analyzed with a FACS caliber instrument (Becton Dickinson, San Jose, CA, USA). The percentage of cells in different cell cycle phases was calculated using DIVA software. 2.7. Phytochemical screening Qualitative screening of BME was made to investigate the major chemical classes of components present. It was screened for the presence of alkaloids, flavonoids, polyuronides, phenols, reducing compounds, saponins and tannins (Canel, 1998; Karumi et al., 2004). 2.8. Estimation of total flavonoids in BME Aluminum chloride colorimetric method was used for flavonoids determination (Chang et al., 2002). BME (0.5 mL of 0.5 mg/mL) in 80% methanol were separately mixed with 0.1 mL
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3. Results 3.1. Effect of BME on MCF-7 cell proliferation
Fig. 1. Effect of BME on MCF-7 cell viability/proliferation. The cell line was treated with BME at different concentrations of 20, 40, 80, 160, 320 g/mL for 48 h and the cell viability was determined by MTT assay. The cell survival was expressed as % over the untreated control. A dose dependent growth inhibition of MCF-7 cells was observed after treatment with BME. Results are mean values ± S.E. of five replicates. * P ≤ 0.05 when compared to untreated control.
of 10% aluminum chloride, 0.1 mL of 1 M potassium acetate and 4.3 mL of 80% methanol. It was kept at room temperature for 40 min; the absorbance of the reaction mixture was measured at 415 nm with a double beam PerkinElmer UV/Visible spectrophotometer (USA). The calibration curve was prepared by preparing quercetin solutions at concentrations 25–200 g/mL in methanol.
MCF-7 cells were treated with or without BME to study its antiproliferative effects. The effect of BME on growth inhibition was assessed as percentage cell viability where DMSO treated cells was taken as 100% viable. MCF-7 cells were treated with increasing concentrations of BME (20–320 g/mL) for 48 h. As shown in Fig. 1 there was a decrease in viability of cells treated with BME in comparison to the DMSO treated control. The cell viability decreased to 46.8% in 320 g/mL, 48 h BME treated cells. It also inhibited the estradiol induced proliferation in MCF-7 cell lines suggesting its potential antiestrogenic properties. 3.2. ER binding ability of BME In an attempt to ascertain whether BME interacts with ER, competitive binding studies were carried out. MCF-7 cytosolic lysate was used as a source of ER. As shown in Fig. 2 BME (20–320 g/mL) inhibited the binding of [3 H]-E2 to cytosolic ER in a dose dependent manner. The IC50 value of binding of BME, i.e. the concentration of the extract required to reduce the specific radioligand binding by 50% is 320 ± 25 g/mL. 3.3. BME downregulates the ER mediated transcription
2.9. Estimation of total phenols in BME Total phenols were determined by Folin Ciocalteu reagent (Mc Donald et al., 2001). A dilute extract of each plant extract (0.5 mL of 0.5 mg/mL) or gallic acid (standard phenolic compound) was mixed with Folin Ciocalteu reagent (5 mL, 1:10 diluted with distilled water) and aqueous Na2 CO3 (4 mL, 1 M). The mixtures were allowed to stand for 15 min and the total phenols were determined by colorimetry at 765 nm. The standard curve was prepared using 25–200 mg/L solutions of gallic acid in methanol:water (50:50, v/v). Total phenol values are expressed in terms of gallic acid equivalent (mg/g of dry mass), which is a common reference compound.
The effect of BME on pS2 gene expression was studied as a model for endogenous estrogen responsive gene expressed by MCF-7 cells. E2 and ICI 182-780 were used as agonistic control and antagonistic control respectively. MCF-7 cells were incubated with BME (100 g/mL) or estradiol (10 nM) or ICI 182-780 (10 nM) for 24 h and RT-PCR was performed to amplify the pS2 mRNA using gapdh as endogenous control. As shown in Fig. 3A, BME decreased the expression of pS2 gene to levels approximately as great as those produced by ICI 182-780. E2 up-regulated the expression of the
2.10. Statistical analysis The experiments were performed in triplicates. All values were expressed as mean ± S.E. and Tukey’s post hoc test was done to analyze significance of difference between different groups using the statistical analysis software package SPSS (Version 16.0). Values with P ≤ 0.05 were considered as significant.
Fig. 2. ER affinity assay of BME. Binding of 20 nM [3 H]-E2 to cytosolic ER in the presence of varying concentrations (20–320 g/mL) of BME. Specific bound radioligand was calculated by subtracting non-specific bound counts from total bound counts. Data presented as mean ± S.E. from three separate experiments for each data point.
Fig. 3. Effects of BME on pS2 gene expression in MCF-7 cells. MCF-7 cells were incubated with the BME (100 g/mL) or estradiol (10 nM) or ICI 182-780 (10 nM) for 24 h (A). The graph (B) shows the ratio of density of pS2 expression to that of endogenous control gapdh and it represents mean ± S.E. of three replicates. * P ≤ 0.05 when compared to untreated control.
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gene. The graph (Fig. 3B) shows the ratio of density of pS2 to that of endogenous control gapdh and it represents mean ± S.E. of three replicates. * P ≤ 0.05 when compared to untreated control. 3.4. Cell cycle analysis by flow cytometry The effect of BME on cell cycle progression of MCF-7 cells was determined by flow cytometry. MCF-7 cells treated with varying concentrations of BME (20–320 g/mL) showed G0-G1 arrest by increasing the population of G0-G1 phase from 69.1% to 75.8% as compared with that of the untreated control (Fig. 4A–F). 3.5. Phytochemical screening Phytochemical investigation has revealed the presence of alkaloids, flavonoids, phenolics and saponins in BME. The flavonoid contents of the extracts in terms of quercetin equivalent (the standard curve equation: y = 0.0096x + 0.0114, r2 = 0.999) was 18.5 ± 1.18 mg/g of dry extract powder. The total phenols measured by Folin Ciocalteu reagent in terms of gallic acid equivalent (standard curve equation: y = 0.0648x + 0.005, r2 = 0.9921) was 24.28 ± 3 mg/g in the extract powder. 4. Discussion and conclusions
Fig. 4. Inhibition of cell cycle progression in breast cancer cell lines as analyzed by flow cytometry. Flow cytometric analysis (propidium iodide staining) of the cell cycle distribution of MCF-7 cells without the extracts (A) and after treatment with 20 g/mL (B), 40 g/mL (C), 80 g/mL (D), 160 g/mL (E) and 320 g/mL (F) of BME for 48 h.
Estrogen level is a critical factor in the human physiology in that it influence the development, sexual differentiation, fertility, control of the female reproductive tract, organ responsiveness and female diseases due to hormone imbalance. In the classical ER mediated pathway, the ligand binds to the ligand-binding domain of the ER followed by the formation of a liganded receptor homodimeric complex. The complex binds to the distal ERE’s in the 5 -promoter regions of the E2-responsive genes, further interacts with the components of the general transcription factor complex and recruit coactivators or adaptors and finally induces transcription (Korach et al., 1997). Antiestrogenic compounds act on one hand by directly competing with E2 for binding to ER, resulting in a functionally inactive ligand bound complex. On the other hand, they can force the metabolism of E2 or restrain the biosynthesis of E2, leading to the depletion of endogenous E2 binding to the ER in an indirect manner (Porter and Safe, 1999). Inhibition of estrogen is a common therapeutic strategy for estrogen dependent breast tumors in postmenopausal females and tumor prevention in premenopausal women (Papoutsi et al., 2007). In this study the antiestrogenic effect of methanol extract of Boerhaavia diffusa (BME) through direct ER binding was analyzed in ER positive human mammary carcinoma cell line MCF-7. Estrogens are known stimulants of breast cancer growth whereas antiestrogens arrest the growth of cells (Pratt and Pollak, 1993). To assess the antiproliferative effects of the extracts we applied MTT assay which depends on the reduction of tetrazolium salt by the mitochondria of living cells to form a blue formazan product (Denizot and Lang, 1986). The results depend on both number of cells present and on the mitochondrial activity per cell and showed a dose dependent decrease in viability of MCF-7 cells treated with BME in comparison to the control. BME inhibited E2 induced proliferation in MCF-7 cells suggesting its possible antiestrogenic role. In support to this, competitive radioactive binding studies revealed that BME binds to ER in a dose dependent manner with an IC50 value of 320 ± 25 g/mL. These findings emphasize further the involvement of ER regulated genes in controlling cell proliferation. Estrogen induces the expression of various genes such as cfos, c-erb-b2, ER, PR, pS2, etc. pS2 gene transcription is a primary response to E2 in human breast cancer cell line MCF-7 (Brown et al.,
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1984). pS2 is an estrogen regulated gene whose expression can be increased by estrogen and inhibited by antiestrogen (Masiakowski et al., 1982). Since BME down-regulated pS2 gene transcription (Fig. 3A and B), it was confirmed that BME has some potential compounds that could down-regulate the ER mediated transcription and cause antiestrogenic effects in breast tissue. E2 via ER influences the genes controlling cell proliferation, cell cycle progression or apoptosis resulting in uncontrolled proliferation leading to cancers. Many antiestrogenic anticancer agents from plants inhibit cancer cell growth through cell cycle regulation including G0/G1 accumulation (Cariou et al., 2000). As shown in Fig 4A–F, MCF-7 cells treated with BME caused G0-G1 arrest by increasing the population of G0-G1 phase from 69.1% to 75.8% as compared with that of the control. The results indicated that the BME could suppress MCF-7 proliferation via cell cycle blockage by down-regulating the ER mediated pathways. In conclusion, the potential antiestrogenic activity of Boerhaavia diffusa against human breast cancer cells was investigated in this experimental study for the first time. Boerhaavia diffusa extracts exhibited a strong inhibitory effect on the proliferation of human breast cancer cells in vitro and the antiestrogenic effects are mediated by ER. Phytochemical studies have revealed the presence of alkaloids, flavonoids, phenols and saponins in BME. The antiestrogenic activity shown by the extract may be attributed to these diverse compounds. However it warrants further identification of the active components of the extract and thorough analysis of its antiestrogenic and health promoting effects in vivo to demonstrate its suitability in hormone dependent breast cancer chemoprevention. Acknowledgements We express our deep sense of gratitude to Dr. M. Radhakrishna Pillai, Director, Rajiv Gandhi Centre for Biotechnology for providing the facilities and encouragement. We thank the members of our laboratory and summer training students for their wholehearted support and help throughout this work. The financial assistance from Indian Council of Medical Research (ICMR), Govt. of India, as Junior Research Fellowship to Ms. S. Sreeja is gratefully acknowledged. References Bharali, R., Azad, M.R., Tabassum, J., 2003. Chemopreventive action of Boerhaavia diffusa on DMBA-induced skin carcinogenesis in mice. Indian Journal of Physiology and Pharmacology 47, 459–464. Brown, A.M., Jeltsch, J.M., Roberts, M., Chambon, P., 1984. Activation of pS2 gene transcription is a primary response to estrogen in the human breast cancer cell line MCF-7. Proceedings of the National Academy of Sciences of the United States of America 81, 6344–6348. Canel, J.P., 1998. Natural products isolation. Methods in Biotechnology 4, 220–222. Cariou, S., Donovan, J.C., Flanagan, W.M., Milic, A., Bhattacharya, N., Slingerland, J.M., 2000. Down-regulation of p21WAF1/CIP1 or p27Kip1 abrogates antiestrogenmediated cell cycle arrest in human breast cancer cells. Proceedings of the National Academy of Sciences of the United States of America 97, 9042–9046. Chakraborti, K.K., Handa, S.S., 1989. Antihepatotoxic activity of Boerhaavia diffusa. Indian drugs 2713, 161–166. Chang, C., Yang, M., Wen, H., Chern, J., 2002. Estimation of total flavonoids content in propolis by two complementary colorimetric methods. Journal of Food Drug Analysis 10, 178–182.
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