Pea lectin inhibits growth of Ehrlich ascites carcinoma cells by inducing apoptosis and [G.sub.2]/M cell cycle arrest in vivo in mice.
ARTICLE INFOKeywords:
Lectin
Pisum sativum
Antitumor
Apoptosis
Caspase
Cell cycle
Gene expression
ABSTRACT
Pea (Pisum sativum L) lectin is known to have interesting pharmacological activities and of great interest on biomedical research. In the current research pea lectin was purified followed by ion exchange chromatography on DEAE column and affinity chromatography on glucose-sepharose column. The lectin shown 11.7-84% inhibitory effect against Ehrlich ascites carcinoma (EAC) cells at the concentration range of 8-120 [micro]g/ml in RPM! 1640 medium as determined by MTT assay. Pea lectin was also shown 63% and 44% growth inhibition against EAC cells in vivo in mice when administered 2.8 mg/kg/day and 1.4 mg/kg/day (i.p.) respectively for five consequent days. When Pea lectin injected into the EAC bearing mice for 10 days its significantly increased the hemoglobin and RBC with the decreased of WBC levels toward the normal. Apoptotic cell morphological change of the treated EAC cells of mice was determined by fluorescence and optical microscope. Interestingly, cell growth inhibition of the lectin was significantly reduced in the presence of caspase inhibitors. Treatment with the lectin caused the cell cycle arrest at G2/M phase of EAC cells which was determined by flow cytometry. The expression of apoptosis-related genes, Bcl-2, Bcl-X and Bax was evaluated by reverse transcriptase polymerase chain reaction (RT-PCR). Intensive increase of Box gene expression and totally despaired of Bcl-2 and Bcl-X gene expression were observed in the cells treated with Pea lectin for five consecutive days.
[c] 2013 Elsevier GmbH. All rights reserved.
Introduction
Lectins are proteins or glycoproteins from non-immune origin that specifically recognize cell surface molecules with at least two binding sites to carbohydrates. They are found in all kinds of organisms, including animals, plants, fungi, bacteria and viruses. Plant lectins represent a unique group of proteins with potent biological activities, such as agglutination, toxicity, anti-proliferation of cancer cells as well as having anti-fungal and anti-bacterial activities (Kabir et al. 2011a, b, 2012; Liu et al. 2010; Sitohy et al. 2007). They can specifically recognize and bind to various sugar structures, and thus trigger several important cellular processes (Sharon and Lis 1989; Sharon 2007). Several plant lectins have been shown to have antitumor activity and induce apoptosis in a series of tumor cell lines (Kabir et al. 2011a, b, 2012; Kim et al. 2000; Liu et al. 2010; De Mejla and Prisecaru 2005).
The lectin from Pisum sativum L. is commonly known as Pea lectin. The amino acid sequence and the crystal structure of the lectin were determined (Einspahr et al. 1986). Pea lectin highly agglutinated human, rabbit and rat erythrocytes but weekly agglutinated chicken erythrocytes. It was also reported that Pea lectin inhibited the growth of Aspergillus flavus, Trichoderma viride and Fusarium oxysporum (Sitohy et al. 2007). The lectin also inhibited the migration of cardiac mesenchymal cells (Sumida et al. 1997). In vitro binding assays, Pisum sativum agglutinin showed high affinity for Ehrlich ascites tumor (Kojima and Jay 1986). In the current article we have focused on the Pea lectin induced apoptosis in Ehrlich ascites carcinoma (EAC) cells and assessed its effect on the expression of different apoptosis-related genes.
Materials and methods
Chemicals and reagents
Sepharose-4B, Hoechst 33342, propidium iodide and RPMI 1640 medium were purchased from Sigma (USA). Fetal calf serum and penicillin-streptomycin from Invitrogen (USA), z-DEVD-fmk and z-IETD-fmk from Biovision (USA). Trypan blue and all other chemicals/reagents were of analytical grade. The Pisum sativum seeds were collected from the local market.
Purification of Pea lectin
50g of Pisum sativum seeds were homogenized in distilled water (DW) and then centrifuged at 10,000 rpm for 30 min at 4 C. The supernatant was dried by freeze dryer and stored at 4 C. Freeze dried powder was dissolved in 10 mM of Tris-HCl buffer pH 7.8 and dialyzed against the same buffer and centrifuged at 10,000 rpm for 10 min. The supernatant was then applied on DEAE cellulose column previously equilibrated with 10 mM of Tris-HCl buffer pH 7.8. Unbound fraction was collected and pH was adjusted to 8.2 by 1M Tris-HCl buffer, pH 8.4. Then the fraction was added to 20m1 of glucose-sepharose gel (glucose linked to epoxy-activated sepharose-4B) previously equilibrated with 10 mM of Tris-HCl buffer pH 8.2. NaCl and Ca[Cl.sub.2] salts solutions were added to the gel to the final concentration of 0.15 M and 2 mM respectively and kept overnight at 4[degrees]C with shaking for effective binding of lectins. A column was prepared with the gel and the unbound proteins were washed out by 10 mM of Tris-HCl buffer saline (TBS) pH 8.2 containing 1 mM of Ca[Cl.sub.2]. Lectin was eluted from the column by TBS containing 20 mM of EDTA or 1 M urea. The eluted fraction was dialyzed against 10 mM of Tris-HCl buffer containing 1 mM of Ca[Cl.sub.2] and the homogenity was checked by using SDS-PAGE in 15% polyacrylamide gel as described by Laemmli (1970). Bovine serum albumin (Mr. 67 kDa), Ovalbumin (Mr. 45 kDa), Carbonic anhydrase (Mr. 29 kDa), Trypsin inhibitor (Mr. 20 kDa) and Lysozyme (Mr. 14.6 kDa) were used as marker proteins. Protein concentration was determined by the Lowry's methods using BSA as the standard protein (Lowry et al. 1951).
Hemmaglutination activity
The hemagglutination assay of Pea lectin was performed in 96-well microtiter U-bottomed plates as described by Kabir et al. (2012). Hemagglutinating inhibition was studied by adding a serially dilution of the following sugars (D-mannose, D- glucose, D-galactose, L-arabinose, D-maltose, methyl-a-D-mannopyranoside, methyl-[alpha]-D-glucopyranoside, N-acetyl-D-glucosamine and methyl-[alpha]-D-galactopyranoside) at the final concentration of 200 mM.
Determination of temperature and pH stability
Pea lectin (0.5 mg/ml in Tris-HC1 buffer saline, pH 7.8) was heated in a water bath for 30 min at different temperatures from 40 to 80C and cooled to room temperature (30[degrees]C) to examine the thermo stability. Then 50 ill of Pea lectin was serially diluted with an equal amount of hemagglutination buffer, pH 7.8 and the hemagglutination titer was performed as described by Kabir et al. (2012). The non-heated lectin sample was used as a control, which denoted 100% activity. The pH stability of Pea lectin was examined by incubating the protein solutions (0.5 mg/m1) against 0.1 M sodium acetate (pH 3.0, 4.0, 5.0 and 6.0), 0.1 M phosphate buffer (pH 7.0), 0.1 M Tris-HCl (pH 8.2) and 0.1 M glycin-NaOH (pH 10.0 and 11.8) for 8 hat room temperature. After 8 h the lectin solutions were dialyzed against 10 mM Tris-HCl buffer, pH 7.8 containing 0.9% NaCl for 12 h.
Treatment with urea and divalent cation
Pea lectin (1 mg/m1) in 0.1 M Tris-HCl buffer saline was incubated at 20[degrees]C with 2 and 4 M of Urea for 2 h. Lectin in the same buffer without denaturants was used as a control and its activity was considered as 100%. Pea lectin (1 mg/ml) was incubated with 20 mM of EDTA for 4 h at 30[degrees]C and then dialyzed against Tris-HC1 buffer pH 7.8. Finally hemagglutination activity was checked in the presence and absence of 10 mM Ca[Cl.sub.2] to determine the dependency on divalent cations.
Cell culture EAC cells were cultured in RPMI-1640 medium containing D-glucose, 2 mM L-glutamine supplemented with 10% fetal calf serum, and 1% (v/v) penicillin-streptomycin, in a humidified atmosphere of 5% [Co.sub.2] at 37 C.
MTT colorimetric assay
MTT colorimetric assay was used to determine EAC cells proliferation. Cells (5 x [10.sup.5] in 200 [mu]l RPMI 1640 media) were plated in the 96-well flat bottom culture plate in the presence and absence of different concentrations (8-120 [micro]g/ml) of Pea lectin and incubated for 24h at 37[degrees]C in [Co.sub.2] incubator. After carefully removing the aliquot, 180 ill of PBS and 20 [micro]l of MIT were added and further incubated for 8 h at 37[degrees]C. The aliquot was removed again and 200 p,1 of acidic isopropanol was added into each well and incubated again at 37[degrees]C for 1 h. Subsequently, absorbance was read at 570 nm using titer plate reader. The following equation was used to calculate the cell proliferation inhibition ratio:
Proliferation inhibition ratio (%) = (A - B) x 100 / A
where A is the [0D.sub.570 nm] of the cellular homogenate (control) without Pea lectin and B is the [0D.sub.570 nm] of the cellular homogenate with Pea lectin.
Animals and ethical clearance
Adult Swiss albino mice which were used throughout the study were collected from the International Center for Diarrheal Diseases Research, Bangladesh. This research work was approved by the Institutional Animal, Medical Ethics. Bio-safety and Bio-security Committee (IAMEBBC) for Experimentations on Animal, Human, Microbes and Living Natural Sources (286/320-IAMEBBC/IBSc), Institute of Biological Sciences, University of Rajshahi, Bangladesh.
Determination of Ehrlich ascites carcinoma (EAC) cell growth inhibition in mice
EAC cells were propagated intraperitonealy in our departmental laboratory biweekly and the cells were collected from a donor Swiss albino mouse bearing 6-7 days old ascites tumors. The cells were adjusted to 3 x106 cells/ml with the dilution of normal saline and counting by using haemocytometer. The viability of tumor cells were observed by trypan blue dye (0.4%) exclusion assay. EAC cells (in 0.1 ml saline) showing above 99% viability were injected intraperitonealy into each Swiss albino mouse. The mice were randomly distributed after 24h into three groups with at least six mice per group. Two groups of mice were treated intraperi-tonealy with Pea lectin at the concentrations of 2.8 mg/kg/day (70 [micro]g/mouse/day) and 1.4 mg/kg/day (35 [micro]g/mouse/day) for five days. Mice in each group were sacrificed on 6th day of the lectin treatment; the total intraperitoneal tumor cells were harvested by normal saline and counted by a haemocytometer. The total numbers of viable cells in every mouse of the treated groups were compared with those of controls (EAC treated only). Following formula was used to calculate the percent of inhibition:
Percent of inhibition
= 100 - {(cells from lectin treated mice/cell from control mice) x 100}.
For hematological parameters, 18 mice were taken and 12 were treated with EAC cells as described above. After 24 h among the 12, six were treated with 2.8 mg/kg/day concentration of Pea lectin for 10 days. Blood was drawn from the tail of each mouse on the 12th day of EAC cells inoculation and the percentage of hemoglobin measured by using hernatometer and the total WBC and RBC were counted by using haemocytometer.
Observation of cell morphologic change and nuclear damage
Cell apoptosis was morphologically observed with and without the Pea lectin under a fluorescence microscope (Olympus iX7l, Korea). EAC cells were collected from the lectin treated (2.8 mg/kg/day) and untreated EAC bearing mice and washed thrice with phosphate buffer saline (PBS). Then the cells were stained with 0.1 [micro]g/ml of Hoechst 33342 at 37[degrees]C for 20 mm in dark and washed again with PBS.
Effect of caspases on Pea lectin-induced cytotoxicity in EAC cells
In order to confirm the involvement of caspases in the lectin-induced cell death, the untreated EAC cells in RPM! 1640 medium were incubated with z-DEVD-fmk (caspase-3 inhibitor, 2 [micro]mol/m1) and z-IETD-fmk (caspase-8 inhibitor, 2 [micro]mol/m1) for 2 h. Then the cells were treated with 120 [micro]g/m1 of lectin and incubated for 24 h at 37[degrees]C in [Co.sub.2] incubator. Finally cell growth inhibition was determined by MIT assay.
Cell cycle analysis
Pea lectin treated (2.8 mg/kg/day) and untreated EAC cells were collected from mice after treatment of five consequent days and washed thrice with the cold PBS. After fixation with 70% ethanol for 24h at 4[degrees]C the cells were then washed thrice with cold PBS. Finally, the cells in 1 ml PBS were treated with 50 [micro].I of RNase A (1 mg/ml) for 30 min at 37[degrees]C followed by staining with 5 [micro]l of Propidium Iodide (1 mg/ml) in dark at 4 C for 5 min before analyzing using flow cytometry. The fractions of cells in [G.sub.0]/[G.sub.1], S and [G.sub.2]/M phase were analyzed by a FACS Flow cytometer (Partec CyFlow SL, Germany).
RT-PCR
Total RNA was extracted using TRIzol method from the Pea lectin treated (2.8 mg/kg/day) and untreated EAC cells after five consequent days of EAC bearing mice. Then 3 [micro]g RNA was reversed transcribed into cDNA in a final volume of 20 [micro]l containing 100 pmol random hexamer, and 50 U of MuLV reverse transcriptase (New England Biolab) according to the manufacturer's guideline. Expression of three tumor related genes namely, Bcl-2, Bax and Bcl-X were studied using these cDNA as template for PCR. [beta]-Actin was used as control. 25111reaction volume were prepared containing 1 x of tag polymerase buffer, 25 pmol each of forwarded and reverse primer, 2.5 mM of each dNTP and 0.25U of platinum Tag polymerase (Tiangen, China). The following specific oligonucleotides (IDT Singapore) were used.
Bc1-2 upstream (5'-GTGGAGGAGCTCTTCAGGGA-3') and bd-2 downstream
(5'-AGGCACCCAGGGTGATGCAA-3'), generating 0.304 kb amplification product;
Bax upstream (5.-GGCCCACCAGCTCTGAGCAGA-3'), and Bax downstream
(5'-GCCACGTGGGCGTCCCAAAGT-3') generating a band of 0.479 kb;
Bcl-X upstream ( 5' -TTG G ACAATG G ACTG ), and Bc1-X downstream
(5'-GTAGAGTGGATGGTCAGTG-3') generating two amplification products of 0.78 kb and 0.591 kb for bcl-[X.sub.L] and bcl-[X.sub.S] isoforms; [beta]-Actin upstream
(5'-ACCCACACTGTGCCCATCTACGA-3'), and [beta]-Actin downstream
(5'-CAGGAGGAGCAATGATCTTGATCTTC-3') generating 0.516 kb amplification product.
Amplifications were performed in a BioRad (USA) gradient thermal cycler. The cycling condition was initial PCR activation step of 3 min at 95[degrees]C, followed by 35 cycles of 95[degrees]C/1 min. 55[degrees]C/1 min, 72[degrees]C/1 min and a final extension of 72[degrees]C/10 min for [beta]-Actin and Bcl-2 gene. In case of Box and Bcl-X the anneling temperature was 54[degrees]C instead of 55[degrees]C. All the PCR reactions were analyzed in 1.0% agarose gel and GeneRular 1 kb DNA ladder (Fermentus, USA) was used as marker.
Statistical analysis
The experimental results are expressed as the mean [+ or -] S.D. (Standard Deviation). Data have been calculated by one way ANOVA followed by Dunnett 't' test using SPSS software of 16 version.
Result
Purification of Pea lectin
The lectin was purified from Pisum sativum seeds followed by ion exchange chromatography on DEAE cellulose column and affinity chromatography on glucose-sepharose column. The unbound fraction of DEAE column (was not shown) was applied on glucose-sepharose column and the protein was eluted by 20 mM of EDTA or by 1 M urea (Fig. 1A). The lectin migrated as two bands in 15% SDS-PAGE and the bands were 5 kDa and 19.5 kDa corresponding to alpha and beta subunits respectively (Fig. 1B). Around 20 mg of lectin was purified from 200g of Pisum sativum seeds (Table 1).
Table 1
Purification of lectin from 200g of Pisum sativum L. seeds.
Protein content Total activity Specific
(mg) (titer/mg x activity
[10.sup.3])
Crude 3200 384 120
Ion exchane on 1600 256 160
chromatography
DEAE-cellulose
Affinity 20 51 2550
chromatography on
glucose-sepharose
Hemagglutination activity
The minimum agglutinating activity of the Pea lectin was found to be 8[micro]g/m1 for rat red blood cells. The hemagglutination inhibition study of the lectin was performed in the presence of different sugars and it was evident that D-glucose and D-mannose were best inhibitors for Pea lectin (Table 2).
Table 2
Hemagglutination inhibition of Pea Lectin by mono- and
oligosaccharides.
Sugar Minimum inhibitory concentration
(mM)
D-Mannose 1.6
D-Glucose 1.6
Methyl-[alpha]-D-mannopyranoside 3.1
Methyl-[alpha]-D-glucopyranoside 6.2
L-Arabinose 6.2
D-Maltose 6.2
N-acetyl-D-glucosamine 6.2
D-Galactose 50
Methyl-[alpha]-D-galactopyranoside 100
D-Melibiose 100
In vivo treatment of Pea lectin inhibit growth of EAC cells
Effect of temperature, pH and denaturants on Pea lectin
The hemagglutination activity of Pea lectin was changed with the change of temperature. Highest activity was observed between 40 and 60[degrees]C and lost the activity 75% at 70[degrees]C and completely at 80[degrees]C. Pea lectin showed the maximum activity at the pH range 6.0-10. At pH 4.0 and 5.0 the lectin lost its activity 50%, whereas 93.7% activity lost at pH 11.8 and completely at pH 3.0. The hemag-glutination activity of Pea lectin was not affected by urea. The lectin showed its activity 100% both in the presence of 2 M and 4 M urea as compared with the control. In the absence of Ca[C1.sub.2], EDTA treated lectin did not show any activity but the lectin recovered its activity fully when 10 mM Ca[C1.sub.2] was added to the hemmaglutination buffer.
MIT assay Effect of Pea lectin on EAC cells was investigated by MU assay. The lectin induced EAC cell death is a dose dependent manner (Fig. 2). The lectin was found to have 84% inhibitory effect at a concentration 120 [micro]g/ml. The effect decreased with the reduction in lectin concentration and it reached to 11.7% at 8 [micro]g/ml.
EAC cells proliferation in mice was effectively inhibited by the lectin in a dose dependent manner. At the dose of 1.4 mg/kg/day, the inhibition of EAC cell growth was 44% but it increased to 63% when the lectin concentration increased to double, i.e. 2.8 mg/kg/day (Fig. 3).
RBC and hematological parameters of normal, EAC inoculated and Pea lectin treated on EAC inoculated mice
Hematological parameters were found to be different between normal, tumor bearing and lectin treated tumor bearing mice. The total WBC and RBC of the normal and lectin treated mice were almost same on the other hand it was half of the tumor bearing mice as compared to treated and normal mice (Fig. 4). The hemoglobin level of EAC bearing mice was 6% but after treatment with lectin it increased significantly to 9.9% where as it was 11.6% for the normal mice (Fig. 4).
Fluorescence morphological examination
After treatment with and without Pea lectin (2.8 mg/kg/day), morphological changes of EAC cells were confirmed by Hoechst 33342 staining. Round and homogeneously stained nuclei was observed in the control group (Fig. 5A) and lectin treated cell exhibited manifest fragmented DNA in nuclei (Fig. 5B). Apoptotic morphologic alterations (e.g. membrane and nuclear condensation) were also observed clearly by optical microscopy (Fig. 5C and D) and these results suggested that Pea lectin could induce apoptosis of EAC cells.
Effect of caspases on Pea lectin-induced cytotoxicity in EAC cells
Caspase inhibitors z-DEVD-fmk (caspase-3 inhibitor) and z-IETD-fmk (caspase-8 inhibitor) were used to detect the involvement of caspases in the apoptotic cell death of EAC cells induced by the treatment of the lectin. In the presence of Pea lectin growth inhibition of EAC cells was 84% and it decreased to 54 and 62% in the presence of z-DEVD-fmk and z-IETD-fmk respectively in the culture medium (Fig. 6).
Cell cycle analysis by FACS
The effect of Pea lectin on the different phases of the cell cycle of EAC cells was studied. The healthy EAC cells exhibited three phases [G.sub.0]/[G.sub.1], S and [G.sub.2]/M in the cell cycle. The percentages of [G.sub.0]/[G.sub.1], S and [G.sub.2]/M phases in the untreated EAC cells were calculated to be 59, 14 and 27% respectively. After treatment with Pea lectin, the [G.sub.2]/M phase population increased to 48% and the [G.sub.0]/[G.sub.1] and S phases decreased to 52 and 0% respectively (Fig. 7). The result suggested that the lectin inhibits the cellular proliferation of EAC cells via [G.sub.2]/M-phase arrest of the cell cycle.
RT-PCR
[beta]-Actin primers were used for an amplification reaction to confirm the suitability of the purified RNAs and the samples were found to be suitable for RT-PCR. Box, Bc1-2 and Bcl-X genes expression were then investigated by RT-PCR and the product give clear band (Fig. 8). In our study, Bcl-2 and Bcl-X genes expressions were observed in Pea lectin untreated control EAC cells. Bcl-X was expressed only as the isoform Bc/-XL with intensive band (Fig. 8). On the other hand no Bax gene expression was appeared in control EAC cells. But after treatment with the lectin, gene expression of Bcl-2 and Bcl-XL was totally omitted and the Bax gene expression was observed.
Discussion
In the present study, Pea lectin was purified by using DEAE cellulose and glucose-sepharose columns that is different from the methods described in previous literatures. Using this method 20 mg of Pea lectin was purified from 3200 mg of crude proteins whereas 16 mg lectin was purified from 4200 mg of crude protein as described by Sitohy et al. (2007). The molecular weight of [alpha]-subunit and [beta]-subunit of Pea lectin was almost the same as described by Sitohy et al. (2007). The minimum concentration of Pea lectin required for hemmaglutination activity of rat red blood cells was 8 [micro]g/ml. D-mannose and D-glucose were the best inhibitory sugars as also shown in other papers. We found a little difference in pH stability and no change in heat stability when compared with Sitohy et al. (2007). The lectin was a divalent cation dependent protein that was stable in urea.
Lectin molecules are known to bind to cancer cell membrane or receptors thereby, causing cytotoxicity, apoptosis and inhibition of tumor growth (De Mejia and Prisecaru 2005). Multiplication of cancer cells was stopped by several kinds of plant lectin (Liu et al. 2010) and due to the differences in their sugar specificity, each lectin exhibits differences in their antiproliferative effect against tumor cell lines. Several experiments were made to study the anticancer effect of lectins on different cancer cells (Liu et al. 2010) but only a few reported against EAC cells (Ahmed et al. 1988: Kabir et al. 2011a, b, 2012: Akev et al. 2007). In the present study MTT assay was performed and the result showed that Pea lectin inhibited EAC cells proliferation in vitro in a dose-dependent manner. The present study also carried out to evaluate the antitumor effect of Pea lectin on EAC bearing mice. The result showed that the lectin also decreased the EAC cell growth by 44 and 63% at 1.4 mg/kg/day (35 [micro]g/mouse/day) and 2.8 mg/kg/day (70 [micro]g/mouse/day) respectively. Growth inhibition of EAC cells was also studied by using different lectins. Jackfruit lectin inhibited only 21.8, 40.2 and 57.5% of EAC cell growth at 50, 100 and 150 [micro]g/day respectively; Kaempferia rotunda lectin inhibited 51 and 67% of cell growth at 1.25 mg/kg/day and 2.5 mg/kg/day respectively; Trichosanthes cucumerina seed lectin (TCSL) showed 28 and 72% growth inhibition against EAC cells at 1 mg/kg/day and 2 mg/kg/day respectively and Nymphaea nouchali tuber lectin (NNTL) showed 56 and 76% growth inhibition against EAC cells at 1.5 mg/kg/day and 3.0 mg/kg/day respectively (Ahmed et al. 1988; Kabir et al. 2011a, b, 2012). In tumor-bearing mice, anemia occurs due to the decrease in RBC or hemoglobin percentage and increased the WBC. Administration of TSCL to EAC bearing mice restored the hemoglobin, WBC and RBC content toward the normal level (Kabir et al. 2012). In the present study the lectin (2.8 mg/kg/day) also increased the RBC and hemoglobin level and decreased the WBC to the normal level. This result supports the suitability of the lectin as an anticancer agent.
Apoptosis, is intrinsic cell suicidal mechanisms that can be regulated by numerous cellular signaling pathways and characterized morphologically by cell shrinkage, apoptotic body formation and condensation of chromatin. These morphological changes and death of apoptotic cells are caused by a series of proteases termed caspases, such as caspase-3, -6, -7, -8 and -9 (Graf et al. 2007). In the present study Pea lectin induced apoptosis was confirmed by the observation of the changes in nuclear morphology and cell shape as compared to that of the control EAC cells. Moreover the activity of the caspase-3 and caspase-8 were blocked significantly by the z-DEVD-fmk and z-IETD-fmk inhibitors respectively. Apoptosis of EAC cells was also studied by using different proteins and plant extracts. A cytotoxic protein BMP1 was purified from the aqueous extract of common Indian toad (Bufo melanostictus Schneider) skin arrested the growth of EAC cells by inducing apoptosis in the caspase dependent pathway (Bhattacharjee et al. 2011). 'Ottelion A' a plant extract inhibited the EAC cells proliferation by apoptosis which mediated by increased of p53 level and CD8+ (EI-Missiry et al. 2012).
Many antitumor agents and DNA-damaging agents induce apoptotic cell death by arresting the cell cycle at the G1, S, or G2/M phase. BMP1 effectively arrested cell cycle progression of EAC cells at the G1 phase (Bhattacharjee et al. 2011). Cell cycle arrest of EAC cells was also found for cape aloe (Kametani et al. 2007), Ottelione A (El-Missiry et al. 2012) and Paullinia cupana Mart. var. sorbilis, Guarana (Fukumasu et al. 2011). G1 & G2 phase arrest of the EAC cells cycle was found when (1'S)-Acetoxychavicol acetate and its enantiomer treated with EAC cells in vitro respectively (Xu et al. 2008). Cell cycle arrest can also be induced by lectin either in one or a combination of different phases during apoptosis. In the present study, Pea lectin induced cell cycle arrest of EAC cells in G2/M phase. It was also reported that Wheat germ lectin and Phaseolus vulgarisc hemag-glutinin also induced G2/M phase arrest in mouse L929 fibroblasts and breast cancer MCF-7 respectively (Liu et al. 2004: Lam and Ng 2010). Furthermore Volvariella volvacea lectin arrests cell proliferation by blocking cell cycle progression in the G2/M phase (Liua et al. 2001).
The mechanism of apoptotic activation has been studied intensively under different pathological conditions, and of the many genes found to participation in the regulation of apoptosis. Among them Bc1-2 (B cell lymphoma gene-2) gene was the first oncogene to be implicated in the regulation of apoptosis. Till now 25 genes in the Bcl-2 family have been identified in humans. This family is divided into a pro-apoptotic class, which includes Bax, Bid and Bak, and an anti-apoptotic class, which includes Bc1-2, Bcl-X and Bcl-W. Bcl-X gene product exists in two forms, Bcl-XL (long), which blocks apoptosis in many systems, and the spliced short from Bc1-Xs, which acts as a dominant inhibitor of Bcl-2 (Gradilone et al. 2003). Changes in the mRNA expression profiling of these genes occurs due to cancer cell response to chemotherapeutic drugs. A decrease in the expression of Bcl-XL and increased in the expression of Bax and Bak was observed after treating of HeLa cells with FRAP (Ju et al. 2012). Such type of results were also reported for Musca domestica Larva lectin (MLL) which induces Bcl-2 decreased during the Bax increased (Zhao et al. 2010). In the present study, intensive bands of Bcl-2 and Bc/-XL gene was observed in control and the bands of these genes were almost disappeared after five days treatment with the lectin. On the other hand intensive band of Bax gene was observed after five days treatment. It was reported that over expression of Bcl-2 prevent the translocation of cytochrome c, thereby block the apoptotic process (Groc et al. 2001). But over expression of Bax does not block apoptosis; instead, it seems to block Bcl-2 functions. The Bax gene encodes a protein that is primarily localized to the cytosol and after apoptotic stimulation it translocated to the mitochondria. At the mitochondria it triggers the release of cytochrome c and forms a complex with other cofactors that triggers the activation of caspase-9, initiating a downstream caspase cascade leading finally to the cell death (Antonsson 2001).
In conclusion, Pea lectin inhibits EAC cell growth and arrest the cell cycle at G2/M phase. This lectin changes the morphology of EAC cells and the cell growth inhibition was reduced in the presence of caspase inhibitors. Gene expression also supports the lectin induces apoptosis in EAC cells.
Funding
This research work was funded by the Faculty of Science, Rajshahi University and Ministry of Science and Technology, Bangladesh.
* Corresponding author at: Department of Biochemistry and Molecular Biology, Faculty of Science, Rajshahi University, Rajshahi 6205,
Bangladesh. Tel.: +880 721 711109; fax: +880 721 711114.
E-mail addresses: [email protected], [email protected] (S.R. Kabir).
0944-7113/$--see front matter [c] 2013 Elsevier GmbH. All rights reserved.
https://dx.doi.org/10.1016/j.phymed.2013.06.010
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Syed Rashel Kabir (a), (*), Md. Mahamodun Nabi (a), Ariful Hague (b), Rokon Uz Zaman (c), Zahid Hayat Mahmud (c), Md. Abu Reza (d)
(a.) Department of Biochemistry and Molecular Biology, Rajshahi University, Rajshahi 6205, Bangladesh
(b.) Institute of Biological Sciences, Rajshahi University. Rajshahi 6205, Bangladesh
(c.) Environmental Microbiology Laboratory, 1CDDR'B, 68 Shaheed Tajudin Ahmed Sarani, Mohakhali, Dhaka 1212, Bangladesh
(d.) Department of Genetic Engineering and Biotechnology, Rajshahi University, Rajshahi 6205, Bangladesh
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| Author: | Kabir, Syed Rashel; Nabi, Mahamodun; Hague, Ariful; Zamanc, Rokon Uz; Mahmud, Zahid Hayat; Reza, Abu |
|---|---|
| Publication: | Phytomedicine: International Journal of Phytotherapy & Phytopharmacology |
| Article Type: | Report |
| Geographic Code: | 9BANG |
| Date: | Nov 15, 2013 |
| Words: | 5899 |
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