Jn Vitro Cell,Dex Biol —Animel (2007) 43:306-314 DOI 10,1007/811626-007-9055-2 Cytotoxic effect of achatininy (lectin) from Achatina fulica against a human mammary carcinoma cell line (MICF7) Indra Dharmu Mary Babu Ramamurty - Ramalingam Kannan + Received: 26 March 2007 /Accepted: 26 July 2007 Published online: 18 September 2007 / Fltor J, Denry Sato (© The Sovity for In Vito Biology 2007 Abstract The hemolymph-lerived achatininy (lectin) from Achatina fulica showed a marked cytotoxic effect on MCF?, a human mammary carcinoma cell line. ICso values as measured by the 3-(4,5-dimethylthiazol-2y1)-2,5-diphe- nyltetrazolium bromide assay for achatinin,, ranged from 6 to 10 jigiml in the MCF7 cells, MCF7 cells showed significant morphological changes leading to cell death. The above cell death was observed afler 48 h of treatment with 8 ug/ml when compared to untreated cells. Alterations jn the tumor marker enzymes, as well as in antioxidant enzymes, were observed afler achatininys treatment. The specificity and purty of the achatininy, was confirmed by the Westem blot assay. Achatininy, binding to MCF7 cells was detected by anti-achatininy, and visualization of the achatininy, binding sites on confluent MCFT cells was confirmed by flourescein isothiocyanate conjugated sec- ondary antibody. MCF7-treated cells fuoresced, indicating the presence of achatininy: binding sites. Fluorescence- 1 Dhama-M. Babu Biomaterials Division, Central Leather Resear Insite, ‘Adyar, Chennai 600 020 Tamil Nadu, India al: babumary2000@yahoo.com 1 Dharm Division of Infectious Diseases, Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan 11529, People’s Republic of China IN, Ramamuny Virology Department, King Istute of Preventive Medicine, Guindy, Chensai 600 032, India Kania PG & Research Department of Zoology, Govt Ants College, Nandazam, Chennat 600035, India D springer activated cell sorting analysis of the cell cycle showed a significant inerease in S-phase in MCF? cells after 48 h of achatinin, treatment. The cells were arrested in Gy/M phase of the cell eycle after 48 bh with significant changes in cell viability. Cellular damage was confirmed by agarose gel electrophoresis with the characteristic appearance of a DNA streak in treated MCF7 cells indicating the ongoing apoptosis, Keywords MCF Achatininy - Tumor marker enzymes Antioxidant enzymes -Ga/M phase arrest Apoptosis Introduction Lectins are proteins or glycoproteins of nonimmune origin ‘that agglutinate cells and precipitate complex carbohydrates, Lectins of both plant and animal origin have been widely used to map cell surface carbohydrates (glycoproteins) and to observe changes oceurring during malignant transforma- tion and development (Sharon and Lis 1989), An array of biological functions is attributed to the binding of lectins to the cell surface sugars. The lectins are thus reckoned with the functions such as tumor cell recognition, cell adhesion, localization, signal transduction across the membrane, ‘mitogenic stimulation, potentiation of host immune de- fense, cytotoxicity, and apoptosis (Mody et al. 1995). Lectins derived from Helix sp., Limax sp., Achatina sp., and their binding to different groups of carbohydrates and their prognostic properties to bind the metastatic cells preferentially have been demonstrated in recent studies (Miller 1982; Iggehi ct al. 1985; Sukhikh ot al. 1985; Fenlon et al. 1987; Schumacher et al. 1995). Lectins or lectin-like proteins endowed with the ability to bind sialoglycoconjugates have been used as recognition mole-EFFECT OF ACHATININ, AGAINST A HUMAN MAMMARY CARCINOMA CELL LINE 307 cules to predict changes in the pattern of sialylation and the gree of O-acetylation during malignant transformation (Cheresh et al. 1984; Basu et al. 1986; Crocker et al. 1991; Fragkiadakis and Stratakis 1997). It is very crucial to identify the reliable markers of metastatie potentials in the development of cancer therapeutics. Several screening protocols may help evaluate the prognostic properties of lectins. In the present study, the cytotoxic effects of hemolymph derived achatininy, (lectin) from Achatina {fulica and its binding properties to the breast cancer cell line MCF7 have been delineated using 3-(4,5-dimethylth- jazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) assay, immunocytochemistry, and floweytometry. It is known that MTT is an established technique to estimate the in vitro cellular viability in the disease-oriented screening protocols in cancer research (Twentyman and Luscombe 1987; Pieters et al, 1990). The MTT assay is based on the fact that only viable cells reduce tetrazolium salts to colored formazan, which can be quantitated spectrophotometrically. Besides measuring viability, activa- tion, and proliferation, the MTT assay has also been recently adopted to measure the clonogenesity of eancer cells in culture system (Van-de-Loosdrecht et al. 1994). The specific binding of achatining, to MCE7 cells observed in our study would provide a viable prognostic marker in tumor cell detection, We have also observed that the increased expression of lactate dehydrogenase (LDII) by tumor cells (Langgut et al. 1993; Reisser etal. 1994) could be suppressed by achatininy, treatment, The cytotoxic potential of achatininy, was evaluated at the cellular level in terms of DNA damage, alterations in cellular morphol- ‘gy, and cell eyele profile Materials and Methods The sialic acid binding achatininy ftom hemolymph of land snail A, filica was purified by affinity chromatography on sheep submaxillary mucin coupled to Sepharose 4B as described previously (Indra et al. 2000). Cell culture. Hurman mammary carcinoma cell line (MCF7) was procured from the National Centre far Cell Sciences, Pune, India, and was adapted to grow in minimum essential medium (MEM) supplemented with 10% fetal calf serum (FCS), 100 mg/ml streptomycin and 100 TUml penicilin. Cell culture was performed in 24 well plates (1% 10° cells! well) in a humidified atmosphere at 37° C with 5% CO>. MIT assay. MUT assay was carried out to evaluate the cell- mediated cytotoxicity as previously described (Mosmann 1983), MCF7 cells were detached from the culture flask by treatment with 0.05% trypsin-EDTA and cell viability was determined by trypan blue. Cells were seeded in 24-well plates (110° cells mi well) and incubated for 48 h. The ‘medium was then withdrawn and fresh medium (1 ml) with different concentrations of achatininy: (6-16 wgiml) in lectin bufler Tris-buffered saline (TBS) (SO mM Tris-HCI pH 7.6 with 1 mM CaCl, and MgCl) was added and incubated for different time intervals (24 and 48 h), Appropriate controls without the addition of achatininys were kept. The plates were observed under a phase-contrast microscope for ‘morphological changes after the specified time intervals, Photomicrographs were taken using @ Leica (Wetzlar, ‘Mannheim, Germany) DMIRB microscope at 10% magnifi- cation. The medium was withdrawn after specified time intervals and fresh medium (200 ul) was added to each well. Then, 50 ul of MTT solution at a concentration of 5 mg/ml [5 mg of MTT/ml of phosphate-buffered saline (PBS) was filter sterilized and kept for not more than 2 wk at 4° C] was added into each well and kept for 4 h at 37° C. The assay is based on cleavage of the tetrazolium salt MIT by ‘mitochondrial dehydrogenase to form a purple-colored formazan complex soluble in DMSO, which was quantified spectrophotometrcally at 540 nm with reference filter at 690 nm using a Perkin-Elmer (Waltham, MA) Lambda 35UV-visible spectrophotometer, Duplicates for each time period were kept in each assay and repeated three times. The viability results are shown as a graph with absorption on the Y axis and achatininyy concentration on the X axis. The percent proliferation and the percent cytotoxicity were calculated by the following equation: 1% Proliferation sts 00Cat et 0 100 % Cytotoxicity = est Fat 00 109 ICs values are defined as the drug concentration needed, to inhibit cell growth to 50% of controls Evaluation of cell proliferation. To evaluate the cell proliferation, 1%10° cells/well were seeded in triplicates in a 24-well plate (Greiner, Frickenhausen, Germany). The plate was incubated at 37°C in a humidified atmosphere of 5% CO, air for 2 d to form a complete monolayer. Then, the medium was removed and fresh maintenance medium MEM with 2% FCS was added. Achatininy. at two different concentrations (8 and 10 ug/ml) was added to cach well and the plate was incubated at 37° C for 24 and 48 b. The cells were detached from the plate using uypsin-EDTA. Cell suspension 0.2 ml and trypan blue 0.2 ml were mixed well and counted using Neubar counting chamber, Development of antibody. Rabbits were immunized by intramuscular injection of achatininy, (protein | mg/ml) mixed with complete Freund's adjuvant and, afler 1 wk, again immunized by intramuscular injection of achatininy, D springs308 DHARMU ET AL. (1 mg/ml) mixed with incomplete Freund’s adjuvant. Finally, booster injection was given with achatininy, without adjuvant and blood was collected after 7 d of the last injection and the antiserum was tested by immunodiffusion. Gel electrophoresis. SDS gel electrophoresis was per formed according to Laemmli (1970) on 7.5% gels using the nonreducing and reducing conditions. The gels were stained with Coomassie brillimt blue. The molecular mass (kDa) was determined using standard protein markers from Sigma, St. Louis, MO. Western blot analysis. Westem blot analysis was performed according to the method of Towbin et al. (1979) using twansblot apparatus (Bio-Rad, Hercules, CA). After separa- tion by SDS-PAGE, the protein (achatinin,;) was transferred onto nitrocellulose (0.45 jum, Hybond-C Super, Amersham, Buckinghamshire, UK). Skimmed milk powder (5% supplemented with 0.1% Tween 20 was used as blocking buffer. The blots were incubated for | h with primary anti- achatiningy antibody (1:500) in PBS Tween and then with anti-rabbit secondary antibody coupled to horseradish peroxidase (Sigma) (1:500) for visualization of the achati- ning transblotted onto the nitrocellulose Tumor markers and antioxidants. Exponentially growing cells were treated with 8 yigiml achatininy, for 48 h, The ‘medium was aspirated and the cells (treated and control) were rinsed with cold PBS, The cells were seraped and homogenized in 1% Iysis buffer (10x lysis buffer: 200 mM Tris-HCl (pH 7.5), 15 M NaCl, 10 mM EDTA, 10% Triton, 25 mM sodium pyrophosphate, 10 mM beta- alycerophosphate, 10 mM NasVO,, 10 g/ml leupeptin and 1 mM phenylmethylsulfonyl fluoride] and centrifuged at 14,000 rpm for 10 min at 4° C, The supematant was aliquoted into a new eppendorf tube and stored at ~80° C. The cell lysate was used for the following analysis, LDH activity was determined by the procedure adapted by King (1965a), Acid and alkaline phosphatases were determined by the method of Moog (1946) and the transaminases viz, alanine transaminase and aspartate transminase were est- mated by the method of King (19655). The protein concentration was determined according to Lowry et a (1951), The enzyme activity is expressed as units per minute per milligram of protein. Appropriate controls without achatining, were kept, The resulis were analyzed using one-way ANOVA (SPSS package student's version, SPSS, Chicago, IL). ‘The level of lipid peroxidase (LPO) was assayed by the method of Obkawa etal. (1979), in which malondialdehyde released served as an index for LPO, The activity of catalase (CAT) was assayed by the method of Sinha (1972). In this method, dichromate in acetic acid was reduced to chromic D springs acetate when heated in the presence of hydrogen peroxide (F103), with the formation of perchloric acid as an unstable intermediate. Chromic acetate thus produced was measured colorimetrically at 610 nm. The activity of superoxide dismutase (SOD) was determined by the method of ‘Marklund and Marklund (1974). The unit of enzyme activity is defined as the enzyme required for 50% inhibition of pyrogallol auto-oxidation, The glutathione peroxidase activ- ity was assayed by the method of Rotruck etal. (1973) based ‘on the reaction between glutathione and 5,5'-dithio bis-(2- nitrobenzoie acid) to form a complex that showed absor- bance maximally at 412 nm, The antioxidant activities were ‘expressed as units per minute per milligram of protein. The values between the control and test were analyzed using one~ way ANOVA (SPSS package student’s version) Binding of achatininy to MCFT cells, MCF7 cells were seeded on slides (1%10° cells/ml) and cultured for 48 h, ‘The slides were incubated with achatininy, (8 pgiml) for 48 h and rinsed with PBS [50 mM PBS pli 7.4, 2% (V/V) FCS and 0.1% (W/V) sodium azide] and fixed with 4% paraformaldehyde in 0.1 M PBS. The slides were washed three times in lectin buffer (TBS), and anti-achatining, antibody (1:500) was added and incubated for I h at room temperature. Slides were washed three times with PBS; cells were incubated with goat anti-rabbit flourescein isothiocyanate (FITC) conjugate (1:500) for 1 b at room temperature. The slides were washed three times with PBS and analyzed by fluorescence microscope (LEICA DMIRB with fluorescence filter). Images were captured with a SPOT digital camera, Cell eyele analysis by fluorescence-activated cell sorting. Flow cytometry was carried out on a fluorescence-activated cell sorting (FACS) caliber instrument (Becton Dickinson, Franklin Lakes, NJ) with argon laser excitation at 488 nm as deseribed previously (Yu et al. 2003). In PBS, 110° cells (Control and achatininyr-treated cells) were washed once and fixed in 70% ethanol at ~20° C overnight, Fixed cells we washed and resuspended in a buffer containing 50 g/ml propidium Iodide and 1% Triton X-100, Pl-stained cells ‘were analyzed in the cytometer and the total DNA content in cach phase of the cell cycle was quantified Agarose-gel electrophoresis, For 20 min, 2*10° cells were lysed in STE buffer (0.9% sodium chloride, 10 mM Tris HCI, 10 mM EDTA, and 0.2% Triton X-100, pH 7.5) and the lysate was incubated with proteinase K overnight at 37° C. DNA was extracted as deseribed previously. Equal amounts of DNA from the control and achatinin-treated cells was Toaded in each lane on a 2% agarose gel and DNA bands were visualized by ethidium bromide staining after electrophoresis (Azmi et al. 2000)EFFECT OF ACHATININ, AGAINST A HUMAN MAMMARY CARCINOMA CELL LINE 309 ‘Statistical analysis, ‘The values are expressed as mean + SE. The results were computed statistically (SPSS software package, version 10.1, SPSS) using one-way analysis of variance. The differences were regarded as statistically significant at P-<0.05, Results Chtotoxicity assay. The cytotoxicity assay by MTT exhibits toxicity in a dose» and time-dependent manner (Fig. 1). In contrast, a significant proliferation was observed with the untreated cells. The reduction in cell proliferation was ob- served at a concentration of 6 ig, while 55% of cell death was observed with a concentration of 8 ug/ml. Complete killing was achieved at a concentration of 20 jig/ml (data not shown), Effect of achatininys on cell viability by cell count. Human mammary carcinoma cell lines (MCF?) treated with two different concentrations of achatininy, indicated a dose- dependent inhibition of cell proliferation, With 8 ug/ml a [oS ~ 2 : fe : ° is 0 2 2 ° Concentration of iy 12 10 Call count (510°) 1 2 3 Concentration of achatininy, Figure 1. Cytotoxic effect of acatininy on MCFT cells. (a) Cells were tsated with diferent concentrations (7 6 wgiml, 2 § sign) 3 10 git, 412 ug/ml, 514 pgm, and 616 usm) of achatininy for 24 and 4% b and analyzed by MTT assay. Test values are compared ‘with contol (1=6). Asterisks represent thatthe p value fess than 0.05 was signficnt. () MCF7 cells were teated with wo diferent concentrations (J contol, 2 ® pgiml, and 3 10 wim) of achat for 26 and 48 hand viable count by trypan blue was made. Test values ae compared with control (n=6). Asterisks represent thatthe p value Tess than 0.05 was significant 20), (@) Congo, (3) ells treated with acatininy for 24 and () lls tried ith achatiniy for 4 concentration, the cell count showed a 62% cell death, ‘whereas the control or untreated cells displayed exponential ‘growth (Fig. 14). Morphological changes were observed in the treated cells; they were shrunken, rounded, and retracted ftom the neighboring cells (Fig. 2). Gel electrophoresis and Western blot assay. The purified achatining, gave a single band on the native gel, and its molecular weight was estimated as 24.2 kDa, In the presence ‘of SDS and B-mercaptoethanol, the protein resolved into identical subunits with molecular weight 15,000 Da (Fig, 3a). In the Western blot, one major band was detected by the specific antibody (anti-achatinin,?) raised in rabbit, and this confirms the purity of the achatininy (Fig, 34) D springera0 DHARMU ET AL. Influence of achatininy, on tumor marker enzymes and antioxidants, The results on the activity of tumor marker en- zymes and antioxidants in both control and achatininy-treated MCF? cells is given in Fig. 4a and b. The results indicate that the activities of both the acid and alkaline phosphatase ‘were reduced to nearly 50% in the treated cells compared to that of untreated control cells. The activities of LDH, aspartate aminotansferase, and alanine aminotransferase were also significantly decreased in the treated cells, On the other hand, activities of glutathione peroxidase and CAT slightly increased in the treated cells compared to that of control. The level of LPO in the achatininy-treated MCF7 cells showed significant increase from 0,240.02 to 0.46 0.05. SOD activity was significantly decreased after achati- ning treatment when compared to control MCF7 eels. A B 212 116 97.2 66.4 14.3, Figure 3. Electrophoresis of purified achotininy, (a) Lane 4, molecular weight markers: 212, myosin; 116, B-galactosidase; 97.2, phosphorylase Bi 66.4, bovine serum albumin; and 14.3, ly ‘Lane B, SDS-PAGE without 8 ME; lane C, SDS-PAGE with B ME. (6) Wester bot of achatininy, with ante-achatinny raised in rabbits D Springer a 1 zoos z 06 aa 2 Bos g & ca 0 b Dtes Units / mg protein la Antioxidants Figure 4. (a) Tumor marker enzymes levels for both control and chatning (& piml}-reated MCF7 breast cancer coll Hine for 48 h. J Alkaline phosphatase, 2 acid phosphetase, 3 LDH, ¢ asparate Sminotransferate, and” 5 alaine aminotransferase, Test values are compared with control (n=3). Asterisk represent that the p value less ‘an 0.05 was significant. (@) Antioxidants levels for both contol and achatniny (8 ygim)}-reated MCF7 breast cance cll line for 48 h. SOD, 2 CAT, 3 glutathione peroxidase, and 4 LPO. Test values are compared with contol (n~3). Asterisk represent thatthe p value less ‘han 0.05 was significant Binding of achatininy; to MCF7, Binding of achatininy t© MCF7 cells is demonstrated in Fig. 5. The fluorescence intensity on the membrane of MCF? cells was visualized by the antilectin antibody (1:500) conjugated to FITC second- ary antibody. Almost all the MCET cells treated with achatininy; Muoresced and demonstrated a moderate to sttong staining (Fig. 5), indicating the presence of the aachatininys binding sites on MCF7 cells Cell evele analysis using flow cytometry. The flowicytom= cetry-hased assay exhibited the changes in cell eyele on tueatment with achatiniy. The percentage of cells in $ Phase after exposure to ICsp (8 pg/ml) of achatininy, was 12%, while it was 36% in untreated (absence of achatiin,) cells (Fig. 6). However, after 48 b, the Ga/M peak declined in the treated MCF7 cells conspicuously. In the untreated cells, the percentage of cells in GyyM phase (mitosis) was 18%. The process of cell division has been halted, as indicated by the presence of only 9% cells in Gy/M phase. It is also observed that 32% of the cells have entered theEFFECT OF ACHATININ,, AGAINST A HUMAN MAMMARY CARCINOMA CELL LINE sul Figure 5. Achatining binding to MCF7 breast cancer cell line detected by immunofuorescence assay. (a) Control, (8) MCFT cells treated with achatining for 48 f (megnficstion 205), and (e) cells treated with achatniny for 4 h (magnitieaion 60>). apoptotic phase, while it was 6% in untreated cells, in- dicating DNA damage. Agarose gel electrophoresis. DNA isolated fiom the achati- niny-treated cells was electrophoresed on a 2% agarose gel ‘to quantify DNA damage. Figure 7 is the clectophoretic profile of DNA, as evidenced by ethidium bromide staining. Lane 1 is the DNA ladder (100 bp), lane 2 is the DNA sample from 24-h achatinin-treated cells, lane 3 is the DNA sample fiom 48-h achatininy-treated cells, and lane 4 is the control MCF cells. The DNA damage in lanes 2 and 3 is nearly the same thus signifying that 24 h is sufficient for achatining to bring about damage of cellular DNA. Discussion In this study, achatininy-mediated cytotoxicity is described with respect to human mammary carcinoma cell line MCFT. Excellent linearity could be observed in the MTT assay between cell number and absorbance (OD) up to 3% 10° cells/well, Reduetion in eell proliferation was observed at a concentration of 6 yg of achatining. At 8 jig/ml concentration, the MTT assay showed 55% cell death, ‘whereas by cell count method the cell death observed was 62%, This small difference may be due to a reduced sensitivity by MTT. The MTT assay depends both on the umber of cells present and on the mitochondrial activity per cell, Earlier studies by Baker etal, (1979) demonstrated Gu/Gi= 4s soquiny 129 “00600 3300 DNA content 8 aaquiny IPD, DNA content lysis of DNA (a) contol MCFT cells and (6) (CFT eal, seo 3000 Figure 6. FACS schatninge-treated D springer32 DHARMU ET AL. Figure 7. Agave gel elcto- phorest af DNA isolated rom. MCT tested with achatining for 24 (lane 2), MCF tected with achatinny for 48 (ane 3), and onto (lane 4, DNA Tadder (100 bp) that concanavalinA-activated T-cells produced much less formazan than EL4, and in our study, achatininy (Iectin)- bound cells produced less formazan, The viability of MCF7 cells was affected inthe presence of achatininy, as it induced cytotoxicity in these cells. The cytotoxic action of achati- ning on MCE7 cells is of interest, due to the selective killing of only transformed cells. Achatininyy had no effe fon normal human fibroblasts even at high doses (data not shown). Similarly, the cytotoxicity of lectins derived from other mollusean species has been demonstrated with other types of cancer cel lines. (Schumacher et al. 1992; Thomas et al, 1993; Chitra et al, 1997) In the present study, the effect of achatininy, on MCFT cells showed increase in LPO indicating activation of the free radical scavenging enzymes such as SOD and CAT. The increase in CAT activity in achatininy-treated MCF? cells indicates that the treatment may help to lower H302 concentration by its decomposition and, hence, reduce D springs oxidative stress. The SOD activity decreased with achati- ning, treatment, It may be construed that the altered antioxidants and enzyme activity may lower the cAMP. concentration and decrease subsequent reduction of deox= yribonucleotides for DNA synthesis, which is a erucial step in neoplastic growth and tumorigenesis (Albert etal. 1990). In the present study, the LDH, acid and alkaline phospha- tase, and alanine aminowansferase activities also signifi- cantly (p<005) decreased in the treated MCF? cells compared to control (Fig, 46), LDU is a tetrameric enzyme and is recognized as a potential tumor marker in assessing the progression of the proliferating malignant cells. The elevated activity of LDH may be due to its overproduction by tumor cells (Mirmomeni et al, 1979), or it may be due to the release of isoenzymes from destroyed tissues (Engan and Hannisdal 1990), Recent studies also showed that LDH activity in the serum of DLA mice was elevated about 14- fold, and treatment with queuine decreased the activity significantly up to fourfold (Pathak and Manjula 2005), ‘The achatininy-caused antimitotic effect on cancer cells is evident in the cell cycle studies with MCF7 cells. Flow cytometry is one of the most powerful and specific methods for the integrated study of molecular and morphological events occurring during cell death and cell proliferation and ‘measures the total amount of DNA in cells (Darz et al 1997), FACS analysis of DNA from untreated cells and DNA from achatininy-treated cells was carried out, and Fig. 6a and b exhibit similar results. MCFT cells were blocked at S-phase after lectin treatment, and the percen age of cells in S-phase was 12%, whereas in untreated cells it was 36% after exposure for 48 h. A previous report indicated that 2-methoxyestradiol treatment of the estrogen- dependent cell line (MCE7) caused GyiM arrest associated ‘with the depolymerization of tubulin (Tishler et al. 1995; Attala et al. 1996), A lower percentage of MCF7 cells in S-phase and a lower percentage in G/M (metaphase) than the eonteols is depicted in Fig, 6. In S-phase, cell death was observed after 48 h of achatininy, exposure, which produces a low cell count in GyM phase (mitotic phase). It may be inferred from the above results that, although lectin binds to the receptor domains on the cell’s surface, the signal transduction occurs ceven in S-phase. One of the major significant observations is the amest in G-M phase, which is considered to be the ‘major checkpoint of the cell cycle. Appearance of the sub- GI peak is characteristic of apoptotic cells The extent of apoptosis is quantified by the formation of the typical DNA ladder (Khodarer et al. 1998), Figure 7 is aan electrophoretogram of DNA isolated from control and achatininy-treated MCF7 cells. Lanes 2 and 3 represent DNA isolated from cells treated with achatining, for different time periods. Some cells are known to show apparent morphological induction of apoptosis in theEFFECT OF ACHATININ, AGAINST A HUMAN MAMMARY CARCINOMA CELL LINE a3 absence of oligonucleosomal DNA fragmentation as a late event in the apoptotic process. There are some reports of MCF? cells producing a DNA ladder but others have demonstrated that MCF7 cells do not undergo apoptotic DNA fragmentation. In an attempt to clarify this confliting information, Gooch and Yee (1999) demonstrated that the ability of MCF? cells to undergo DNA laddering appears to be strain-speeitic. Taking cue from the above observations and reports, the present scenario signifies that achatinin, is very potent in inducing cellular damage as reflected by variation in total DNA content, The finding also suggests that achatinins may offer therapeutic utility as a new type of anticancer agent as it selectively inhibited mitosis in the fast-growing. ‘malignant cells. The ligand-binding function of achatinin,, as a homing molecule and molecular probe and the complement-mediated cell lysis of lectins have already been established (Chitra et al. 1997; Indra et al. 2000; Ramalingam et al. 2005), Acknowledgements One ofthe authors (D.L) thanks the Council of Sciemifie and Industrial Research for providing a fellowship to caryout the research work. The authors are thank to Dr. Rajeswa, Cancer Institute, WIA (Chenna), for her kind help in carrying aut FACS analysis The authors thank Dr. T. Ramasam:, Director, Central Leather Research Insitute, forhis kind permission to publish tis work. 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