Leaf extracts from Nitraria retusa promote cell population growth of human cancer cells by inducing apoptosis
© Boubaker et al; licensee BioMed Central Ltd. 2011
Received: 3 June 2011
Accepted: 31 October 2011
Published: 31 October 2011
In this report the phytochemical profile of Nitraria. Retusa (N. Retusa) leaf extracts were identified and their ability to induce apoptosis in human chronic myelogenous erythroleukaemia (K562) was evaluated.
Apoptosis of the human chronic myelogenous erythroleukaemia (K562) was evidenced by investigating DNA fragmentation, PARP cleavage and caspases 3 and 8 inducing activities, in the presence of N. retusa extracts.
Our study revealed that the tested extracts from N. Retusa contain many useful bioactive compounds. They induced in a time-dependent manner the apoptosis the tested cancerous our cell line. This result was confirmed by ladder DNA fragmentation profile and PARP cleavage, as well as a release in caspase-3 and caspase-8 level.
Our results indicate that the tested compounds have a significant antiproliferative effect which may be due to their involvement in the induction of the extrinsic apoptosic pathway.
Apoptosis is a form of cell death in which a programmed sequence of events eliminates cells without damaging neighbouring cells. Apoptosis is triggered through either a death receptor mediated extrinsic pathway or a mitochondrial intrinsic pathway. Phytotherapy is considered as an alternative, to mitigate side effects due the indiscriminate use of synthetic drugs. For many years, the antiproliferative actions of chemotherapeutic drugs were ascribed solely to their ability to induce genotoxic damage . Therefore, the role of plant derived polyphenols in chemoprevention of cancer has emerged as an interesting area of research. To date, many anticancer drugs have been developed and applied by clinical doctors . In addition flavonoids have been shown to cause apoptosis through induction of Bax with concomitant suppression of Bcl-2, or through other molecules and pathways including up-regulation of death receptor 5, modulation of IGFBP-3, involvement of p38-MAPK, and inhibition of PI-3-kinase/Akt and ERK pathways . In our case, we were interested with leaf extracts from Nitraria retusa in order to investigate an alternative phytoterapy solution to current anticancerous treatments. Its fleshy red fruits are eaten by humans and are used to prepare drinks. The leaves serve as supplement for the tea and are used as poultice . The ashes of this species have the ability to remove fluids of infected wounds . Belkadhar  indicates that a decoction of fresh leaves of Nitraria retusa is used in Morocco in case of poisoning, upset stomach, ulcers, gastritis, enteritis, heartburn, colitis, colonic abdominal pain. In this study, we analyzed and compared cytotoxic effects of hexane, chloroform and methanol extracts, on a human chronic myelogenous erythroleukaemia (K562) cell line. We attempt to elucidate the apoptotic pathway and molecular mechanisms responsible for their cytotoxic and apoptotic activities.
All the organic solvents were obtained from Carlo ERBA (Paris, France). L-glutamine was purchased from GIBCO BRL Life technologies (Grand Island, NY, USA). The chromatographic columns were performed with silica gel 60 (Pharmacia Biotech, Uppsala, Sweden), reverse phase C18 column (Merck, Darmstadt, Hesse, Germany). The N-(1-naphtyl) ethlenediaminedihydrochloride (EDTA) was purchased from Sigma-Aldrich (Steinheim, Germany). Dimethylsulfoxide (DMSO), monoclonal antibody i.e anti poly ADP-ribose polymerase (anti-PARP), goat anti mouse alkaline phosphtase conjugated antibody, caspase-3 and caspase-8 colorimetric assay kits and 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium) (MTT) were purchased from Sigma RBI, (St.Louis, MO, USA). RPMI-1640, foetal bovine serum and gentamicin were bought from GIBCO BRL Life technologies (Grand Island, NY, USA). The proteinase K, the sodium dodecyl sulfate (SDS), ribonuclease (RNase), Sarkosyl, Thiobarbituric Acid (TBA), and pyridine were purchased from Sigma Aldrich Co (St. Louis, MO, USA). Acrylamide and bisacrylamide, 5-bromo-4 chloro-3 indolyl phosphate (BCIP)/nitro blue tetrazolium (NBT) and tween 20, were purchased from promega (Madison, Wisconsin, USA). Ethidium bromide (EtBr) and bromophenol blue were purchased from Merck (Darmstadt, Hesse, Germany). Agarose and ployvinylidene difluoride (PVDF) membranes were obtained from Invitrogen, life technologies (Glasgow, UK). Acetic acid was procured from Panreac (Barcelone, Espagne).
Leaves of N. retusa were collected from saline soils in Sahline, a region situated in mid-Tunisia, in December 2006. Identification was carried out by Pr. M. Cheieb (Department of Botany, Faculty of Sciences, University of Sfax, Sfax, Tunisia), according to the Flora of Tunisia  and Contribution to ethnobotanical study of the flora of Tunisia . A voucher specimen (N.r-12.06) was kept in our laboratory for future reference. The leaves were hade dried, powdered, and stored in a tightly closed container for further use.
Preparation of plant extracts
Three hundred and fifty grams of powder, from dried leaves, were sequentially extracted in a Soxhlet apparatus (6 h) (AM Glassware, Aberdeen, Scotland, United Kingdom) with hexane, chloroform, ethyl acetate and methanol solvents. We obtained the correspondent extracts for each solvent. Hexane (Hex), chloroform (Chl) and methanol (MeOH) extracts, with different polarities, were concentrated to dryness and the residues were kept at 4°C. Then, each extract was resuspended in dimethyl sulfoxide solvent (DMSO).
Preliminary phytochemical analysis and determination of Total Polyphenol, Flavonoid, Tannins and Sterol Contents
However, flavonoid content was determined according to the modified method of Zhishen et al.. The Quercetin (0.05 mg/ml) was used as a standard compound.
MO: Weight filter (mg), Mf: Weight of filter with the precipitate (mg).
where ε; molar extinction coefficient (= l g-1 cm-1) of tannic acid (= 3.27 l g-1 cm-1)
Human chronic myelogenous leukemia cell line K562 was obtained from the American Type Culture Collection (Rockville, MD). Cells were cultivated in RPMI-1640 medium supplemented with 10% (v/v) foetal calf serum, 0.1 mg/ml gentamicin and 2 mM L-glutamine as a complete growth medium and were incubated at 37)°C in an incubator with 5% CO2 in a humidified atmosphere. Every two days the cells were subcultured by splitting the culture with fresh medium.
Assay for cytotoxic activity
Cytotoxicity of Nitraria retusa extracts against K562 leukemia cells was estimated by the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay, based on the reduction of the MTT by mitochondrial dehydrogenases in viable cells. The resulting blue formazan product is measured spectrophotometrically . Cells were seeded in a 96-well plate at a concentration of 5 × 104 cells/well and incubated at 37°C for 24 h in a 5% CO2 enriched atmosphere. The extracts were firstly dissolved in 1% DMSO, then in the cell growth medium. Cells were incubated again at 37°C for 48 h with each of the tested extract at concentrations ranging from 10 to 800 μg/ml. Next, the medium was removed and cells in each well were incubated with 50 μl of MTT solution (5 mg/ml) at 37°C for 4 h. MTT solution was then discarded and 50 μl of 100% DMSO were added to dissolve the insoluble formazan crystal. The optical density was measured at 540 nm. Each drug concentration was tested in triplicate.
The cytotoxic effects of the extracts were estimated in terms of cell population growth inhibition percentage and expressed as IC50 which is the concentration of extract that reduces the absorbance of the treated cells by 50% with reference to the control (cells treated with DMSO). The IC50 values were graphically obtained from the dose-response curves. We determined IC50 values when cytotoxicity resulted more than 50% at screening concentrations.
Evaluation of lipid peroxidation induction provoked by H2O2, using the thiobarbituric acid reactive substances (TBARS) assay
The method known as thiobarbituric acid reactive species (TBARS) assay, concerns the spectrophotometric measurement of the pink pigment produced through reaction of thiobarbituric acid (TBA) with malondialdehyde (MDA) and other secondary lipid peroxidation products. TBARS were determined by previously described assay . The cells (3.5 107 cells/ml) were exposed to various concentrations of each compounds (200, 400 and 800 μg/ml of MeOH extract, 150, 300 and 600 μg/ml of hexane extract and 190, 380 and 760 μg/ml of chloroform extract) in the incubation medium during 2 h, followed by incubation with 75 mM H2O2 for 2 h. The ranges of doses of different tested compounds were chosen on basis of their cytotoxic activity. The cells were washed with PBS, pelleted and homogenized in 1.15% KCl. Samples were combined with 0.2 ml of 8.1% SDS, 1.5 ml of 20% acetic acid and 1.5 ml of 0.8% thiobarbituric acid. The mixture was brought to a final volume of 4.0 ml with distilled water and heated to 95°C for 120 min. After cooling for 10 min on ice, 5.0 ml of a mixture of n-butanol and pyridine (15:1 v/v) were added to each sample, and the mixture was shaken vigorously. After centrifugation at 825 g for 10 min, the supernatant fraction was isolated and the absorbance was measured at 532 nm. Lipid peroxidation effect was expressed as equivalent of MDA. Data were reported as mean ± SD for triplicate determinations.
DNA fragmentation analysis
DNA fragmentation was analysed by agarose gel electrophoresis as described by Wang et al, with slight modifications. K562 cells (1.5 106 cells/ml) were exposed to various concentrations of each compounds (200, 400 and 800 μg/ml of MeOH extract, 150, 300 and 600 μg/ml of hexane extract and 190, 380 and 760 μg/ml of chloroform extract) for 24 and 48 h and harvested by centrifugation. Cell pellets were disolvedin 200 μl of lysis buffer (50 mM Tris-HCl, pH 8.0, 10 mM EDTA, 0.5% N-Lauroyl Sarcosine Sodium Salt) at room temperature for 1 h then centrifuged at 12 000 g for 20 min at 4°C. The supernatant was incubated overnight at 56°C with 250 μg/ml proteinase K. Cell lysates were then treated with 2 mg/ml RNase A and incubated at 56°C for 2 h. DNA was extracted with chloroform/phenol/isoamyl alcohol (24/25/1, v/v/v) and precipitated from the aqueous phase by centrifugation at 14 000 g for 30 min at 0°C. The DNA solution was transferred to 1.5% agarose gel and electrophoresis was carried out at 67 V for 3/4 h with TAE (Tris 40 mM, sodium acetate 20 mM, EDTA 1 mM) as the running buffer. DNA in the gel was visualized with ethidium bromide (0.5 μg/ml) under UV light.
Western blot analysis
K562 cells (1.5 106 cells/ml) were exposed to various concentrations of each compounds (200, 400 and 800 μg/ml of MeOH extract, 150, 300 and 600 μg/ml of hexane extract and 190, 380 and 760 μg/ml of chloroform extract) for 6, 24 and 48 h. Cells were centrifuged at 3000 rpm for 8 min at 25°C and lysed with a lysis buffer (62.5 mM Tris Hcl and 6 mM urea, pH = 6.8). Protein concentrations were determined in cell lysates using the Bradford method . Equal amounts of proteins (40 mg) were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), and transferred into PVDF membrane, which was blocked with 5% non-fat milk in 0.1% Tween 20-phosphate buffer salin (PBST) overnight at 4°C. Membranes were then incubated with a primary antibody anti-PARP at a 1:100 dilution for 2 h at room temperature. The membrane was then washed and incubated with a goat anti-mouse alkaline phosphatase-conjugated antibody at 1:7500 dilution for 1 h.
Next, the membrane was washed and the chromogenic substrate BCIP/NBT was added to localise antibody binding proteins. Protein levels were determined by computer assisted densitometric analysis (Densitometer, GS-800, Bio.Rad Quantity One).
Investigation of caspase-3 and caspase-8 induction
The cells were cultured (106 cells/ml) in 25 cm2 flasks for 24 h in the absence or the presence of extracts at 37°C. Controls were performed at the same time with 0, 5% DMSO. The cells were harvested and centrifuged at 600 × g and the pellets were incubated in ice cold lysis buffer (250 mM HEPES, pH 7.4, 25 mM CHAPS, 25 mM DTT) for 15 min, then they were centrifuged at 16000 × g for 20 min. Supernatants (cell extracts eventually containing caspase-3 and caspase-8) were retrieved and aliquots corresponding to 50 μg total protein were incubated along with acetylated tetrapeptide Ac-DEVD substrate labelled with the chromophore p-nitroaniline (p- NA), 2 mM, for caspase 3 assay, or Ac-IETD-p-NA substrate, 2 mM, for caspase 8 assay, in the presence of each caspase buffer in a 96-well flat bottomed microplate.
In the presence of active caspase-3 and caspase-8, cleavage and release of p-NA from the substrate occurs. Free p- NA produces a yellow colour detected spectrophotometrically at 405 nm against a blank performed at the same time and containing assay buffer (200 mM HEPES, pH 7.4, 1% CHAPS, 50 mM DTT, 20 mM EDTA, for caspase 3 assay) and (200 mM HEPES, pH 7.4, 1% CHAPS, 50 mM DTT, 20 mM EDTA, 50% sucrose for caspase 8 assay) and substrate but without cell lysate. A standard curve was performed in order to determine the correspondence between absorbance and p- NA concentration, then the results were expressed as caspase-3 and caspase-8 specific activity (μmol p-NA per min/ml protein) calculated as indicated by the manufacturer (Caspase-3, caspase-8, assay kit colorimetric, Sigma.)
Data were collected and expressed as the mean ± standard deviation of three independent experiments and analyzed for statistical significance from control. The data were tested for statistical differences by one-way ANOVA followed by student test using statistica. The criterion for significance was set at p < 0.05.
Phytochemical study and determination of extract yield, total polyphenol, flavonoid, tannin and sterol contents of Nitraria retusa leaf extracts
Phytochemical screening of extracts from Nitraria retusa:
Quantitative phytochemical screening of extracts from Nitraria retusa leaves
Extract content (%)
18.84 ± 0.002
14.82 ± 0.009
10.03 ± 0.008
3.3 ± 0.006
31 ± 0.02
11.75 ± 0.007
Evaluation of the cytotoxicity of extracts
Effect of extracts on lipid peroxidation induced by H2O2
Lipid peroxidation inhibitory activity in K562 cells treated with Hex, Chl and MeOH extracts against H2O2 (50 μM) induced peroxidation.
Concentration (μg/ml) a
Concentration of MDA (nM)
260 ± 16
230 ± 11
150 ± 10
245 ± 15
220 ± 14
190 ± 20
250 ± 10
265 ± 5
315 ± 10
H 2 O 2
225 ± 5
Induction of apoptotic DNA fragmentation by Nitraria retusa extracts on leukemia cells
Effect of Nitraria retusa extracts on the proteolysis of PARP
DNA fragmentation is often associated with the activation of a family of cysteine proteases, the caspases. Caspase-3, in particular, seems to play an important role in several models of apoptosis . To confirm the apoptotic process, generally admitted when a ladder DNA fragmentation profile is observed, we investigated the enzymatic activation of apoptotic proteins by measuring the cleavage of PARP (116 kDa), which is a caspase-3 substrate, into fragments of 85 and 31 kDa.
Caspase-3 and caspase-8 activation assay
The relationship between concentration of extracts and their antiproliferative effect on K562 cells was investigated by MTT assay. Hex, MeOH and Chl extracts possess an inhibitory effect on K562 cell proliferation. The strong antiproliferative activity of Hex may be due to the presence of sterols, which are known to induce antiproliferative effect . In fact, Phytosterols seem to act through multiple mechanisms of action, including inhibition of carcinogen production, cancer-cell growth, angiogenesis, invasion and metastasis, and through the promotion of apoptosis of cancerous cells stress . Inhibition of proliferation of K562 cells exhibited by Chl extract may be attributed to the presence of specific components such as polyphenols . Besides, some studies have shown that polyphenols are able to influence a variety of cell function by modulating cell signalling , altering proliferation and induction anti-proliferative effect in cancer cell lines . In fact, polyphenols exhibited antiproliferative effects on various cancerous human cell lines, for example leukaemia cells  and ovarian cancer cells . However, minor components could also contribute to the antiproliferative effect of these extracts; they may be involved in some types of synergism with other active compounds . The weak antiproliferative activity exhibited by the MeOH extract should be ascribed to its low polyphenol content fraction, if compared to Hex and Chl extracts. Nonetheless, polyphenols contained in MeOH extract should be different from those Chl extract as far as they were extracted with solvents having different polarities.
Membrane lipids are rich in unsaturated fatty acids that are most susceptible to oxidative processes. It is generally thought that the inhibition of lipid peroxidation by antioxidants may be due to their free radical-scavenging activities. The data obtained showed that Hex and Chl extracts exhibited better antioxidant activity at the highest tested concentrations than MeOH extract. We can deduce that sterols which are the main constituents of Hex extract, and sterol and polyphenolic compounds which are the main constituents of Chl extract, should participate in the protective effect, of these two extracts at the highest tested concentration, against lipid peroxidation induced by H2O2 in K562 cells. This protective effect is absent at the lowest tested concentrations of the above mentioned extracts. In fact the lowest tested concentrations of both extracts did not exhibit a protective effect against lipid peroxidation induced by H2O2, as far as the rates of MDA formation obtained when cells are incubated with both H2O2 and each of these concentrations are in the same range as those obtained when cells are incubated with H2O2 alone. These results are in accordance with those of Mahoney and Graf  who showed that ascorbic acid initiates the formation of OH• at low concentrations and scavenges radicals at high concentrations. It is possible that these compounds inhibit the free radicals and ROS produced by oxidation and redox-cycling started by H2O2 and leading to cell lipid peroxidation. Although MeOH extract contains flavonoids, tannins and polyphenols, it exhibits no protective effect against lipid peroxidation induces by H2O2. We believe that its weak polyphenol content and absence of sterols may explain the absence of protective effect against lipid peroxidation. In fact, polyphenols are an important group of pharmacologically active compounds, they are considered to be the most active antioxidant derivatives in plants. However, it has been shown that the phenolic content does not necessarily follow the antioxidant activity. Antioxidant activity is generally the result of the combined activity of a wide range of compounds, including phenolics, peptides, organic acids and other components .
However some flavonoids and polyphenols as catechol or pyrogallol may exhibit a prooxidant activity by generating free radicals, under certain conditions .
Our hypothesis that sterol contents of both Chl and Hex extracts are involved in their antioxidant effect is in accordance with the results described by Wang et al., who reported the antioxidant capacity of some plant sterols. In fact, it is possible that sterols inhibit free radicals and ROS produced by oxidation and redox-cycling, as reported by Argolo et al.  and Ben Mansour et al. .
An oxidative stress probably provoked by extracts would lead to the formation of free radicals being able to induce cellular stress, as cell DNA degradation and membrane permeabilization especially mitochondrial membranes, provoking liberation of pro-apoptotic proteins as pro-caspases, cytochrome C, which interact with proteins as Apaf-1 and pro-caspase-9 forming, in the presence of ATP, a multi-protein complex named "apoptosome". This complex should allow the cleavage of caspase-9. This later will activated executive caspases as capases-3, 7 and 8, involved in the induction of apoptotic process. On the other hand, cell stress induced by free radicals should also activate a pro-apoptotic gene family (Bax, Bak, Bid, Bad, Bim) or inactivate anti-apoptotic genes as Bcl-2, Bcl-xL , inducing thus cell apoptotic process.
The antiproliferative activity of MeOH extract from N. retusa should be attributed to the presence of specific types of flavonoids and polyphenols . In fact, some studies have shown that flavonoids  and tannins  are able to altering proliferation in cancer cell lines. Previous studies have shown that flavonoids induce apoptosis of various tumor cells including K562. This effect has also been observed in other tumor cell lines from gastric, colon and lung carcinomas . In addition, flavonoids also inhibited tumor growth through cell cycle arrest and induced apoptosis through a p53-dependent mechanism .
Although N. retusa extracts should contain some antioxidant entities (revealed by antiradical properties of the same N. retusa extracts against several free radicals; data not shown), we believe that this dual property, reported also in other works  is not in contradiction with our aforementioned deduction, as several researchers have shown that antioxidants, such as retinoids and vitamin E, produce genetic changes that cause apoptosis in cancer cells by mechanisms other than a direct antioxidant effect .
The typical DNA fragmentation pattern which is considered as the hallmark of apoptosis, was observed in cancerous cells treated with the tested N.retusa leaf extracts. As far as the extracts tested in the present study were in crude form and probably contained many compounds which may well act synergistically. It is not possible to say which compounds are responsible for the observed effects. However, our data suggest that the biological effects exhibited by this plant, under these experimental conditions, could be related to an overall effect of the tannins, flavonoids, sterols and coumarins present in these extracts. Phenolic compounds were postulated as effective in inducing apoptosis and as anticancer agents .
Likewise steroids showed growth inhibition of human prostate cancer PC-3 cells, being effective in inducing apoptosis .
As far as we obtained at all tested concentrations of different extracts, the activation of caspases 3 and 8, as well as the more effective PARP cleavage effect, after 48 h of incubation, we can deduce that the tested extracts should provoke a cytotoxic effect towards K562 cells by activating the extrinsic pathway of apoptosis. In fact, activation of caspase 8 leads to the activation of caspase 3 and subsequently induces PARP cleavage (the 116 kDa band disappears in favour of 85 kDa band) and DNA fragmentation (ladder electrophoretic profile). However we can not exclude the participation of other pathways in the apoptotic effect exhibited by these extracts.
In summary, N.retusa leaf extracts appear to contain compounds with, antiproliferative and apoptotic properties. The three tested extracts induced apoptotic effect by the activation of the extrinsic apoptotic pathway. As apoptosis has become a new therapeutic target in cancer research, it appears reasonable to suggest that N.retusa may have potential as an agent of chemotherapeutic and cytostatic activity in human leukaemia lymphoma.
The authors acknowledge the ''Ministry of Higher Education, Scientific Research and Technology, Tunisia", for the support of this study and to the Pr Lotfi Chouchene and all members of the laboratory of Immunology, Faculty of Medicine, Monastir, Tunisia.
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