Printer Friendly

Inhibitory effect of Abrus abrin-derived peptide fraction against Dalton's lymphoma ascites model.


Peptides derived from larger molecules that are important modulators in cancer regression are becoming leads for development of therapeutic drugs. It has been reported that Abrus abrin, isolated from the seeds of Abrus precatorius, showed in vitro and in vivo antitumor properties by the induction of apoptosis. The present study was designed to evaluate the in vivo therapeutic effectiveness of abrin-derived peptide (ABP) fraction in Dalton's lymphoma (DL) mice model. The lethal dose ([LD.sub.50]) of ABP was found to be 2.25mg/kg body weight and further the acute toxicity was determined with sublethal doses in normal mice. The acute toxicity like body weight, peripheral blood cell count, lympho-hematological and biochemical parameters remained unaffected till 200ug/kg body weight of ABP. The sublethal doses of ABP showed very significant growth inhibitory properties in vivo DL mice model. There were 24%, 70.8% and 89.7% reductions in DL cell survival in 25, 50 and 100 [micro]g/kg body weight of ABP, respectively. Analysis of the growth inhibitory mechanism in DL cells revealed nuclear fragmentation, and condensation with the appearance of the sub-[G.sub.0]/[G.sub.1] peak is indicative of apoptosis. Further, the Western blotting showed that apoptosis was mediated by the reduction in the ratio of Bcl-2 and Bax protein expression, and activation of caspase-3 through the release of cytochrome c in DL cells. Kaplan-Meier survival analysis showed an effective antitumor response (104.6 increase in life span (ILS) %) with a dose of 100 [micro]g/kg body weight.

[C] 2008 Elsevier GmbH. All rights reserved.

Keywords: Abrin-derived peptide (ABP); Abrus precatorius: Dalton's lymphoma (DL); Apoptosis


Abrus abrin, isolated from the seeds of Abrus precatorius, is a hetero-dimeric glycoprotein of 63-kDa molecular weight, composed of two nonidentical polypeptide chains (A- and B-chain) cross-linked through a single disulfide bond (Tahirov et al., 1995). It belongs to the type II ribosome inactivating protein family (RIPII) with a protein synthesis inhibitory concentration ([IC.sub.50]) of 10ng/ml and a lethal dose ([LD.sub.50]) of 20 [micro]g/kg body weight in mice (Hegde et al., 1991; Stirpe et al., 1992).The abrin binds to the cell-surface receptors containing terminal galactose through the B subunit, enter cells by receptor-mediated endocytosis and A subunit inhibits the protein synthesis by modification of the ribosomal subunits of the cells. In addition, abrin induces apoptosis followed by the inhibition of protein synthesis. The apoptosis is triggered through intrinsic mitochondrial pathway by caspase 3-activation involving mitochondrial membrane potential damage and reactive oxygen species production (Narayanan et al., 2004, 2005). Abrin is 10-100 times more toxic to some transformed cell lines than to normal cells. The selective antiproliferative properties of abrin are attractive as a potential anticancer agent (Moriwaki et al., 2000; De Mejia and Prisecaru, 2005). The antitumor properties of abrin have been reported in different models (Lin et al., 1982; Ramnath et al., 2002). The in vivo tumoricidal property of abrin against Dalton's lymphoma (DL)-and Ehrlich's ascites carcinoma-induced tumors is thought to mediate through apoptosis (Ramnath et al., 2007).

Peptides derived from larger molecules that are important modulators in cancer regression are becoming leads for the development of therapeutic drugs (Bhutia and Maiti, 2008). In our previous study, we have reported that Abrus agglutinin-derived peptide fraction named 10kMPP induced apoptosis in cervical cancer cells through the intrinsic pathway (Bhutia et al., 2008). Here we have explored the antitumor activity of a peptide fraction isolated from toxic lectin abrin present in the seed of Abrus. The study was designed to evaluate the in vivo antitumor properties of abrin-derived peptides (ABP) fraction obtained from 10 kD molecular weight cut-off membrane permeate of tryptic-digested abrin in a DL mice model. Further, we have investigated to determine the possible mechanisms of cell death elicited by ABP on DL cells.

Material and methods


MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetra-zolium), dimethylsulfoxide (DMSO), propidium iodide (PI), RNase A, trypsin, rhodamine123, dihydrorhoda-mine 123 (DRH123), and agarose were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Amicon Ultra (10 kDa) was purchased from Millipore, India. Fetal bovine serum (FBS) was from Hyclone, and RPMI-1640 media, was from Invitrogen, India.

Enzymic hydrolysate of abrin and isolation ABP

The ABP was isolated and characterized as previously reported (Bhutia et al., 2008). Briefly, trypsin (10 x [10.sup.3] BAEEunit/mg of protein) was added at a ratio of 1:50 to abrin solution (1.0 mg protein/ml in 0.01 M phosphate buffer saline [PBS]). Trypsinization was carried out at a temperature of 37 [degrees]C overnight. ABP was isolated using a 10-kDa molecular weight cutoff membrane (Amicon Ultra, Millipore). The peptide fractions were lyophilized and the concentration was quantified using Fluorescamine with glycine as standard (Udenfriend et al., 1972). MALDI-ToF mass spectrum was obtained on a Voyager-DE[TM] PRO (Applied Biosystem) mass spectrometer equipped with a nitrogen laser operating at 337 nm. Mass spectra were recorded in a linear mode in positive ion detection using [alpha]-cyano-4-hydroxycinnamic acid (10 mg/ml) as the matrix.

MTT assay for DL cell viability

DL cells were harvested and the cell concentration was adjusted to 1 x [10.sup.5] cells/ml and cells were plated in 96-well flat-bottom culture plates and incubated with various concentrations of ABP. All cultures were incubated for 24, 48 and 72 h at 37 [degrees]C in a humidified incubator, which maintained a constant 5% [CO.sub.2]. Cell concentration was checked by MTT assay (Mosmann, 1983).

Animal experiment

Mice and tumor system

Female Swiss albino mice (20 [+ or -] 2 g, 6-8 weeks old) were used for acute toxicity and anticancer study. Mice were housed in open-top cages and maintained on food and water ad labium. Room temperature was maintained at 22 [+ or - ] 2[degrees]C with light and dark cycle of 14/10 h. All animal experiments were performed according to the rules of "Committee for the purpose of control and supervision of experiments on animals, Ministry of Environment and Forests, government of India" and Institutional Animal Ethics Committee, Indian Institute of Technology, Kharagpur, PIN-721302. DL is maintained in ascetic form by serial transplantation in Swiss albino mice or in vitro cell culture system by serial passage. Irrespective of whether the cells are obtained from in vitro culture or from ascetic fluid, they exhibited typical phenotypic features.

Assay of acute toxicity

The [LD.sub.50] of ABP was determined by the method of Litchfield and Wilcox (Litchfied and Wilcoxon, 1949) and [LD.sub.50] was found to be 2.25 mg/kg body weight. Later, acute toxicity assay was performed with sublethal doses according to the method described in Ghosh et al, (2006). Female mice (5-7 weeks old, 20-22 g) were randomly divided into 5 groups with a control and 5 in mice each group. Different sublethal doses of ABP in the range of 25-500 [micro]g/kg body weight were given intraper-itoneally (i.p.) everyday to each group till 10 days and the control mice received PBS. The body weight of mice was taken daily to monitor the toxicity effect. On the 11th day, all mice were sacrificed and different parameters of toxicity like blood cell count, total hemoglobin content and serum enzymes were measured. To examine the effect of ABP administration on spleen and thymus, these organs are harvested and the number of nucleated cells determined with a hemocytometer.

Evaluation of antitumor activity in DL-bearing mice model

The tumor growth inhibition study was carried out in a DL-bearing mice model (Ghosh and Maiti, 2007). Under sterile condition, 0.5 ml of DL cell suspension (2 x [10.sup.6] cells/ml) was inoculated i.p. to each mouse at day zero. The inoculated mice were divided into four groups (6 mice in each group) having a DL-bearing control group and three treatment groups. After 3 days of tumor inoculation, different doses (25-100 [micro]g/kg body weight) of ABP were administrated to each treatment group for 6 days and the control mice received PBS. Cyclophosphamide (Ledoxan[R], Batch No. 40108) from Dabur Pharma Limited, New Delhi (6 mg/kgb.wt., i.p. for 6 days) was used as the standard reference drug (Ghosh et al., 1997; Kumar and Kuttan. 2005). On day 10, all mice were sacrificed and the total accumulated ascites fluid volume and tumor cells in the peritoneal cavity of each animal were harvested and total ascites cell count was determined.

4',6-Diamidino-2-phenylindole dihydrochloride (DAPI) staining of in vivo DL cells

Nuclear staining was performed according to the method previously described (Kim et al., 2006). In brief, DL cells either untreated or treated group of mice with ABP were smeared on a clean glass slide, cells were fixed with 3.7% formaldehyde for 15min. permeabilized with 0.1% Triton X-100 and stained with 1 [micro]g/ml DAPI for 5 min at 37 [degrees]C. The cells were then washed with PBS and examined by fluorescence microscopy (Olympus IX 70).

Cell-cycle analysis of in vivo DL cells

The cells were harvested and fixed in 70% ethanol (stored at -20 [degrees]C). Then, the cells were washed with ice-cold PBS (10 mM, pH 7.4) and resuspended in 200 ul of PBS followed by incubation with 20 [micro]l DNase-free RNase (10 mg/ml) and 20 [micro]l of DNA intercalating dye PI (1 mg/ml) at 37 [degrees]C for 1 h in dark. Apoptotic cells were determined by their hypochromic sub-diploid staining profiles. The distribution of cells in the different cell-cycle phases was analyzed from the DNA histogram using a Becton-Dickinson FACS Caliber flow cytometer and Cell Quest software (Evans et al., 2000).

Western blot analysis

DL cells were harvested from DL-bearing mice, followed by the extraction of proteins. A lysis buffer containing 50 mM Tris-HCl (pH 7.6), 25 mM NaCl, 0.5% Triton X-100 and 2mM dithiothreitol (DTT) was used to extract the cytosolic proteins. The extraction buffer was supplemented with 1 x cocktail protease inhibitors (Roche Applied Science), 1 mM phenyl-methylsulfonyl fluoride (PMSF), lOmM sodium fluoride and 1 mM sodium orthovanadate. Proteins (20-50 (ig) were subjected to electrophoresis through the distinct percentages of SDS polyacrylamide gel, followed by the transfer of proteins onto the poly-vinylidene difluoride membranes. The membranes were blocked with a buffer containing 5% non-fat milk in PBST (1 x PBS, 0.2% Tween 20) at room temperature for 1 h, and subsequently incubated in the same buffer containing various primary antibodies (Bcl-2, Bax, caspase-3, cytochrome c, [beta]-actin from BD Bioscience) at 1-2 [micro]g/ml. Membranes were then incubated with anti-rabbit and/or anti-mouse antibodies conjugated with a horseradish peroxidase at room temperature for 1 h. The proteins of interest were detected using the chemilumi-nescence method (Perkin-Elmer Life Sciences) (Mandal et al., 2006).

DNA fragmentation assay

For the DNA fragmentation assay, low-molecular-weight DNA was isolated (Mandal et al., 2006). Briefly, about 1 x [10.sup.7] DL cells from both control and treated groups were washed three times with PBS, and resuspended in 1 ml of lysis buffer (20 mM Tris-HCl [pH 8], 10 mM ethylenediaminetetraacetie acid [pH 8] and 0.5% Triton X-100). After incubation in ice for 30 min, the lysates were spun at 12,000 rpm in a microcentrifuge for 10 min. Low-molecular-weight DNA in the supernatant was extracted with equal volumes of phenol and chloroform for 1h at 4 [degrees]C. Ammonium acetate (2M) was added to the aqueous phase, and DNA was precipitated with two volumes of ethanol at -20 [degrees]C overnight. DNA was treated with RNase A (1 mg/ml) at 37 [degrees]C for 1 h, and total DNA was analyzed using 1.5% agarose gel and visualized by ethidium bromide staining of the gel.

Study of survival of tumor-bearing host

Mice were transplanted i.p. with DL cells (1 x [10.sup.6] cells/mouse) with 10 in each group. The effect of i.p. administration of ABP on survival and progression of tumor on DL-bearing mice was investigated. After 3 days of tumor transplantation, ABP was administrated to DL-bearing mice through the i.p. route at a dose of 100 ug/kg body weight on a daily basis for 6 days and left till death. Animal survival was recorded till 50 days. Median survival time (MST) and % increase in life span (ILS) were calculated from the mortality data within the observation period. The ILS was evaluated by calculating the MST of the treated (T) and control (C) groups and expressed as ILS value [(T/C - 1) x 100]. The ILS value of >25% is considered for significant activity in these tumors. The survival curve was plotted using the Kaplan Meier method. For this experiment, mice were weighed periodically during and after the therapy. The results are expressed as percentage increase in the body weight using the relationship [(a - b/a) x 100] where a and b are the final body weight and initial body weight of tumor transplantation. The survival curve was plotted using the Kaplan-Meier method (Ghosh and Maiti, 2007; Ghosh et al., 2006).

Statistical analysis

All data were given as the mean + S.D. The survival study was analyzed by the Kaplan-Meier method. Experimental results were analyzed by Student's t test. p < 0.05 was considered as the level of significance for values obtained for treated compound to control.


Cytotoxic activity of ABP from Abrus abrin against DL cells

The ABP fraction was separated from intact abrin by 10kDa molecular weight cut-off membrane and this peptide fraction, which was about 30-40% of the total protein content, was assayed for biological activity. Fig. 1 showed the positive mass spectrum of ABP fraction. The accurate relative molecular mass of the peptides present in this fraction, deduced from the m/z value of (M + H) + by subtraction of one mass unit for the attached proton, were in the range of 600-1500 Da. In a primary screening test, the effect of ABP on the survival rate of DL cells was assessed by MTT assay. We found ABP induced a distinct dose- and time-dependent diminution of cell viability (Fig. 2A).



Effect of ABP on general toxicity in mice

To investigate the effect of ABP on clinically relevant parameters of toxicity, such as body weight, peripheral blood cell count, cellularity of lympho-hematopoietic tissues, biochemical parameters in sera, mice were i.p. administered different sublethal doses of ABP for 10 days. The body weight, peripheral blood cell count, lympho-hematological, biochemical parameter and nucleated cells in different organs remained unaffected with 200 [micro]g/kg body weight and lower. At an administration of 500 [micro]g/kg body weight the weight lost was 48% of their body weight prior to the administration of ABP. Similarly, the hematological parameters like RBC count, WBC count and hemoglobin content were significantly decreased about 35%, 42% and 47%, respectively. Further, biochemical parameters like serum glutamic oxaloacetic transaminase (SGOT), serum glutamic pyruvic transaminase (SGPT), serum urea and serum glucose were significantly elevated at this dose (Table 1).
Table 1. Effect of sublethal doses of ABP on general toxicity in mice

Group               Control            Treated ([micro]g/kg body wt)

                                           25                  50

Initial body    20.2 [+ or -] 3.2    19.64 [+ or -]       22.3 [+ or -]
weight (g)                           1.5                  1.9

Final body     22.87 [+ or -]         21.2 [+ or -] 3.3  24.34 [+ or -]
weight (g)       2.5                                     1.80

RBC(x           8.31 [+ or -] 0.5     8.35 [+ or -]      11.67 [+ or -]
[10.sup.12]                           0.27               0.4

WBC(x            5.0 [+ or -] 0.22    6.55 [+ or -] 0.3    9.1 [+ or -]
[10.sup.9]                                                 0.45

Splenocytes     0.75 [+ or -] 0.2     0.87 [+ or -]       1.23 [+ or -]
(x [10.sup.8]                         0.24                0.40

Thymocytes (x   0.52 [+ or -]         1.24 [+ or -] 0.2   2.49 [+ or -]
[10.sup.8]      0.24                                      0.12

Hemoglobin     10.15 [+ or -]         14.0 [+ or -] 0.2   18.4 [+ or -]
(g/dl)          0.87                                      0.6

Serum glucose   10.3 [+ or -]         9.80 [+ or -]      11.54 [+ or -]
(mg/dl)          0.37                0.56                0.76

Serum urea      16.9 [+ or -]         17.0 [+ or -]      16.22 [+ or -]
(mg/dl)         0.84                 0.55                0.43

SGOT(IU/L)      88.0 [+ or -] 9.7    102.0 [+ or -]       99.6 [+ or -]
                                     8.4                  7.8

SGPT(IU/L)       117 [+ or -] 7.5      103 [+ or -] 9.8  105.7 [+ or -]

Group                       Treated ([micro]g/kg body wt)

                      100                200                500

Initial body    20.3 [+ or -] 2.8   22.8 [+ or -] 0.8    23.4 [+ or -]
weight (g)                                               1.5

Final body      21.9 [+ or -] 1.2   25.0 [+ or -]        12.2 [+ or -]
weight (g)                          0.75                 1.45

RBC(x           8.58 [+ or -]       10.3 [+ or -] 0.5     5.4 [+ or -]
[10.sup.12]     0.25                                      0.77

WBC(x            6.7 [+ or -] 0.33   5.9 [+ or -] 0.55    2.9 [+ or -]
[10.sup.9]                                                0.27

Splenocytes      1.6 [+ or -] 0.09  0.78 [+ or -]        0.52 [+ or -]
(x [10.sup.8]                       0.17                 0.08

Thymocytes (x    1.4 [+ or -] 0.12  2.21 [+ or -]0.11     0.3 [+ or -]
[10.sup.8]                                                0.13

Hemoglobin      19.3 [+ or -]       19.9 [+ or -]        5.32 [+ or -]
(g/dl)          0.33                0.43                 0.25

Serum glucose  10.89 [+ or -]       11.0 [+ or -]        17.8 [+ or -]
(mg/dl)        0.4                  0.64                 0.37

Serum urea      18.5 [+ or -] 0.5   17.9 [+ or -] 0.8    27.6 [+ or -]
(mg/dl)                                                   0.33

SGOT(IU/L)       103 [+ or -] 9.5   95.8 [+ or -] 7.8     121 [+ or -]

SGPT(IU/L)       118 [+ or -] 12.5   110 [+ or -] 11.7  170.5 [+ or -]

In vivo tumor growth inhibition through the induction of apoptosis

The antitumor effect of ABP was checked in vivo in a DL-bearing ascites mice model. The inhibitory effect of ABP on DL cells in vivo was examined in terms of the total number of cells and volume of ascites in mice treated with vehicle or compounds. The compound treatments showed significant decrease in ascites volume and cell number compared to control (p < 0.01) (Table 2). It was found that there was 24%, 70.8% and 89.7% reduction in cancer cell survival on the 10th day in dose of 25, 50 and 100 [micro]g/kg body weight, respectively, as compared to 65.4% cancer cell inhibition with 6 mg/kg body weight of cyclophosphamide (Fig. 2B).
Table 2. Antitumor properties of ABP on DL bearing mice

Group                 Total tumor           Packed cell         % of
                       cell count           volume (ml)      inhibition
                     (x [10.sup.7])

Control              25.7 [+ or -] 1.3    4.8 [+ or -] 0.55     100

Cyclophosphamide (6  8.89 [+ or -] 1.5   1.77 [+ or -] 0.4      65.4
mg/kg body wt)

25 [micro]g/kg body  19.5 [+ or -] 2.15   3.9 [+ or -] 0.38     24.0

50 [micro]g/kg body  7.50 [+ or -] 0.67  1.24 [+ or -] 0.3      70.8

100 [micro]g/kg      2.64 [+ or -] 0.35   0.4 [+ or -] 0.14     89.7
body weight

An attempt was made to identify whether the ABP-induced inhibition of DL cell growth was through the induction of apoptosis. The DAPI staining of treated DL cells showed typical apoptotic morphology with condensed nuclei, membrane blebbing and formation of apoptotic bodies compared to the untreated DL cells (Fig. 3). Further, apoptotic cells can be recognized by flow cytometry through their diminished stainability with the DNA-specific fluorochrome PI, in which the hypodiploid population can be quantified by DNA content frequency histograms (sub-G1 peak). As shown in Fig. 4, the mean apoptotic population of DL cells was 12.54% under control conditions, while it was increased to 24.42%, 39.74% and 96.20% after treatment with a dose of 25, 50 and 100 [micro]g/kg body weight, respectively. These results suggested that ABP retards the growth of DL cells by arresting cell-cycle progression and apoptosis induction.



The Western analysis was carried out in apoptosis-induced DL cells. The Western blot indicated that ABP was involved in the increase in the levels of pro-apoptotic protein Bax and a concomitant decrease in the levels of anti-apoptotic protein Bcl-2, thereby increasing the Bax/Bcl-2 ratio that favors apoptosis. Cytochrome c is localized in the intermembrane space and loosely attached to the surface of the inner mitochondrial membrane. In response to a variety of apoptosis-inducing agents, cytochrome c is released from mitochondria to the cytosol and activates the downstream molecules. The Western blot showed apoptosis was mediated through the release of cytochrome c from mitochondria to cytocol of treated DL cells. The involvement of caspase-3 was also demonstrated in Western blot and was found to be activated in the treated group (Fig. 5A).


The final step is the internucleosomal degradation of DNA, with the appearance of DNA ladders due to the activation of nuclear endonucleases. Cell genomic DNA showed the typical formation of DNA fragments as ladders in a dose-dependent manner, which is the biochemical hallmark of apoptosis (Fig. 5B).

ABP increased the survival rate of DL tumor-bearing mice

To determine the survival effect of ABP, DL tumor-bearing mice were treated with 100 [micro]g/kg body weight for 6 days starting from 3 days after inoculation of tumor cells and left until death. When compared against the control group, the treatment group demonstrated a significant survival advantage (p < 0.0001 group 1, log rank test). All the animals (TV - 10) in controls developed tumor and died within 18-25 days whereas the treated group with restricted tumor growth died in within 39-49 days. The MST for control was 21.5 days. All the treatments produced significant increase in MST and % ILS compared to control (Table 3). The percentage of weight changes was not significant in the treated groups compared to the control. Survivals with associated p values are graphically depicted in Fig. 6.

Table 3. Effect of 10kMPP on survival rate of DL tumor-bearing mice

Group             MST (days)         AST (days)        ILS   p Value
                                                       (%)     (vs.

Control       21.5 [+ or -] 2.0    21.6 [+ or -] 1.5   -     1

Treated (100    44 [+ or -] 2.55  44.41 [+ or -] 1.9  104.6  < 0.0001
body wt)


Finding better candidates through activity-guided isolation of bioactive fractions and compounds from natural products using kinds of in vitro and in vivo bioassay systems is an efficient way of discovering new drugs from medicinal plants. In the present study, we investigated the antitumor activity of the Abrus ABP fraction in vitro as well as in vivo DL mice model. It was observed that antitumor activity was mediated through the induction of apoptosis. In addition, the peptide fraction has no general toxic effect to normal mice when the dose anticancer properties were reported.

Initially, it was shown that ABP decreased the viability of DL cells in a time- and dose-dependent manner in vitro. We have also found that ABP showed growth inhibition in several tumor and transformed cell lines by the induction of apoptosis (Unpublished data). Next, we investigated the general acute toxicity of ABP with sublethal dose in a mice model. Our studies demonstrate that treatment with lower and 200 [micro]g/kg body weight for 10 days does not induce any change in body weight, hematological toxicity or immune cells. But all these properties were affected at a dose of 500 [micro]g/kg body weight. The biochemical parameters like SGOT and SGPT were found to have no change at a dose of 200 [micro]g/kg body weight and lower, whereas these parameters were elevated at 500 [micro]g/kg body weight. These enzymes have their origin in the liver and release into blood in stressful conditions by tissue damage. The levels of serum urea and serum glucose are common indexes clinically, and reflect impaired renal function/ glomerular function. This result suggested that the toxic effect of the peptide fraction was reflected at a dose of 500 [micro]g/kg body weight without any susceptible death (Kumar et al., 2004; Ghosh et al., 2006).

Antitumor activities were well documented with peptides from different synthetic as well as natural sources (Liu et al., 2002; Papo et al., 2003; Floquet et al., 2004; Mizejewshi et al., 2006) in different tumor models. Our results on DL ascites tumor showed that the administration of the ABP fraction (100 [micro]g/kg body weight) for 6 days after 3 days of tumor transplantation produced effective antitumor response (104.6 ILS%). The compounds also exhibited corresponding reduction in mean ascites volume and cell number. The compound effectively reduced the ascites tumor burden and produced no side effects. Morphology of DL cells in the treated groups exhibited apoptotic bodies, nuclear condensation and internucleosomal fragmentation. Further, it was confirmed from the apoptotic phase in cell-cycle analysis and DNA fragmentation assay. All these results indicate that ABP inhibition of DL cell growth was due to the induction of apoptosis in DL cells (Bhutia et al., 2008).

In Western blot analysis, ABP was shown to activate a series of proteins involved in apoptosis. In our study, it was found that the reduced expression of Bcl-2 and increased expression of Bax protein, and activation of caspase-3 through the release of cytochrome c in DL cells resulting in functional alteration of the mitochondria, may be potentially involved in ABP-induced apoptotic responses (Prabhakar et al., 2006; Thippes-wamy and Salimath, 2007).

In conclusion, ABP decreases the tumor burden in DL mice model by the induction of apoptosis without having any side effect. Abrus ABP fraction contains some pro-apoptotic peptides, which are to be characterized along with its selective growth inhibitory mechanism.


National Doctoral Fellowship to Sujit K Bhutia from All council of Technical Education (AICTE), India [Award No. 1-10/FD/NDF-PG/(IIT-KH (01)/2005-06, 26th 2006] is acknowledged.


Bhutia, S.K., Maiti, T.K., 2008. Targeting tumors with peptides from natural sources. Trends Biotechnol., 210-217.

Bhutia, S.K., Mallick, S.K., Stevens, S.M., Prokai, L., Vishwanatha, J.K., Maiti, T.K., 2008. Induction of mitochondria-dependent apoptosis by Abrus agglutinin derived peptides in human cervical cancer cell. Toxicol. In Vitro 22, 344-351.

De Mejia, E.G., Prisecaru, V.I., 2005. Lectins as bioactive plant proteins: a potential in cancer treatment. Crit. Rev. Food Sci. Nutr. 45, 425-445.

Evans, D.L., Bishop, G.R., Jaso-Fricdmann, L., 2000. Methods for cell cycle analysis and detection of apoptosis of teleost cells. Methods Cell Sci. 22, 225-231.

Floquet, N., Pasco, S., Ramont, L., Derreumaux, P., Laronze, J.Y., Nuzillard, J.M., Maquart, F.X., Mix, A.J., Mon-boisse, J.C., 2004. The antitumor properties of the alpha3 (IV)-(185-203) peptide from the NCI domain of type IV collagen (tumstatin) are conformation-dependent. J. Biol. Chem. 279, 2091-2100.

Ghosh, D., Maiti, T.K., 2007. Effects of native and heat-denatured AAG on tumor-associated macrophages in Dalton's lymphoma mice, lmmunobiology 212, 667-673.

Ghosh, M., Bhattacharya, S., Sadhu, U., Dutta, S., Sanyal, U., 1997. Evaluation of beta-tethymustine, a new anticancer compound, in murine tumour models. Cancer Lett. 119, 7-12.

Ghosh, M., Talukdar, D., Ghosh, S., Bhattacharyya, N., Ray, M., Ray, S., 2006. In vivo assessment of toxicity and pharmacokinetics of methylglyoxal: augmentation of the curative effect of methylglyoxal on cancer-bearing mice by ascorbic acid and creatine. Toxicol. Appl. Pharmacol. 21, 45-58.

Hegde, R., Maiti, T.K., Podder, S.K., 1991. Purification and characterization of three toxins and two agglutinins from Abrus precatorhis seeds by using lactamyl-sepharose affinity chromatography. Anal. Biochem. 194, 101-109.

Kim, M.J., Kim, Y.J., Park, H.J., Chung, J.H., Leem, K.H., Kim, H.K., 2006. Apoptotic effect of red wine polyphenols on human colon cancer SNU-C4 cells. Food Chem. Toxicol. 4, 898-902.

Kumar, G., Banu, G.S., Pappa, P.V., Sundararajan, M., Pandian, M.R., 2004. Hepatoprotective activity of Tri-anthema portulacastrum L. against paracetamol and thioa-cetamide intoxication in albino rats. J. Ethnopharmacol. 92, 37-10.

Kumar, K.B.H., Kuttan, R., 2005. Chemoprotective activity of an extract of Phyllanthus amanis against cyclophosphamide induced toxicity in mice. Phytomedicine 12, 494-500.

Lin, J.Y., Lee, T.C., Tung, T.C., 1982. Inhibitory effects of four isoabrins on the growth of sarcoma 180 cells. Cancer Res. 42, 276-279.

Litchfied Jr., J.T., Wilcoxon, F.A., 1949. Simplified method of evaluating dose-effect experiments. J. Pharmacol. Exp. Ther. 96, 99-113.

Liu, Y.F., Hu, J., Zhang, J.H., Wang, S.L., Wu, C.F., 2002. Isolation, purification, and N-terminal partial sequence of an antitumor peptide from the venom of the Chinese scorpion Buthus martensii Karsch. Prep. Biochem. Biotcch-nol. 32, 317-327.

Mandal, M., Younes, M., Swan, E.A., Jasser, S.A., Doan, D., Yigitbasi, O., McMurphey, A., Ludwick, J., El-Naggar, A.K., Bucana, C, Mills, G.B., Myers, J.N., 2006. The Akt inhibitor KP372-1 inhibits proliferation and induces apoptosis and anoikis in squamous cell carcinoma of the head and neck. Oral Oncol. 42 (4), 430-439.

Mizejewshi, G.J., Muehlemann, M., Dauphinee, M., 2006. Update of alpha fetoprotein growth inhibitory peptide as biotherapeutic agents for tumor growth and metastasis. Chemotherapy 52, 83-90.

Moriwaki, S., Ohba, H., Nakamura, O., Sallay, I., Suzuki, M., Tsubouchi, H., Yamasaki, N., Itoh, K., 2000. Biological activities of the lectin, abrin-a, against human lymphocytes and cultured leukemic cell lines. J. Hematother. Stem Cell Res. 9, 47-53.

Mosmann, T., 1983. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods 65, 55-63.

Narayanan, S., Surolia, A., Karande, Anjali, A.A., 2004. Ribosome-inactivating protein and apoptosis: abrin causes cell death via mitochondrial pathway in Jurkat cells. Biochem. J. 377, 233-240.

Narayanan, S., Surendranath, K., Bora, N., Surolia, A., Karande, A.A., 2005. Ribosome inactivating proteins and apoptosis. FEBS Lett. 579, 1324-1331.

Papo, N, Shahar, M., Eisenbach, L., Shai, Y., 2003. A novel lytic peptide composed of DL-amino acids selectively kills cancer cells in culture and in mice. J. Biol. Chem. 278, 21018-21023.

Prabhakar, B.T., Khanum, S.A., Jayashree, K., Salimath, B.P.. Shashikanth, S., 2006. Anti-tumor and proapop-totic effect of novel synthetic benzophenone analogues in Ehrlich ascites tumor cells. Bioorg. Med. Chem. 14, 435-446.

Ramnath, V., Kuttan, G., Kuttan, R., 2002. Antitumour effect of abrin on transplanted tumours in mice. Indian J. Physiol. Pharmacol. 46, 69-77.

Ramnath, V., Rekha, P.S., Kuttan, G., Kuttan, R., 2007. Regulation of caspase-3 and Bcl-2 expression in Dalton's lymphoma ascites cells by abrin. Evid. Based Compl. Alt. Med. 4, 1-6.

Stirpe, F., Barbieri, L., Battelli, M.G., Soria, M., Lappi, D.A., 1992. Ribosome-inactivating proteins from plants: present status and future prospects. Bio/Technology 10, 405-412.

Tahirov, T.H., Lu, T.H., Liaw, Y.C., Chen, Y.L., Lin, J.Y., 1995. Crystal structure of abrin-a at 2.14A[degrees]. J. Mol. Biol. 250, 354-367.

Thippeswamy, G., Salimath, B.P., 2007. Induction of caspase-3 activated DNase mediated apoptosis by hexane fraction of Tinospora cordifolia in EAT cells. Environ. Toxicol. Pharmacol. 23, 212-220.

Udenfriend, S., Stein, S., Bohlen, P., Dairman, W., 1972. Fluorescamine: a reagent for assay of amino acids, peptides, proteins, and primary amines in the picomole range. Science 178, 871-872.

Sujit K. Bhutia, Sanjaya K. Mallick, Swatilekha Maiti, Tapas K. Maiti *

Department of Biotechnology, Indian Institute of Technology, Kharagpur 721302, West Bengal, India

* Corrcsponding author. Tel: +91 3222283766; fax: +913222255303.

E-mail address: (T.K. Maiti).

COPYRIGHT 2009 Urban & Fischer Verlag
No portion of this article can be reproduced without the express written permission from the copyright holder.
Copyright 2009 Gale, Cengage Learning. All rights reserved.

Article Details
Printer friendly Cite/link Email Feedback
Author:Bhutia, Sujit K.; Mallick, Sanjaya K.; Maiti, Swatilekha; Maiti, Tapas K.
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:9INDI
Date:Apr 1, 2009
Previous Article:In vitro evaluation of antibacterial, anticollagenase, and antioxidant activities of hop components (Humulus lupulus) addressing acne vulgaris.
Next Article:Vasorelaxant effects of forsythide isolated from the leaves of forsythia viridissima on NE-induced aortal contraction.

Terms of use | Privacy policy | Copyright © 2018 Farlex, Inc. | Feedback | For webmasters