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Betanin a betacyanin pigment purified from fruits of Opuntia ficus-indica induces apoptosis in human chronic myeloid leukemia Cell line-K562.

Abstract

Betalains are water-soluble nitrogenous vacuolar pigments present in flowers and fruits of many caryophyllales with potent antioxidant properties. In the present study the antiproliferative effects of betanin, a principle betacyanin pigment, isolated from the fruits of Opuntia ficus-indica, was evaluated on human chronic myeloid leukemia cell line (K562). The results show dose and time dependent decrease in the proliferation of K562 cells treated with betanin with an [IC.sub.50] of 40 [micro]M. Further studies involving scanning and transmission electron microscopy revealed the apoptotic characteristics such as chromatin condensation, cell shrinkage and membrane blebbing. Agarose electrophoresis of genomic DNA of cells treated with betanin showed fragmentation pattern typical for apoptotic cells. Flow cytometric analysis of cells treated with 40 [micro]M betanin showed 28.4% of cells in sub G0/G1 phase. Betanin treatment to the cells also induced the release of cytochrome c into the cytosol, poly (ADP) ribose polymerase (PARP) cleavage, down regulation Bcl-2, and reduction in the membrane potentials. Confocal microscopic studies on the cells treated with betanin suggest the entry of betanin into the cells. These studies thus demonstrate that betanin induces apoptosis in K562 cells through the intrinsic pathway and is mediated by the release of cytochrome c from mitochondria into the cytosol, and PARP cleavage. The antiproliferative effects of betanin add further value to the nutritional characteristics of the fruits of O. ficus-indica.

[c] 2007 Elsevier GmbH. All rights reserved.

Keywords: Opuntia ficus-indica; Betanin; Apoptosis; Chronic myeloid leukemia

Introduction

Natural products with high medicinal values are gaining much importance in light of serious side effects posed by the medicinal derivatives from chemical origin. Natural pigments are another class of natural products that have attracted considerable attention as antioxidants that induce beneficial effects on human health and disease prevention. Carotenoids (Sies and Stahl, 2003) and anthocyanins (Matsumoto et al., 2001) are such natural pigments that have proven to possess health benefits. Recently we have reported that C-Phycocyanin, a natural pigmented antioxidant, isolated from Spirulina platensis induces apoptosis in mouse macrophage cell line RAW 264.7 stimulated with LPS (Reddy et al., 2000) and human chronic myeloid leukemia cell line-K 562 (Subhashini et al., 2004). Betalins are another class of natural pigments with reddish purple (betacyanins) or yellow (betaxanthins) nitrogenous vacuolar components that are widely used as food colorants. In contrast to other natural pigments such as carotenoids and anthocyanins, the physiological effects of betalains are not well studied. In a recent study the antiviral and antimicrobial effects of betalain pigments have been reported (Strack et al., 2003). Furthermore, the antioxidant properties of betalins has been demonstrated in a wide range of assays (Zakharova and Petrova, 1998; Kanner et al., 2001; Gentile et al., 2004) and it was reported that enrichment of human low-density lipoproteins by betalains, effectively increased resistance to oxidation (Tesoriere et al., 2003). In addition, a role for betalain pigments in the chemoprevention against lung and skin cancers has been documented (Kapadia et al., 1996). It is recently documented that natural food colors such as betanin can inhibit the cell proliferation of a variety of human tumor cells (Muntha Reddy et al., 2005), however; the exact mechanism of action for the betalain pigments has not been addressed in any of the foregoing studies. In the present study we report antiproliferative effects of betanin, a principle betacyanin pigment isolated from the fruits of Opuntia ficus-indica, on human chronic myeloid leukemia cell line-K562 and elucidate the molecular mechanisms involved.

Materials and methods

Phosphate buffered saline (PBS), RPMI medium, fetal bovine serum (FBS) were purchased from GIBCO Ltd. (BRL Life Technologies Inc., Grand Island, NY). MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide], proteinase K, RNase A, propidium iodide were from Sigma Chemical Co. (St Louis, MO). Nitrocellulose membranes were from Amersham Life Sciences; monoclonal antibodies against cytochrome c and Bcl-2 were from Upstate Technologies. monoclonal Bax antibodies were from Santa Cruz, CA, USA and polyclonal PARP antibodies were from R & D Systems, USA.

Extraction, purification and characterization of betanin from fruits of O. ficus-indica

O. ficus-indica (prickly pear) fruit pulp (100g) was homogenized, extracted into 100 ml of aqueous methanol and concentrated in vacuo at 25[degrees]C to 3-4 ml.

Prior to preparative HPLC, the concentrated fractions were purified by gel filtration on sephadex-LH 20 column (40 x 2.2 cm). The betacyanin fractions thus obtained were lyophilized. The purified betacyanin fractions were subjected to preparative HPLC on Shimadzu ODS-HPLC column (20 mm x 25 cm) and elution was carried out following a gradient program (Strack et al., 1987). The structural identity of betanin was further confirmed by NMR and LC-MS studies (data not shown).

Cell culture and treatment

K562 cells were grown in suspension cultures employing RPMI 1640 medium supplemented with 10% heat inactivated fetal bovine serum (FBS), 100IU/ml penicillin, 100mg/ml streptomycin and 2mM L-glutamine. Cultures were maintained in a humidified atmosphere with 5% C[O.sub.2] at 37[degrees]C. The cultured cells were passed twice each week, seeding at a density of about 2 x [10.sup.5] cells/ml. For treatments, exponentially growing K562 cells were collected and re-suspended in fresh culture medium. A stock solution of 40 mM betanin was prepared in PBS from the cryodessicated purified samples and used in the experiments.

Cell proliferation assay

Cell proliferation was assessed using the 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) staining method (Mossman, 1983). Briefly, 5 x [10.sup.3] cells (K562) were incubated in 96-well plates in the presence or absence of betanin (10, 20, 40, 80 [micro]M) for various time points in a final volume of 100 [micro]l. At the end of the treatment, 20 [micro]l of MTT (5 mg/ml in PBS) was added to each well and incubated for an additional 4 h at 37[degrees]C. The purple-blue MTT formazan precipitate was dissolved in 50 [micro]l of lysis buffer (12mM HC1, 5% isobutanol and 10% SDS). The activity of the mitochondria, reflecting cellular growth and viability, was evaluated by measuring the optical density at 570 nm on micro titer plate reader (Microscan MS 5608A, ECIL Instruments).

Scanning electron microscopy

After treatment with betanin (40 [micro]M, 24 h) the cells were collected, washed with PBS and concentrated to 1 x [10.sup.5] cells/ml. One drop of this suspension was placed on to a plastic coverslip that is previously coated with 1% poly-L-Lysine. Cells were fixed with glutaraldehyde for 1 h and post fixed with 1% osmium tetroxide for 1 h. Cells were dehydrated by passing through graded alcohols and dried by the critical-point technique. After trimming, mounting, and coating with gold-platinum, the specimens were observed on SEM (JSM-5600, JEOL Co.)

Transmission electron microscopy

Cell pellets were rinsed rapidly with PBS and fixed for 12 h at 4[degrees]C with 2% vol/vol glutaraldehyde in 0.1% sodium cacodylate buffer, pH 7.4. Fixation was followed by 3-5 min rinses with 0.1% sodium cacodylate buffer, pH 7.4. Cells were post fixed with a solution containing 1% osmium tetroxide (w/v) and 2% K4Fe(CN) (w/v), and stained with 1% uranyl acetate, and pelleted in 2% agar. Pellets were dehydrated in graded ethanol solutions and embedded in spur resin. Ultra thin (60 nm) sections were cut on a Reichert Ultra cut microtome collected on Rhodanium 400-mesh grids, post-stained with uranyl acetate followed by lead citrate, and rinsed with water. The sections were examined in Philips CM-12 electron microscope at 80 kV.

Cell cycle analysis

To quantitate apoptosis, flow cytometric analysis was performed using propidium iodide as described previously (Reddy et al., 2003).

Detection of DNA fragmentation

Cells were treated with betanin (0, 20, 40, 80 [micro]M) for 24 h and the fragmented DNA was isolated using the SDS/Proteinase K/RNase A extraction method, which allows the isolation of only fragmented DNA without contaminating genomic DNA (Reddy et al., 2003). The presence of apoptosis was indicated by the appearance of a ladder of oligonucleosomal DNA fragments that are approximately 180-200 bp multiples on the agarose gel.

Flow cytometric analysis of mitochondrial membrane potential

K562 cells were cultured with betanin (40 [micro]M for 24h). The cells were harvested and changes in the mitochondrial membrane potentials were measured by flow cytometric analysis (Seuduto and Grotyohann, 1999).

Western blot analysis

Cells were exposed to 40 [micro]M betanin for various time points (0, 4, 8, 12, 24 h) and whole cell extracts were prepared based on the method of Pardhasaradhi et al. (2003). To prepare the whole cell extract, cells were washed with PBS and suspended in a lysis buffer (20 mM Tris, 1mM EDTA, 150mM NaCl, 1% Triton X-100, 0.5% deoxycholic acid, 1 mM glycerophosphate, 1mM sodium orthovanadate, 1mM PMSF, 10mg/ml leupeptin, 20 mg/ml aprotinin). After 30 min of shaking at 4[degrees]C, the mixtures were centrifuged (10,000 x g) for 10min, and the supernatants were collected as the whole-cell extracts. The protein content was determined according to the Bradford (1976) method. An equal amount of total cell lysate (50 [micro]g) was resolved on 8-12% SDS-PAGE gels and then transferred on to nitrocellulose membranes. Membranes were stained with 0.5% ponceau in 1% acetic acid to confirm equal loading. The membranes were blocked with 5% w/v non-fat dry milk and then incubated with the primary antibodies (Cyt-c, Bcl-2, Bax and PARP) in 10 ml of antibody-dilution buffer (Tris-buffered saline and 0.05% Tween-20 with 5% milk) with gentle shaking at 4[degrees]C for 8-12 h and then incubated with respective conjugated secondary antibodies. Signals were detected using Western blot detection reagents.

Intracellular localization of betanin in K562 cells

The K562 cells treated with betanin (40 [micro]M) for 24 h were washed in PBS and fixed in 4% formaldehyde, pH 7.4 for 20 min at 4[degrees]C. After fixation, cells were washed twice in PBS and water. The cells were then viewed under confocal microscope with 506 and 529 nm of excitation and emission wavelengths.

Statistical analysis

Data reported as the mean [+ or -] SD of three independent experiments. Statistical analysis of differences was carried out by one-way analysis of variance (ANOVA). P-value of less than 0.05 was considered as significant.

Results

Isolation and identification of betanin

The betalains from O. ficus-indica fruit pulp were extracted and purified by gel filtration chromatography and then on HPLC to yield a prominent betacyanin pigment (data not shown). The prominent betacyanin pigment was identified as betanin-betanidin 5-O-beta glucoside (Fig. 1) based on mass spectral and NMR studies. The absorption spectrum of purified betanin showed maximum absorbance at 535 nm (data not shown). This purified betanin pigment with a molecular weight of 551 was employed in the present study.

[FIGURE 1 OMITTED]

Betanin inhibited the K562 cell growth

The effect of betanin on growth of K562 cells was evaluated by MTT assay. We observed a dose dependent decrease in K562 cell growth until 48 h after betanin treatment (Fig. 2), with 50% decrease in cell proliferation at 40 [micro]M betanin for 24 h. Further studies were carried out to analyze the mode of cell death on cells exposed to 40 [micro]M betanin for 24 h.

Betanin induced ultra structural changes in the K562 cells

Scanning and transmission electron microscopic studies were carried out to observe the morphological and ultra structural changes induced by betanin in the K562 cells. Morphological features pertaining to apoptotic cell death such as membrane blebbing was clearly observed (data not shown). The transmission electron microscopic studies revealed the condensation of chromatin and loss of intra cellular details (Fig. 3).

[FIGURE 2 OMITTED]

[FIGURE 3 OMITTED]

Betanin induced DNA fragmentation of K562 cells

DNA extracted from K562 cells treated with betanin, at indicated concentrations (0, 20 40, 80 [micro]M) for 24 h revealed a progressive increase in the 180-200 bp ladder fragments. Such a pattern corresponds to inter nucleo-somal cleavage, which is characteristic of apoptosis. Control cells did not exhibit any such DNA fragmentation (data not shown).

Effect of betanin on cell cycle profile of K562 cells

The induction of apoptosis in cells treated with betanin was further confirmed by flow cytometric analysis of DNA content. Fig. 4 illustrates the DNA content histograms of K562 cells treated with (40 [micro]M) or without betanin for 24 h. These studies showed 28.4% of betanin treated cells at sub G0/G1 phase, when compared to 3% in untreated cells (Fig. 4).

Betanin caused reduction in cell mitochondrial membrane potentials (flow cytometric analysis)

The decrease in mitochondrial membrane potential ([DELTA][PSI]m) is associated with mitochondrial dysfunction (Seuduto and Grotyohann, 1999). In the present study we investigated the changes in the membrane potential of K562 cells exposed to 40 [micro]M betanin. The cells were harvested and changes in the mitochondrial membrane potentials were measured by the uptake of lipophilic cation Rhodamine 123 into mitochondria (Kroemer and Reed, 2000). Untreated control cells were used to determine the normal uptake of this cation, and the percentage of treated cells with low membrane potentials was calculated. Shift in percentage of fluorescence intensity from 96.1% in control to 89.1% in the cells treated with 40 [micro]M betanin was observed (data not shown).

[FIGURE 4 OMITTED]

Betanin effects on cytochrome c release, Bcl-2 and Bax proteins in K562 cells

The process of apoptosis is associated with the disruption of mitochondrial membrane potential which results from the opening of permeability transition pores in the mitochondrial membrane leading to the release of cytochrome c. To determine whether there is any release of cytochrome c from the mitochondria into the cytosol, cytosolic fractions from the cells treated with 40 [micro]M betanin for indicated times, were subjected to Western blot analysis. We observed a time dependent elevation in the cytosolic levels of cytochrome c with maximum increase at 12 and 24 h after betanin treatment (Fig. 5A). Furthermore, the expression levels of Bcl-2 and Bax proteins are associated with mitochondrial membrane integrity and play a crucial role in the regulation of apoptosis. Time-dependent decrease of Bcl-2 protein levels was observed after betanin treatment (Fig. 5B), but no appreciable change in the levels of Bax protein was observed at all the time periods (Fig. 5C). These results suggest a disturbed Bcl-2/Bax ratio in the cells treated with betanin.

PARP cleavage in response to betanin treatment

PARP, poly (ADP ribose) polymerase, is a nuclear enzyme implicated in many cellular processes including apoptosis and DNA repair. During apoptosis PARP (116kDa) is cleaved to yield two fragments of 85 and 23kDa. To determine whether PARP is cleaved in betanin induced cell death, we treated K562 cells with 40 [micro]M betanin for indicated times (0, 4, 8, 12, 24 h) and PARP cleavage was monitored by employing PARP antibodies that recognize the 23 kDa fragment of cleaved PARP and uncleaved 116kDa full length PARP. The data presented in Fig. 5D show the gradual increase in the appearance of 23 kDa signature fragment of PARP cleavage at all the indicated times (lanes 2-5). However, in control cells (lane 1) no 23 kDa fragment of PARP was observed, except the uncleaved 116 kDa protein.

Intracellular localization of betanin in K562 cells

In order to find out whether betanin enters the K562 cells, confocal microscopic studies were undertaken on cells exposed to betanin (40 [micro]M) for 24 h. These studies showed a strong fluorescence signal in the cytoplasm of the cells treated with the betanin, with no such signal in the untreated cells (Fig. 6).

Discussion

Cancer chemoprevention is defined as inhibition of tumor initiation, promotion and progression by employing pharmacologic or natural agents that prevent the metabolic activation of procarcinogens (Hong and Sporn, 1997). A number of natural phytochemicals (Katdare et al., 1998) and natural antioxidants such as C-Phycocyanin (Subhashini et al., 2004; Pardhasaradhi et al., 2005), Anthocyanidins (Hou et al., 2001; Meiers et al., 2001; Nagase et al., 1998; Bomser et al., 1996; Kamei et al., 1995), Curcumin (Kuo et al., 1996; Jiang et al., 1996), fungal cytochalasins (Nagasawa et al., 2000) etc., were shown to have anticancer properties. The mechanisms responsible for executing the antiproliferative effects include: (i) induction of alterations in the cell differentiation pattern, which plays a vital role in the invasiveness and metastatic progression of the tumors, (ii) blockade of pre neoplastic cell expansion or induction of apoptosis, and (iii) intervention of metabolic activation of carcinogens by scavenging ROS. In the present study we have analyzed the effects of betanin, a principle betacyanin pigment isolated from O. ficus-indica, on human chronic myeloid leukemia cell line, K562.

[FIGURE 5 OMITTED]

[FIGURE 6 OMITTED]

O. ficus-indica is a perennial plant that grows all over the world and belongs to the family of Cactaceae. A wide range of studies demonstrated the usage of this herbal extracts in management of many disorders. Ethanol extracts of O. ficus-indica showed potent anti-inflammatory properties (Park et al., 2001). Pretreatment with lyophilized cladode extracts in the rats revealed a protective action against ethanol-induced ulcer formation (Galati et al., 2001). In another study the methanolic extracts of stems reported to possess wound-healing properties (Park and Chun, 2001). The fruits of O. ficus-indica are very good source of water-soluble nitrogenous chromo alkaloids called betalains. Betalains find their importance as food colorants as they provide attractive bright hues for food products (Stinzing, 1999). In a recent study the pharmacokinetic effects of these pigments in human subjects was demonstrated (Netzel et al., 2005). There has been growing interest in the use of these natural pigments for food coloring, since synthetic dyes are becoming more and more critically assessed by the consumer, both from the ends of quality and health. Apart from serving as food additives, the natural pigments also play a vital nutraceutical role in circumventing the health hazards posed by synthetic and chemical colorants. Betalains are one such nutraceutical nitrogenous cationic natural pigments that are widely used as food additives. Unlike other pigments betalains are not very well studied with regard to their pharmacological properties and mechanism of action.

In the present study we evaluated the anti proliferative effects of betanin, a principle betacyanin pigment isolated from the fruits of O. ficus-indica, on chronic myeloid leukemia cell line, K562. Further in-depth studies were taken up to understand the molecular mechanisms involved in betanin-induced effects. We found that betanin induced dose dependent and time dependent inhibition in the growth of K562 cells, with [IC.sub.50] of 40 [micro]M. Apoptosis is a process of cell death that is critically regulated based on the expression of cell's intrinsic suicide machinery (Vaux and Korsmeyer, 1999), which further leads to the characteristic pattern of morphological, biochemical, and molecular changes. The present study showed typical apoptotic characteristics such as membrane blebbing, laddering of DNA and chromatin condensation in the cells treated with betanin. The flow cytometric analysis of betanin treated cells showed an increase in the hypo diploid apoptotic DNA content with a decrease in the number of cells at the S and G2 phases of the cell cycle. Accumulating scientific evidences suggest the pivotal role of mitochondria in the execution of apoptosis of the cells exposed to various stimuli (Desagher and Martinou, 2000; Green and Reed, 1998). Our present studies showed decreased mitochondrial membrane potential with simultaneous appearance of cytochrome c in the cytosolic fractions of cells exposed to betanin. We hypothesize that betanin, after gaining entry into the cell, induced the mitochondrial disruption leading to decreased membrane potentials and leakage of mitochondrial cytochrome c into the cytosol. Confocal microscopic studies support such a possibility of entry of betanin into the cells. Further decrease in expression of Bcl-2 protein and the alterations in the Bcl-2/Bax ratios strongly support mitochondrial membrane alterations in the cells treated with betanin. Caspase-3, an executioner caspase in apoptotic cascades is implicated in the cleavage of a numberf proteins including Poly (ADP-ribose) polymerase (PARP), which is a hallmark of apoptosis. In the present study a time dependent increase in the PARP cleavage in cells treated with betanin (40 [micro]M) was observed, suggesting the activation of caspase-3 activity. The foregoing studies clearly demonstrate that betanin induces apoptosis in K562 cells by intrinsic death pathway. Further studies, however, are required to understand the mechanisms involved in betanin-induced alterations in mitochondrial membrane integrity in K562 cells.

In conclusion, the present study demonstrates that betanin, a natural betacyanin pigment isolated from the fruits of O. ficus-indica, enters K562 cells and alters mitochondrial membrane integrity, leading to cyto-chrome c leakage, activation of caspases and nuclear disintegration. These biochemical alterations are reflected in ultra structural changes, typical of cells undergoing apoptosis. These findings on anticancer effects of betanin, further add value to the nutritional characteristics of the fruits of Opuntia ficus-indica.

Acknowledgments

Financial support in the form of research fellowships to Mr. D. Sreekanth (Andhra Pradesh-Netherlands Biotechnology program and Universities Grants Commission) and Mr. Karnati R. Roy (Council for Scientific and Industrial Research) is gratefully acknowledged.

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Devalraju Sreekanth, M.K. Arunasree, Karnati R. Roy, T. Chandramohan Reddy, Gorla V. Reddy, Pallu Reddanna*

School of Life Sciences, University of Hyderabad, Hyderabad 500 046, India

*Corresponding author. Tel./fax: +91 40 23010745.

E-mail address: prsl@uohyd.ernet.in (P. Reddanna).
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Author:Sreekanth, Devalraju; Arunasree, M.K.; Roy, Karnati R.; Reddy, T. Chandramohan; Reddy, Gorla V.; Red
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Geographic Code:9INDI
Date:Nov 1, 2007
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